Crystal Structure of a Human IκB Kinase β Asymmetric Dimer

PubMed Central

Liu, Shenping; Misquitta, Yohann R.; Olland, Andrea; Johnson, Mark A.; Kelleher, Kerry S.; Kriz, Ron; Lin, Laura L.; Stahl, Mark; Mosyak, Lidia

2013-01-01

Phosphorylation of inhibitor of nuclear transcription factor κB (IκB) by IκB kinase (IKK) triggers the degradation of IκB and migration of cytoplasmic κB to the nucleus where it promotes the transcription of its target genes. Activation of IKK is achieved by phosphorylation of its main subunit, IKKβ, at the activation loop sites. Here, we report the 2.8 Å resolution crystal structure of human IKKβ (hIKKβ), which is partially phosphorylated and bound to the staurosporine analog K252a. The hIKKβ protomer adopts a trimodular structure that closely resembles that from Xenopus laevis (xIKKβ): an N-terminal kinase domain (KD), a central ubiquitin-like domain (ULD), and a C-terminal scaffold/dimerization domain (SDD). Although hIKKβ and xIKKβ utilize a similar dimerization mode, their overall geometries are distinct. In contrast to the structure resembling closed shears reported previously for xIKKβ, hIKKβ exists as an open asymmetric dimer in which the two KDs are further apart, with one in an active and the other in an inactive conformation. Dimer interactions are limited to the C-terminal six-helix bundle that acts as a hinge between the two subunits. The observed domain movements in the structures of IKKβ may represent trans-phosphorylation steps that accompany IKKβ activation. PMID:23792959

N-glycosylation of the β2 adrenergic receptor regulates receptor function by modulating dimerization.

PubMed

Li, Xiaona; Zhou, Mang; Huang, Wei; Yang, Huaiyu

2017-07-01

N-glycosylation is a common post-translational modification of G-protein-coupled receptors (GPCRs). However, it remains unknown how N-glycosylation affects GPCR signaling. β 2 adrenergic receptor (β 2 AR) has three N-glycosylation sites: Asn6, Asn15 at the N-terminus, and Asn187 at the second extracellular loop (ECL2). Here, we show that deletion of the N-glycan did not affect receptor expression and ligand binding. Deletion of the N-glycan at the N-terminus rather than Asn187 showed decreased effects on isoproterenol-promoted G-protein-dependent signaling, β-arrestin2 recruitment, and receptor internalization. Both N6Q and N15Q showed decreased receptor dimerization, while N187Q did not influence receptor dimerization. As decreased β 2 AR homodimer accompanied with reduced efficiency for receptor function, we proposed that the N-glycosylation of β 2 AR regulated receptor function by influencing receptor dimerization. To verify this hypothesis, we further paid attention to the residues at the dimerization interface. Studies of Lys60 and Glu338, two residues at the receptor dimerization interface, exhibited that the K60A/E338A showed decreased β 2 AR dimerization and its effects on receptor signaling were similar to N6Q and N15Q, which further supported the importance of receptor dimerization for receptor function. This work provides new insights into the relationship among glycosylation, dimerization, and function of GPCRs. Peptide-N-glycosidase F (PNGase F, EC 3.2.2.11); endo-β-N-acetylglucosaminidase A (Endo-A, EC 3.2.1.96). © 2017 Federation of European Biochemical Societies.

The Globular Tail Domain of Myosin-5a Functions as a Dimer in Regulating the Motor Activity.

PubMed

Zhang, Wen-Bo; Yao, Lin-Lin; Li, Xiang-Dong

2016-06-24

Myosin-5a contains two heavy chains, which are dimerized via the coiled-coil regions. Thus, myosin-5a comprises two heads and two globular tail domains (GTDs). The GTD is the inhibitory domain that binds to the head and inhibits its motor function. Although the two-headed structure is essential for the processive movement of myosin-5a along actin filaments, little is known about the role of GTD dimerization. Here, we investigated the effect of GTD dimerization on its inhibitory activity. We found that the potent inhibitory activity of the GTD is dependent on its dimerization by the preceding coiled-coil regions, indicating synergistic interactions between the two GTDs and the two heads of myosin-5a. Moreover, we found that alanine mutations of the two conserved basic residues at N-terminal extension of the GTD not only weaken the inhibitory activity of the GTD but also enhance the activation of myosin-5a by its cargo-binding protein melanophilin (Mlph). These results are consistent with the GTD forming a head to head dimer, in which the N-terminal extension of the GTD interacts with the Mlph-binding site in the counterpart GTD. The Mlph-binding site at the GTD-GTD interface must be exposed prior to the binding of Mlph. We therefore propose that the inhibited Myo5a is equilibrated between the folded state, in which the Mlph-binding site is buried, and the preactivated state, in which the Mlph-binding site is exposed, and that Mlph is able to bind to the Myo5a in preactivated state and activates its motor function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A cyano-terminated dithienyldiketopyrrolopyrrole dimer as a solution processable ambipolar semiconductor under ambient conditions.

PubMed

Wang, Li; Zhang, Xiaojie; Tian, Hongkun; Lu, Yunfeng; Geng, Yanhou; Wang, Fosong

2013-12-14

A cyano-terminated dimer of dithienyldiketopyrrolopyrrole (TDPP), DPP2-CN, is a solution processable ambipolar semiconductor with field-effect hole and electron mobilities of 0.066 and 0.033 cm(2) V(-1) s(-1), respectively, under ambient conditions.

Structural basis for ligand-dependent dimerization of phenylalanine hydroxylase regulatory domain

PubMed Central

Patel, Dipali; Kopec, Jolanta; Fitzpatrick, Fiona; McCorvie, Thomas J.; Yue, Wyatt W.

2016-01-01

The multi-domain enzyme phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of dietary I-phenylalanine (Phe) to I-tyrosine. Inherited mutations that result in PAH enzyme deficiency are the genetic cause of the autosomal recessive disorder phenylketonuria. Phe is the substrate for the PAH active site, but also an allosteric ligand that increases enzyme activity. Phe has been proposed to bind, in addition to the catalytic domain, a site at the PAH N-terminal regulatory domain (PAH-RD), to activate the enzyme via an unclear mechanism. Here we report the crystal structure of human PAH-RD bound with Phe at 1.8 Å resolution, revealing a homodimer of ACT folds with Phe bound at the dimer interface. This work delivers the structural evidence to support previous solution studies that a binding site exists in the RD for Phe, and that Phe binding results in dimerization of PAH-RD. Consistent with our structural observation, a disease-associated PAH mutant impaired in Phe binding disrupts the monomer:dimer equilibrium of PAH-RD. Our data therefore support an emerging model of PAH allosteric regulation, whereby Phe binds to PAH-RD and mediates the dimerization of regulatory modules that would bring about conformational changes to activate the enzyme. PMID:27049649

Insights into the Structure of Dimeric RNA Helicase CsdA and Indispensable Role of Its C-Terminal Regions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Ling; Wang, Lijun; Peng, Junhui

CsdA has been proposed to be essential for the biogenesis of ribosome and gene regulation after cold shock. However, the structure of CsdA and the function of its long C-terminal regions are still unclear. For this study, we solved all of the domain structures of CsdA and found two previously uncharacterized auxiliary domains: a dimerization domain (DD) and an RNA-binding domain (RBD). Small-angle X-ray scattering experiments helped to track the conformational flexibilities of the helicase core domains and C-terminal regions. Biochemical assays revealed that DD is indispensable for stabilizing the CsdA dimeric structure. We also demonstrate for the first timemore » that CsdA functions as a stable dimer at low temperature. The C-terminal regions are critical for RNA binding and efficient enzymatic activities. CsdA_RBD could specifically bind to the regions with a preference for single-stranded G-rich RNA, which may help to bring the helicase core to unwind the adjacent duplex.« less

Insights into the Structure of Dimeric RNA Helicase CsdA and Indispensable Role of Its C-Terminal Regions.

PubMed

Xu, Ling; Wang, Lijun; Peng, Junhui; Li, Fudong; Wu, Lijie; Zhang, Beibei; Lv, Mengqi; Zhang, Jiahai; Gong, Qingguo; Zhang, Rongguang; Zuo, Xiaobing; Zhang, Zhiyong; Wu, Jihui; Tang, Yajun; Shi, Yunyu

2017-12-05

CsdA has been proposed to be essential for the biogenesis of ribosome and gene regulation after cold shock. However, the structure of CsdA and the function of its long C-terminal regions are still unclear. Here, we solved all of the domain structures of CsdA and found two previously uncharacterized auxiliary domains: a dimerization domain (DD) and an RNA-binding domain (RBD). Small-angle X-ray scattering experiments helped to track the conformational flexibilities of the helicase core domains and C-terminal regions. Biochemical assays revealed that DD is indispensable for stabilizing the CsdA dimeric structure. We also demonstrate for the first time that CsdA functions as a stable dimer at low temperature. The C-terminal regions are critical for RNA binding and efficient enzymatic activities. CsdA_RBD could specifically bind to the regions with a preference for single-stranded G-rich RNA, which may help to bring the helicase core to unwind the adjacent duplex. Copyright © 2017 Elsevier Ltd. All rights reserved.

Insights into the Structure of Dimeric RNA Helicase CsdA and Indispensable Role of Its C-Terminal Regions

DOE PAGES

Xu, Ling; Wang, Lijun; Peng, Junhui; ...

2017-12-05

CsdA has been proposed to be essential for the biogenesis of ribosome and gene regulation after cold shock. However, the structure of CsdA and the function of its long C-terminal regions are still unclear. For this study, we solved all of the domain structures of CsdA and found two previously uncharacterized auxiliary domains: a dimerization domain (DD) and an RNA-binding domain (RBD). Small-angle X-ray scattering experiments helped to track the conformational flexibilities of the helicase core domains and C-terminal regions. Biochemical assays revealed that DD is indispensable for stabilizing the CsdA dimeric structure. We also demonstrate for the first timemore » that CsdA functions as a stable dimer at low temperature. The C-terminal regions are critical for RNA binding and efficient enzymatic activities. CsdA_RBD could specifically bind to the regions with a preference for single-stranded G-rich RNA, which may help to bring the helicase core to unwind the adjacent duplex.« less

Mass spectrometric characterization of human serum albumin dimer: A new potential biomarker in chronic liver diseases.

PubMed

Naldi, Marina; Baldassarre, Maurizio; Nati, Marina; Laggetta, Maristella; Giannone, Ferdinando Antonino; Domenicali, Marco; Bernardi, Mauro; Caraceni, Paolo; Bertucci, Carlo

2015-08-10

Human serum albumin (HSA) undergoes several structural alterations affecting its properties in pro-oxidant and pro-inflammatory environments, as it occurs during liver cirrhosis. These modifications include the formation of albumin dimers. Although HSA dimers were reported to be an oxidative stress biomarker, to date nothing is known about their role in liver cirrhosis and related complications. Additionally, no high sensitive analytical method was available for HSA dimers assessment in clinical settings. Thus the HSA dimeric form in human plasma was characterized by mass spectrometry using liquid chromatography tandem mass spectrometry (LC-ESI-Q-TOF) and matrix assisted laser desorption time of flight (MALDI-TOF) techniques. N-terminal and C-terminal truncated HSA, as well as the native HSA, undergo dimerization by binding another HSA molecule. This study demonstrated the presence of both homo- and hetero-dimeric forms of HSA. The dimerization site was proved to be at Cys-34, forming a disulphide bridge between two albumin molecules, as determined by LC-MS analysis after tryptic digestion. Interestingly, when plasma samples from cirrhotic subjects were analysed, the dimer/monomer ratio resulted significantly increased when compared to that of healthy subjects. These isoforms could represent promising biomarkers for liver disease. Additionally, this analytical approach leads to the relative quantification of the residual native HSA, with fully preserved structural integrity. Copyright © 2014 Elsevier B.V. All rights reserved.

N-terminal domains of fibrillin 1 and fibrillin 2 direct the formation of homodimers: a possible first step in microfibril assembly.

PubMed Central

Trask, T M; Ritty, T M; Broekelmann, T; Tisdale, C; Mecham, R P

1999-01-01

Aggregation of fibrillin molecules via disulphide bonds is postulated to be an early step in microfibril assembly. By expressing fragments of fibrillin 1 and fibrillin 2 in a mammalian expression system, we found that the N-terminal region of each protein directs the formation of homodimers and that disulphide bonds stabilize this interaction. A large fragment of fibrillin 1 containing much of the region downstream from the N-terminus remained as a monomer when expressed in the same cell system, indicating that this region of the protein lacks dimerization domains. This finding also confirms that the overexpression of fibrillin fragments does not in itself lead to spurious dimer formation. Pulse-chase analysis demonstrated that dimer formation occurred intracellularly, suggesting that the process of fibrillin aggregation is initiated early after biosynthesis of the molecules. These findings also implicate the N-terminal region of fibrillin 1 and fibrillin 2 in directing the formation of a dimer intermediate that aggregates to form the functional microfibril. PMID:10359653

F99 is critical for dimerization and activation of South African HIV-1 subtype C protease.

PubMed

Naicker, Previn; Seele, Palesa; Dirr, Heini W; Sayed, Yasien

2013-10-01

HIV-1 protease (PR) is an obligate homodimer which plays a pivotal role in the maturation and hence propagation of HIV. Although successful developments on PR active site inhibitors have been achieved, the major limiting factor has been the emergence of HIV drug-resistant strains. Disruption of the dimer interface serves as an alternative mechanism to inactivate the enzyme. The terminal residue, F99, was mutated to an alanine to investigate its contribution to dimer stability in the South African HIV-1 subtype C (C-SA) PR. The F99A PR and wild-type C-SA PR were overexpressed and purified. The activities of the PRs and their ability to bind an active site inhibitor, acetyl-pepstatin, were determined in vitro. The F99A PR showed no activity and the inability to bind to the inhibitor. Secondary and quaternary structure analysis were performed and revealed that the F99A PR is monomeric with reduced β-sheet content. The mutation of F99 to alanine disrupted the presumed 'lock-and-key' motif at the terminal dimer interface, in turn creating a cavity at the N- and C-terminal antiparallel β-sheet. These findings support the design of inhibitors targeting the C-terminus of the C-SA PR, centered on interactions with the bulky F99.

A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis.

PubMed

Duggin, Iain G; Matthews, Jacqueline M; Dixon, Nicholas E; Wake, R Gerry; Mackay, Joel P

2005-04-01

Two dimers of the replication terminator protein (RTP) of Bacillus subtilis bind to a chromosomal DNA terminator site to effect polar replication fork arrest. Cooperative binding of the dimers to overlapping half-sites within the terminator is essential for arrest. It was suggested previously that polarity of fork arrest is the result of the RTP dimer at the blocking (proximal) side within the complex binding very tightly and the permissive-side RTP dimer binding relatively weakly. In order to investigate this "differential binding affinity" model, we have constructed a series of mutant terminators that contain half-sites of widely different RTP binding affinities in various combinations. Although there appeared to be a correlation between binding affinity at the proximal half-site and fork arrest efficiency in vivo for some terminators, several deviated significantly from this correlation. Some terminators exhibited greatly reduced binding cooperativity (and therefore have reduced affinity at each half-site) but were highly efficient in fork arrest, whereas one terminator had normal affinity over the proximal half-site, yet had low fork arrest efficiency. The results show clearly that there is no direct correlation between the RTP binding affinity (either within the full complex or at the proximal half-site within the full complex) and the efficiency of replication fork arrest in vivo. Thus, the differential binding affinity over the proximal and distal half-sites cannot be solely responsible for functional polarity of fork arrest. Furthermore, efficient fork arrest relies on features in addition to the tight binding of RTP to terminator DNA.

Solution structure of the c-terminal dimerization domain of SARS coronavirus nucleocapsid protein solved by the SAIL-NMR method.

PubMed

Takeda, Mitsuhiro; Chang, Chung-ke; Ikeya, Teppei; Güntert, Peter; Chang, Yuan-hsiang; Hsu, Yen-lan; Huang, Tai-huang; Kainosho, Masatsune

2008-07-18

The C-terminal domain (CTD) of the severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP) contains a potential RNA-binding region in its N-terminal portion and also serves as a dimerization domain by forming a homodimer with a molecular mass of 28 kDa. So far, the structure determination of the SARS-CoV NP CTD in solution has been impeded by the poor quality of NMR spectra, especially for aromatic resonances. We have recently developed the stereo-array isotope labeling (SAIL) method to overcome the size problem of NMR structure determination by utilizing a protein exclusively composed of stereo- and regio-specifically isotope-labeled amino acids. Here, we employed the SAIL method to determine the high-quality solution structure of the SARS-CoV NP CTD by NMR. The SAIL protein yielded less crowded and better resolved spectra than uniform (13)C and (15)N labeling, and enabled the homodimeric solution structure of this protein to be determined. The NMR structure is almost identical with the previously solved crystal structure, except for a disordered putative RNA-binding domain at the N-terminus. Studies of the chemical shift perturbations caused by the binding of single-stranded DNA and mutational analyses have identified the disordered region at the N-termini as the prime site for nucleic acid binding. In addition, residues in the beta-sheet region also showed significant perturbations. Mapping of the locations of these residues onto the helical model observed in the crystal revealed that these two regions are parts of the interior lining of the positively charged helical groove, supporting the hypothesis that the helical oligomer may form in solution.

Insights into PG-binding, conformational change, and dimerization of the OmpA C-terminal domains from Salmonella enterica serovar Typhimurium and Borrelia burgdorferi: Characterization of OmpA C-Terminal Domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Kemin; Deatherage Kaiser, Brooke L.; Wu, Ruiying

S. Typhimurium can induce both humoral and cell-mediated responses when establishing itself in the host. These responses are primarily stimulated against the lipopolysaccharide and major outer membrane (OM) proteins of the bacterium. OmpA is one of these major OM proteins. It comprises a N-terminal eight-stranded -barrel membrane domain and a C-terminal so-called OmpA C-terminal domain (OmpACTD). The OmpACTD and its homologs are believed to bind to peptidoglycan (PG) within the periplasm, maintaining bacterial osmotic homeostasis and modulating the permeability and integrity of the outer membrane. Here we present the structures of two forms of the OmpACTD of S. Typhimurium (STOmpACTD)more » and one structure of the less-studied OmpACTD of Borrelia burgdorferi (BbOmpACTD). In the open form of STOmpACTD, an aspartic acid residue from a long 2-3 loop points into the binding pocket, suggesting that an anion group such as a carboxylate group from PG is favored at the binding site. In the closed form of STOmpACTD and in the structure of BbOmpACTD, a sulfate group from the crystallization buffer is tightly bound at the equivalent site. The differences between the closed and open forms of STOmpACTD, suggest a large conformational change that includes an extension of 3 helix by ordering a part of 2-3 loop. We suggest that the sulfate anion observed in these structures mimics the carboxylate group of PG when bound to STOmpACTD. In addition, the binding of PG or a ligand mimic may enhance dimerization of STOmpACTD, or possibly that of full length STOmpA.« less

Effect of sodium chloride on the structure and stability of spider silk's N-terminal protein domain.

PubMed

Gronau, Greta; Qin, Zhao; Buehler, Markus J

2013-03-01

A spider's ability to store silk protein solutions at high concentration is believed to be related to the protein's terminal domains. It has been suggested that a shift in salt concentration and pH can have a significant influence on the assembly process. Based on experimental data, a model has been proposed in which the N-terminal domain exists as a monomer during storage and assembles into a homodimer upon spinning. Here we perform a systematic computational study using atomistic, coarse-grained and well-tempered metadynamics simulation to understand how the NaCl concentration in the solution affects the N-terminal domain of the silk protein. Our results show that a high salt concentration, as found during storage, weakens key salt bridges between the monomers, inducing a loss in bond energy by 28.6% in a single salt bridge. As a result dimer formation is less likely as 35.5% less energy is required to unfold the dimer by mechanical force. Conversely, homodimer formation appears to be more likely at low salt concentrations as the salt bridge stays at the lower energy state. The link between salt concentration, structure and stability of the N-terminal domain provides a possible mechanism that prevents premature fiber formation during storage.

Light activation of the LOV protein vivid generates a rapidly exchanging dimer.

PubMed

Zoltowski, Brian D; Crane, Brian R

2008-07-08

The fungal photoreceptor Vivid (VVD) plays an important role in the adaptation of blue-light responses in Neurospora crassa. VVD, an FAD-binding LOV (light, oxygen, voltage) protein, couples light-induced cysteinyl adduct formation at the flavin ring to conformational changes in the N-terminal cap (Ncap) of the VVD PAS domain. Size-exclusion chromatography (SEC), equilibrium ultracentrifugation, and static and dynamic light scattering show that these conformational changes generate a rapidly exchanging VVD dimer, with an expanded hydrodynamic radius. A three-residue N-terminal beta-turn that assumes two different conformations in a crystal structure of a VVD C71V variant is essential for light-state dimerization. Residue substitutions at a critical hinge between the Ncap and PAS core can inhibit or enhance dimerization, whereas a Tyr to Trp substitution at the Ncap-PAS interface stabilizes the light-state dimer. Cross-linking through engineered disulfides indicates that the light-state dimer differs considerably from the dark-state dimer found in VVD crystal structures. These results verify the role of Ncap conformational changes in gating the photic response of N. crassa and indicate that LOV-LOV homo- or heterodimerization may be a mechanism for regulating light-activated gene expression.

Diacylglycerol Acyltransferase 1 Is Regulated by Its N-Terminal Domain in Response to Allosteric Effectors.

PubMed

Caldo, Kristian Mark P; Acedo, Jeella Z; Panigrahi, Rashmi; Vederas, John C; Weselake, Randall J; Lemieux, M Joanne

2017-10-01

Diacylglycerol acyltransferase 1 (DGAT1) is an integral membrane enzyme catalyzing the final and committed step in the acyl-coenzyme A (CoA)-dependent biosynthesis of triacylglycerol (TAG). The biochemical regulation of TAG assembly remains one of the least understood areas of primary metabolism to date. Here, we report that the hydrophilic N-terminal domain of Brassica napus DGAT1 (BnaDGAT1 1-113 ) regulates activity based on acyl-CoA/CoA levels. The N-terminal domain is not necessary for acyltransferase activity and is composed of an intrinsically disordered region and a folded segment. We show that the disordered region has an autoinhibitory function and a dimerization interface, which appears to mediate positive cooperativity, whereas the folded segment of the cytosolic region was found to have an allosteric site for acyl-CoA/CoA. Under increasing acyl-CoA levels, the binding of acyl-CoA with this noncatalytic site facilitates homotropic allosteric activation. Enzyme activation, on the other hand, is prevented under limiting acyl-CoA conditions (low acyl-CoA-to-CoA ratio), whereby CoA acts as a noncompetitive feedback inhibitor through interaction with the same folded segment. The three-dimensional NMR solution structure of the allosteric site revealed an α-helix with a loop connecting a coil fragment. The conserved amino acid residues in the loop interacting with CoA were identified, revealing details of this important regulatory element for allosteric regulation. Based on these results, a model is proposed illustrating the role of the N-terminal domain of BnaDGAT1 as a positive and negative modulator of TAG biosynthesis. © 2017 American Society of Plant Biologists. All Rights Reserved.

The Nudix Hydrolase CDP-Chase, a CDP-Choline Pyrophosphatase, Is an Asymmetric Dimer with Two Distinct Enzymatic Activities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duong-Ly, Krisna C.; Gabelli, Sandra B.; Xu, WenLian

2011-09-06

A Nudix enzyme from Bacillus cereus catalyzes the hydrolysis of CDP-choline to produce CMP and phosphocholine. Here, we show that in addition, the enzyme has a 3{prime} {yields} 5{prime} RNA exonuclease activity. The structure of the free enzyme, determined to a 1.8-{angstrom} resolution, shows that the enzyme is an asymmetric dimer. Each monomer consists of two domains, an N-terminal helical domain and a C-terminal Nudix domain. The N-terminal domain is placed relative to the C-terminal domain such as to result in an overall asymmetric arrangement with two distinct catalytic sites: one with an 'enclosed' Nudix pyrophosphatase site and the othermore » with a more open, less-defined cavity. Residues that may be important for determining the asymmetry are conserved among a group of uncharacterized Nudix enzymes from Gram-positive bacteria. Our data support a model where CDP-choline hydrolysis is catalyzed by the enclosed Nudix site and RNA exonuclease activity is catalyzed by the open site. CDP-Chase is the first identified member of a novel Nudix family in which structural asymmetry has a profound effect on the recognition of substrates.« less

Effect of sodium chloride on the structure and stability of spider silk’s N-terminal protein domain

PubMed Central

Gronau, Greta; Qin, Zhao; Buehler, Markus J.

2013-01-01

A spider’s ability to store silk protein solutions at high concentration is believed to be related to the protein’s terminal domains. It has been suggested that a shift in salt concentration and pH can have a significant influence on the assembly process. Based on experimental data, a model has been proposed in which the N-terminal domain exists as a monomer during storage and assembles into a homodimer upon spinning. Here we perform a systematic computational study using atomistic, coarse-grained and well-tempered metadynamics simulation to understand how the NaCl concentration in the solution affects the N-terminal domain of the silk protein. Our results show that a high salt concentration, as found during storage, weakens key salt bridges between the monomers, inducing a loss in bond energy by 28.6% in a single salt bridge. As a result dimer formation is less likely as 35.5% less energy is required to unfold the dimer by mechanical force. Conversely, homodimer formation appears to be more likely at low salt concentrations as the salt bridge stays at the lower energy state. The link between salt concentration, structure and stability of the N-terminal domain provides a possible mechanism that prevents premature fiber formation during storage. PMID:23833703

Active Site Flexibility of Mycobacterium tuberculosis Isocitrate Lyase in Dimer Form.

PubMed

Lee, Yie-Vern; Choi, Sy Bing; Wahab, Habibah A; Choong, Yee Siew

2017-09-25

Tuberculosis (TB) still remains a global threat due to the emergence of a drug-resistant strain. Instead of focusing on the drug target of active stage TB, we are highlighting the isocitrate lyase (ICL) at the dormant stage TB. ICL is one of the persistent factors for Mycobacterium tuberculosis (MTB) to survive during the dormant phase. In addition, the absence of ICL in human has made ICL a potential drug target for TB therapy. However, the dynamic details of ICL which could give insights to the ICL-ligand interaction have yet to be solved. Therefore, a series of ICL dimer dynamics studies through molecular dynamics simulation were performed in this work. The ICL active site entrance gate closure is contributed to by hydrogen bonding and electrostatic interactions with the C-terminal. Analysis suggested that the open-closed behavior of the ICL active site entrance depends on the type of ligand present in the active site. We also observed four residues (Ser91, Asp108, Asp153, and Cys191) which could possibly be the nucleophiles for nucleophilic attack on the cleavage of isocitrate at the C 2 -C 3 bond. We hope that the elucidation of ICL dynamics can benefit future works such as lead identification or antibody design against ICL for TB therapeutics.

Structural and functional insights into the role of the N-terminal Mps1 TPR domain in the SAC (spindle assembly checkpoint).

PubMed

Thebault, Philippe; Chirgadze, Dimitri Y; Dou, Zhen; Blundell, Tom L; Elowe, Sabine; Bolanos-Garcia, Victor M

2012-12-15

The SAC (spindle assembly checkpoint) is a surveillance system that ensures the timely and accurate transmission of the genetic material to offspring. The process implies kinetochore targeting of the mitotic kinases Bub1 (budding uninhibited by benzamidine 1), BubR1 (Bub1 related) and Mps1 (monopolar spindle 1), which is mediated by the N-terminus of each kinase. In the present study we report the 1.8 Å (1 Å=0.1 nm) crystal structure of the TPR (tetratricopeptide repeat) domain in the N-terminal region of human Mps1. The structure reveals an overall high similarity to the TPR motif of the mitotic checkpoint kinases Bub1 and BubR1, and a number of unique features that include the absence of the binding site for the kinetochore structural component KNL1 (kinetochore-null 1; blinkin), and determinants of dimerization. Moreover, we show that a stretch of amino acids at the very N-terminus of Mps1 is required for dimer formation, and that interfering with dimerization results in mislocalization and misregulation of kinase activity. The results of the present study provide an important insight into the molecular details of the mitotic functions of Mps1 including features that dictate substrate selectivity and kinetochore docking.

The N-terminal domain of GluR6-subtype glutamate receptor ion channels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Janesh; Schuck, Peter; Jin, Rongsheng

2009-09-25

The amino-terminal domain (ATD) of glutamate receptor ion channels, which controls their selective assembly into AMPA, kainate and NMDA receptor subtypes, is also the site of action of NMDA receptor allosteric modulators. Here we report the crystal structure of the ATD from the kainate receptor GluR6. The ATD forms dimers in solution at micromolar protein concentrations and crystallizes as a dimer. Unexpectedly, each subunit adopts an intermediate extent of domain closure compared to the apo and ligand-bound complexes of LIVBP and G protein-coupled glutamate receptors (mGluRs), and the dimer assembly has a markedly different conformation from that found in mGluRs.more » This conformation is stabilized by contacts between large hydrophobic patches in the R2 domain that are absent in NMDA receptors, suggesting that the ATDs of individual glutamate receptor ion channels have evolved into functionally distinct families.« less

Critical Structural and Functional Roles for the N-Terminal Insertion Sequence in Surfactant Protein B Analogs

PubMed Central

Walther, Frans J.; Waring, Alan J.; Hernandez-Juviel, Jose M.; Gordon, Larry M.; Wang, Zhengdong; Jung, Chun-Ling; Ruchala, Piotr; Clark, Andrew P.; Smith, Wesley M.; Sharma, Shantanu; Notter, Robert H.

2010-01-01

Background Surfactant protein B (SP-B; 79 residues) belongs to the saposin protein superfamily, and plays functional roles in lung surfactant. The disulfide cross-linked, N- and C-terminal domains of SP-B have been theoretically predicted to fold as charged, amphipathic helices, suggesting their participation in surfactant activities. Earlier structural studies with Mini-B, a disulfide-linked construct based on the N- and C-terminal regions of SP-B (i.e., ∼residues 8–25 and 63–78), confirmed that these neighboring domains are helical; moreover, Mini-B retains critical in vitro and in vivo surfactant functions of the native protein. Here, we perform similar analyses on a Super Mini-B construct that has native SP-B residues (1–7) attached to the N-terminus of Mini-B, to test whether the N-terminal sequence is also involved in surfactant activity. Methodology/Results FTIR spectra of Mini-B and Super Mini-B in either lipids or lipid-mimics indicated that these peptides share similar conformations, with primary α-helix and secondary β-sheet and loop-turns. Gel electrophoresis demonstrated that Super Mini-B was dimeric in SDS detergent-polyacrylamide, while Mini-B was monomeric. Surface plasmon resonance (SPR), predictive aggregation algorithms, and molecular dynamics (MD) and docking simulations further suggested a preliminary model for dimeric Super Mini-B, in which monomers self-associate to form a dimer peptide with a “saposin-like” fold. Similar to native SP-B, both Mini-B and Super Mini-B exhibit in vitro activity with spread films showing near-zero minimum surface tension during cycling using captive bubble surfactometry. In vivo, Super Mini-B demonstrates oxygenation and dynamic compliance that are greater than Mini-B and compare favorably to full-length SP-B. Conclusion Super Mini-B shows enhanced surfactant activity, probably due to the self-assembly of monomer peptide into dimer Super Mini-B that mimics the functions and putative structure of

Structures of the N-acetyltransferase domain of Xylella fastidiosa N-acetyl-L-glutamate synthase/kinase with and without a His tag bound to N-acetyl-L-glutamate.

PubMed

Zhao, Gengxiang; Jin, Zhongmin; Allewell, Norma M; Tuchman, Mendel; Shi, Dashuang

2015-01-01

Structures of the catalytic N-acetyltransferase (NAT) domain of the bifunctional N-acetyl-L-glutamate synthase/kinase (NAGS/K) from Xylella fastidiosa bound to N-acetyl-L-glutamate (NAG) with and without an N-terminal His tag have been solved and refined at 1.7 and 1.4 Å resolution, respectively. The NAT domain with an N-terminal His tag crystallized in space group P4(1)2(1)2, with unit-cell parameters a=b=51.72, c=242.31 Å. Two subunits form a molecular dimer in the asymmetric unit, which contains ∼41% solvent. The NAT domain without an N-terminal His tag crystallized in space group P21, with unit-cell parameters a=63.48, b=122.34, c=75.88 Å, β=107.6°. Eight subunits, which form four molecular dimers, were identified in the asymmetric unit, which contains ∼38% solvent. The structures with and without the N-terminal His tag provide an opportunity to evaluate how the His tag affects structure and function. Furthermore, multiple subunits in different packing environments allow an assessment of the plasticity of the NAG binding site, which might be relevant to substrate binding and product release. The dimeric structure of the X. fastidiosa N-acetytransferase (xfNAT) domain is very similar to that of human N-acetyltransferase (hNAT), reinforcing the notion that mammalian NAGS is evolutionally derived from bifunctional bacterial NAGS/K.

Dimerization and phosphatase activity of calcyclin-binding protein/Siah-1 interacting protein: the influence of oxidative stress

PubMed Central

Topolska-Woś, Agnieszka M.; Shell, Steven M.; Kilańczyk, Ewa; Szczepanowski, Roman H.; Chazin, Walter J.; Filipek, Anna

2015-01-01

CacyBP/SIP [calcyclin-binding protein/Siah-1 [seven in absentia homolog 1 (Siah E3 ubiquitin protein ligase 1)] interacting protein] is a multifunctional protein whose activity includes acting as an ERK1/2 phosphatase. We analyzed dimerization of mouse CacyBP/SIP in vitro and in mouse neuroblastoma cell line (NB2a) cells, as well as the structure of a full-length protein. Moreover, we searched for the CacyBP/SIP domain important for dimerization and dephosphorylation of ERK2, and we analyzed the role of dimerization in ERK1/2 signaling in NB2a cells. Cell-based assays showed that CacyBP/SIP forms a homodimer in NB2a cell lysate, and biophysical methods demonstrated that CacyBP/SIP forms a stable dimer in vitro. Data obtained using small-angle X-ray scattering supported a model in which CacyBP/SIP occupies an anti-parallel orientation mediated by the N-terminal dimerization domain. Site-directed mutagenesis established that the N-terminal domain is indispensable for full phosphatase activity of CacyBP/SIP. We also demonstrated that the oligomerization state of CacyBP/SIP as well as the level of post-translational modifications and subcellular distribution of CacyBP/SIP change after activation of the ERK1/2 pathway in NB2a cells due to oxidative stress. Together, our results suggest that dimerization is important for controlling phosphatase activity of CacyBP/SIP and for regulating the ERK1/2 signaling pathway.—Topolska-Woś, A. M., Shell, S. M., Kilańczyk, E., Szczepanowski, R. H., Chazin, W. J., Filipek, A. Dimerization and phosphatase activity of calcyclin-binding protein/Siah-1 interacting protein: the influence of oxidative stress. PMID:25609429

C-terminal Lysine-Linked Magainin 2 with Increased Activity Against Multidrug-Resistant Bacteria.

PubMed

Lorenzón, Esteban N; Santos-Filho, Norival A; Ramos, Matheus A S; Bauab, Tais M; Camargo, Ilana L B C; Cilli, Eduardo M

2016-01-01

Due to the growing problem of antibiotic-resistant microorganisms, the development of novel antimicrobial agents is a very important challenge. Dimerization of cationic antimicrobial peptides (cAMPs) is a potential strategy for enhancing antimicrobial activity. Here, we studied the effects of magainin 2 (MG2) dimerization on its structure and biological activity. Lysine and glutamic acid were used to synthesize the C- and N-terminal dimers of MG2, respectively, in order to evaluate the impact of linker position used to obtain the dimers. Both MG2 and its dimeric versions showed a random coil structure in aqueous solution. However, in the presence of a structure-inducing solvent or a membrane mimetic, all peptides acquired helical structure. N-terminal dimerization did not affect the biological activity of the peptide. On the other hand, the C-terminal dimer, (MG2)2K, showed antimicrobial activity 8-16 times higher than that of MG2, and the time required to kill Escherichia coli was lower. The enhanced antimicrobial activity was related to membrane permeabilization. (MG2)2K was also more active against multidrug-resistant bacteria of clinical origin. Overall, the results presented here demonstrate that C-terminal lysine-linked dimerization improve the activity of MG2, and (MG2)2K can be considered as a potential antimicrobial agent.

Elucidation of the Hsp90 C-terminal Inhibitor Binding Site

PubMed Central

Matts, Robert L.; Dixit, Anshuman; Peterson, Laura B.; Sun, Liang; Voruganti, Sudhakar; Kalyanaraman, Palgunan; Hartson, Steve D.; Verkhivker, Gennady M.; Blagg, Brian S. J.

2011-01-01

The Hsp90 chaperone machine is required for the folding, activation and/or stabilization of more than 50 proteins directly related to malignant progression. Hsp90 contains small molecule binding sites at both its N- and C-terminal domains, however, limited structural and biochemical data regarding the C-terminal binding site is available. In this report, the small molecule binding site in the Hsp90 C-terminal domain was revealed by protease fingerprinting and photoaffinity labeling utilizing LC-MS/MS. The identified site was characterized by generation of a homology model for hHsp90α using the SAXS open structure of HtpG and docking the bioactive conformation of NB into the generated model. The resulting model for the bioactive conformation of NB bound to Hsp90α is presented herein. PMID:21548602

A Designed Angiopoietin-1 Variant, Dimeric CMP-Ang1 Activates Tie2 and Stimulates Angiogenesis and Vascular Stabilization in N-glycan Dependent Manner

PubMed Central

Oh, Nuri; Kim, Kangsan; Jin Kim, Soo; Park, Intae; Lee, Jung-Eun; Suk Seo, Young; Joo An, Hyun; Min Kim, Ho; Young Koh, Gou

2015-01-01

Angiopoietin-1 (Ang1), a potential growth factor for therapeutic angiogenesis and vascular stabilization, is known to specifically cluster and activate Tie2 in high oligomeric forms, which is a unique and essential process in this ligand-receptor interaction. However, highly oligomeric native Ang1 and Ang1 variants are difficult to produce, purify, and store in a stable and active form. To overcome these limitations, we developed a simple and active dimeric CMP-Ang1 by replacing the N-terminal of native Ang1 with the coiled-coil domain of cartilage matrix protein (CMP) bearing mutations in its cysteine residues. This dimeric CMP-Ang1 effectively increased the migration, survival, and tube formation of endothelial cells via Tie2 activation. Furthermore, dimeric CMP-Ang1 induced angiogenesis and suppressed vascular leakage in vivo. Despite its dimeric structure, the potencies of such Tie2-activation-induced effects were comparable to those of a previously engineered protein, COMP-Ang1. We also revealed that these effects of dimeric CMP-Ang1 were affected by specified N-glycosylation in its fibrinogen-like domain. Taken together, our results indicate that dimeric CMP-Ang1 is capable of activating Tie2 and stimulating angiogenesis in N-glycan dependent manner. PMID:26478188

Fe65-PTB2 Dimerization Mimics Fe65-APP Interaction.

PubMed

Feilen, Lukas P; Haubrich, Kevin; Strecker, Paul; Probst, Sabine; Eggert, Simone; Stier, Gunter; Sinning, Irmgard; Konietzko, Uwe; Kins, Stefan; Simon, Bernd; Wild, Klemens

2017-01-01

Physiological function and pathology of the Alzheimer's disease causing amyloid precursor protein (APP) are correlated with its cytosolic adaptor Fe65 encompassing a WW and two phosphotyrosine-binding domains (PTBs). The C-terminal Fe65-PTB2 binds a large portion of the APP intracellular domain (AICD) including the GYENPTY internalization sequence fingerprint. AICD binding to Fe65-PTB2 opens an intra-molecular interaction causing a structural change and altering Fe65 activity. Here we show that in the absence of the AICD, Fe65-PTB2 forms a homodimer in solution and determine its crystal structure at 2.6 Å resolution. Dimerization involves the unwinding of a C-terminal α-helix that mimics binding of the AICD internalization sequence, thus shielding the hydrophobic binding pocket. Specific dimer formation is validated by nuclear magnetic resonance (NMR) techniques and cell-based analyses reveal that Fe65-PTB2 together with the WW domain are necessary and sufficient for dimerization. Together, our data demonstrate that Fe65 dimerizes via its APP interaction site, suggesting that besides intra- also intermolecular interactions between Fe65 molecules contribute to homeostatic regulation of APP mediated signaling.

Fe65-PTB2 Dimerization Mimics Fe65-APP Interaction

PubMed Central

Feilen, Lukas P.; Haubrich, Kevin; Strecker, Paul; Probst, Sabine; Eggert, Simone; Stier, Gunter; Sinning, Irmgard; Konietzko, Uwe; Kins, Stefan; Simon, Bernd; Wild, Klemens

2017-01-01

Physiological function and pathology of the Alzheimer’s disease causing amyloid precursor protein (APP) are correlated with its cytosolic adaptor Fe65 encompassing a WW and two phosphotyrosine-binding domains (PTBs). The C-terminal Fe65-PTB2 binds a large portion of the APP intracellular domain (AICD) including the GYENPTY internalization sequence fingerprint. AICD binding to Fe65-PTB2 opens an intra-molecular interaction causing a structural change and altering Fe65 activity. Here we show that in the absence of the AICD, Fe65-PTB2 forms a homodimer in solution and determine its crystal structure at 2.6 Å resolution. Dimerization involves the unwinding of a C-terminal α-helix that mimics binding of the AICD internalization sequence, thus shielding the hydrophobic binding pocket. Specific dimer formation is validated by nuclear magnetic resonance (NMR) techniques and cell-based analyses reveal that Fe65-PTB2 together with the WW domain are necessary and sufficient for dimerization. Together, our data demonstrate that Fe65 dimerizes via its APP interaction site, suggesting that besides intra- also intermolecular interactions between Fe65 molecules contribute to homeostatic regulation of APP mediated signaling. PMID:28553201

The tripartite motif coiled-coil is an elongated antiparallel hairpin dimer.

PubMed

Sanchez, Jacint G; Okreglicka, Katarzyna; Chandrasekaran, Viswanathan; Welker, Jordan M; Sundquist, Wesley I; Pornillos, Owen

2014-02-18

Tripartite motif (TRIM) proteins make up a large family of coiled-coil-containing RING E3 ligases that function in many cellular processes, particularly innate antiviral response pathways. Both dimerization and higher-order assembly are important elements of TRIM protein function, but the atomic details of TRIM tertiary and quaternary structure have not been fully understood. Here, we present crystallographic and biochemical analyses of the TRIM coiled-coil and show that TRIM proteins dimerize by forming interdigitating antiparallel helical hairpins that position the N-terminal catalytic RING domains at opposite ends of the dimer and the C-terminal substrate-binding domains at the center. The dimer core comprises an antiparallel coiled-coil with a distinctive, symmetric pattern of flanking heptad and central hendecad repeats that appear to be conserved across the entire TRIM family. Our studies reveal how the coiled-coil organizes TRIM25 to polyubiquitylate the RIG-I/viral RNA recognition complex and how dimers of the TRIM5α protein are arranged within hexagonal arrays that recognize the HIV-1 capsid lattice and restrict retroviral replication.

The tripartite motif coiled-coil is an elongated antiparallel hairpin dimer

PubMed Central

Sanchez, Jacint G.; Okreglicka, Katarzyna; Chandrasekaran, Viswanathan; Welker, Jordan M.; Sundquist, Wesley I.; Pornillos, Owen

2014-01-01

Tripartite motif (TRIM) proteins make up a large family of coiled-coil-containing RING E3 ligases that function in many cellular processes, particularly innate antiviral response pathways. Both dimerization and higher-order assembly are important elements of TRIM protein function, but the atomic details of TRIM tertiary and quaternary structure have not been fully understood. Here, we present crystallographic and biochemical analyses of the TRIM coiled-coil and show that TRIM proteins dimerize by forming interdigitating antiparallel helical hairpins that position the N-terminal catalytic RING domains at opposite ends of the dimer and the C-terminal substrate-binding domains at the center. The dimer core comprises an antiparallel coiled-coil with a distinctive, symmetric pattern of flanking heptad and central hendecad repeats that appear to be conserved across the entire TRIM family. Our studies reveal how the coiled-coil organizes TRIM25 to polyubiquitylate the RIG-I/viral RNA recognition complex and how dimers of the TRIM5α protein are arranged within hexagonal arrays that recognize the HIV-1 capsid lattice and restrict retroviral replication. PMID:24550273

The N-terminal tropomyosin- and actin-binding sites are important for leiomodin 2's function.

PubMed

Ly, Thu; Moroz, Natalia; Pappas, Christopher T; Novak, Stefanie M; Tolkatchev, Dmitri; Wooldridge, Dayton; Mayfield, Rachel M; Helms, Gregory; Gregorio, Carol C; Kostyukova, Alla S

2016-08-15

Leiomodin is a potent actin nucleator related to tropomodulin, a capping protein localized at the pointed end of the thin filaments. Mutations in leiomodin-3 are associated with lethal nemaline myopathy in humans, and leiomodin-2-knockout mice present with dilated cardiomyopathy. The arrangement of the N-terminal actin- and tropomyosin-binding sites in leiomodin is contradictory and functionally not well understood. Using one-dimensional nuclear magnetic resonance and the pointed-end actin polymerization assay, we find that leiomodin-2, a major cardiac isoform, has an N-terminal actin-binding site located within residues 43-90. Moreover, for the first time, we obtain evidence that there are additional interactions with actin within residues 124-201. Here we establish that leiomodin interacts with only one tropomyosin molecule, and this is the only site of interaction between leiomodin and tropomyosin. Introduction of mutations in both actin- and tropomyosin-binding sites of leiomodin affected its localization at the pointed ends of the thin filaments in cardiomyocytes. On the basis of our new findings, we propose a model in which leiomodin regulates actin poly-merization dynamics in myocytes by acting as a leaky cap at thin filament pointed ends. © 2016 Ly, Moroz, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Identification of succinimide sites in proteins by N-terminal sequence analysis after alkaline hydroxylamine cleavage.

PubMed Central

Kwong, M. Y.; Harris, R. J.

1994-01-01

Under favorable conditions, Asp or Asn residues can undergo rearrangement to a succinimide (cyclic imide), which may also serve as an intermediate for deamidation and/or isoaspartate formation. Direct identification of such succinimides by peptide mapping is hampered by their lability at neutral and alkaline pH. We determined that incubation in 2 M hydroxylamine, 0.2 M Tris buffer, pH 9, for 2 h at 45 degrees C will specifically cleave on the C-terminal side of succinimides without cleavage at Asn-Gly bonds; yields are typically approximately 50%. N-terminal sequence analysis can then be used to identify an internal sequence generated by cleavage of the succinimide, hence identifying the succinimide site. PMID:8142891

Influence of Dimerization of Lipopeptide Laur-Orn-Orn-Cys-NH2 and an N-terminal Peptide of Human Lactoferricin on Biological Activity.

PubMed

Kamysz, Elżbieta; Sikorska, Emilia; Dawgul, Małgorzata; Tyszkowski, Rafał; Kamysz, Wojciech

Lactoferrin (LF) is a naturally occurring antimicrobial peptide that is cleaved by pepsin to lactoferricin (LFcin). LFcin has an enhanced antimicrobial activity as compared to that of LF. Recently several hetero- and homodimeric antimicrobial peptides stabilized by a single disulfide bond linking linear polypeptide chains have been discovered. We have demonstrated that the S-S bond heterodimerization of lipopeptide Laur-Orn-Orn-Cys-NH 2 (peptide III) and the synthetic N -terminal peptide of human lactoferricin (peptide I) yields a dimer (peptide V), which is almost as microbiologically active as the more active monomer and at the same time it is much less toxic. Furthermore, it has been found that the S-S bond homodimerization of both peptide I and peptide III did not affect antimicrobial and haemolytic activity of the compounds. The homo- and heterodimerization of peptides I and III resulted in either reduction or loss of antifungal activity. This work suggests that heterodimerization of antimicrobial lipopeptides via intermolecular disulfide bond might be a powerful modification deserving consideration in the design of antimicrobial peptides.

Solution structure of dimeric Mnt repressor (1-76).

PubMed

Burgering, M J; Boelens, R; Gilbert, D E; Breg, J N; Knight, K L; Sauer, R T; Kaptein, R

1994-12-20

Wild-type Mnt repressor of Salmonella bacteriophage P22 is a tetrameric protein of 82 residues per monomer. A C-terminal deletion mutant of the repressor denoted Mnt (1-76) is a dimer in solution. The structure of this dimer has been determined using NMR. The NMR assignments of the majority of the 1H, 15N, and 13C resonances were obtained using 2D and triple-resonance 3D techniques. Elements of secondary structure were identified on the basis of characteristic sequential and medium range NOEs. For the structure determination more than 1000 NOEs per monomer were obtained, and structures were generated using distance geometry and restrained simulated annealing calculations. The discrimination of intra- vs intermonomer NOEs was based upon the observation of intersubunit NOEs in [15N,13C] double half-filtered NOESY experiments. The N-terminal part of Mnt (residues 1-44), which shows a 40% sequence homology with the Arc repressor, has a similar secondary and tertiary structure. Mnt (1-76) continues with a loop region of irregular structure, a third alpha-helix, and a random coil C-terminal peptide. Analysis of the secondary structure NOEs, the exchange rates, and the backbone chemical shifts suggests that the carboxy-terminal third helix is less stable than the remainder of the protein, but the observation of intersubunit NOEs for this part of the protein enables the positioning of this helix. The rsmd's between the backbone atoms of the N-terminal part of the Mnt repressor (residues 5-43, 5'-43') and the Arc repressor is 1.58 A, and between this region and the corresponding part of the MetJ repressor 1.43 A.

High-resolution Crystal Structure of Dimeric VP40 From Sudan ebolavirus.

PubMed

Clifton, Matthew C; Bruhn, Jessica F; Atkins, Kateri; Webb, Terry L; Baydo, Ruth O; Raymond, Amy; Lorimer, Donald D; Edwards, Thomas E; Myler, Peter J; Saphire, Erica Ollmann

2015-10-01

Ebolaviruses cause severe hemorrhagic fever. Central to the Ebola life cycle is the matrix protein VP40, which oligomerizes and drives viral budding. Here we present the crystal structure of the Sudan virus (SUDV) matrix protein. This structure is higher resolution (1.6 Å) than previously achievable. Despite differences in the protein purification, we find that it still forms a stable dimer in solution, as was noted for other Ebola VP40s. Although the N-terminal domain interface by which VP40 dimerizes is conserved between Ebola virus and SUDV, the C-terminal domain interface by which VP40 dimers may further assemble is significantly smaller in this SUDV assembly. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Role of Protein Dimeric Interface in Allosteric Inhibition of N-Acetyl-Aspartate Hydrolysis by Human Aspartoacylase.

PubMed

Kots, Ekaterina D; Lushchekina, Sofya V; Varfolomeev, Sergey D; Nemukhin, Alexander V

2017-08-28

The results of molecular modeling suggest a mechanism of allosteric inhibition upon hydrolysis of N-acetyl-aspartate (NAA), one of the most abundant amino acid derivatives in brain, by human aspartoacylase (hAsp). Details of this reaction are important to suggest the practical ways to control the enzyme activity. Search for allosteric sites using the Allosite web server and SiteMap analysis allowed us to identify substrate binding pockets located at the interface between the subunits of the hAsp dimer molecule. Molecular docking of NAA to the pointed areas at the dimer interface predicted a specific site, in which the substrate molecule interacts with the Gly237, Arg233, Glu290, and Lys292 residues. Analysis of multiple long-scaled molecular dynamics trajectories (the total simulation time exceeded 1.5 μs) showed that binding of NAA to the identified allosteric site induced significant rigidity to the protein loops with the amino acid side chains forming gates to the enzyme active site. Application of the protein dynamical network algorithms showed that substantial reorganization of the signal propagation pathways of intersubunit communication in the dimer occurred upon allosteric NAA binding to the remote site. The modeling approaches provide an explanation to the observed decrease of the reaction rate of NAA hydrolysis by hAsp at high substrate concentrations.

Implication of the oligomeric state of the N-terminal PTX3 domain in cumulus matrix assembly

PubMed Central

Ievoli, Elena; Lindstedt, Ragnar; Inforzato, Antonio; Camaioni, Antonella; Palone, Francesca; Day, Anthony J.; Mantovani, Alberto; Salvatori, Giovanni; Salustri, Antonietta

2011-01-01

Pentraxin 3 (PTX3) plays a key role in the formation of the hyaluronan-rich matrix of the cumulus oophorus surrounding ovulated eggs that is required for successful fertilization and female fertility. PTX3 is a multimeric protein consisting of eight identical protomers held together by a combination of non-covalent interactions and disulfide bonds. Recent findings suggest that the oligomeric status of PTX3 is important for stabilizing the cumulus matrix. Because the role of PTX3 in the cumulus resides in the unique N-terminal sequence of the protomer, we investigated further this issue by testing the ability of distinct Cys/Ser mutants of recombinant N-terminal region of PTX3 (N_PTX3) with different oligomeric arrangement to promote in vitro normal expansion in cumuli from Ptx3-null mice. Here we report that the dimer of the N_PTX3 is unable to rescue cumulus matrix organization, and that the tetrameric assembly of the protein is the minimal oligomeric state required for accomplishing this function. We have previously demonstrated that PTX3 binds to HCs of IαI and TSG-6, which are essential for cumulus matrix formation and able to interact with hyaluronan. Interestingly, here we show by solid-phase binding experiments that the dimer of the N_PTX3 retains the ability to bind to both IαI and TSG-6, suggesting that the octameric structure of PTX3 provides multiple binding sites for each of these ligands. These findings support the hypothesis that PTX3 contributes to cumulus matrix organization by cross-linking HA polymers through interactions with multiple HCs of IαI and/or TSG-6. The N-terminal PTX3 tetrameric oligomerization was recently reported to be also required for recognition and inhibition of FGF2. Given that this growth factor has been detected in the mammalian preovulatory follicle, we wondered whether FGF2 negatively influences cumulus expansion and PTX3 may also serve in vivo to antagonize its activity. We found that a molar excess of FGF2, above PTX3

Functional Roles of the Non-Catalytic Calcium-Binding Sites in the N-Terminal Domain of Human Peptidylarginine Deiminase 4

PubMed Central

Liu, Yi-Liang; Tsai, I-Chen; Chang, Chia-Wei; Liao, Ya-Fan; Liu, Guang-Yaw; Hung, Hui-Chih

2013-01-01

This study investigated the functional roles of the N-terminal Ca2+ ion-binding sites, in terms of enzyme catalysis and stability, of peptidylarginine deiminase 4 (PAD4). Amino acid residues located in the N-terminal Ca2+-binding site of PAD4 were mutated to disrupt the binding of Ca2+ ions. Kinetic data suggest that Asp155, Asp157 and Asp179, which directly coordinate Ca3 and Ca4, are essential for catalysis in PAD4. For D155A, D157A and D179A, the k cat/K m,BAEE values were 0.02, 0.63 and 0.01 s−1mM−1 (20.8 s−1mM−1 for WT), respectively. Asn153 and Asp176 are directly coordinated with Ca3 and indirectly coordinated with Ca5 via a water molecule. However, N153A displayed low enzymatic activity with a k cat value of 0.3 s−1 (13.3 s−1 for wild-type), whereas D176A retained some catalytic power with a k cat of 9.7 s−1. Asp168 is the direct ligand for Ca5, and Ca5 coordination by Glu252 is mediated by two water molecules. However, mutation of these two residues to Ala did not cause a reduction in the k cat/K m,BAEE values, which indicates that the binding of Ca5 may not be required for PAD4 enzymatic activity. The possible conformational changes of these PAD4 mutants were examined. Thermal stability analysis of the PAD4 mutants in the absence or presence of Ca2+ indicated that the conformational stability of the enzyme is highly dependent on Ca2+ ions. In addition, the results of urea-induced denaturation for the N153, D155, D157 and D179 series mutants further suggest that the binding of Ca2+ ions in the N-terminal Ca2+-binding site stabilizes the overall conformational stability of PAD4. Therefore, our data strongly suggest that the N-terminal Ca2+ ions play critical roles in the full activation of the PAD4 enzyme. PMID:23382808

Insights into PG-binding, conformational change, and dimerization of the OmpA C-terminal domains from Salmonella enterica serovar Typhimurium and Borrelia burgdorferi: Characterization of OmpA C-Terminal Domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Kemin; Deatherage Kaiser, Brooke L.; Wu, Ruiying

S. Typhimurium can induce both humoral and cell-mediated responses when establishing itself in the host. These responses are primarily stimulated against the lipopolysaccharide and major outer membrane (OM) proteins. OmpA is one of these major OM proteins. It comprises a N-terminal eight-stranded b-barrel trans membrane domain and a C-terminal domain (OmpACTD). The OmpACTD and its homologs are believed to bind to peptidoglycan (PG) within the periplasm, maintaining bacterial osmotic homeostasis and modulating the permeability and integrity of the OM. Here we present the first crystal structures of the OmpACTD from two pathogens: S. Typhimurium (STOmpACTD) in open and closed formsmore » and causative agent of Lyme Disease Borrelia burgdorferi (BbOmpACTD), in closed form. In the open form of STOmpACTD, an aspartic acid residue from a long b2-a3 loop points into the binding pocket, suggesting that an anion group such as a carboxylate group from PG is favored at the binding site. In the closed form of STOmpACTD and in the structure of BbOmpACTD, a sulfate group from the crystallization buffer is tightly bound at the binding site. The differences between the closed and open forms of STOmpACTD, suggest a large conformational change that includes an extension of a3 helix by ordering a part of b2-a3 loop. We propose that the sulfate anion observed in these structures mimics the carboxylate group of PG when bound to STOmpACTD suggesting PG-anchoring mechanism. In addition, the binding of PG or a ligand mimic may enhance dimerization of STOmpACTD, or possibly that of full length STOmpA.« less

Dimerization of nitrophorin 4 at low pH and comparison to the K1A mutant of nitrophorin 1.

PubMed

Berry, Robert E; Yang, Fei; Shokhireva, Tatiana K; Amoia, Angela M; Garrett, Sarah A; Goren, Allena M; Korte, Stephanie R; Zhang, Hongjun; Weichsel, Andrzej; Montfort, William R; Walker, F Ann

2015-01-20

Nitrophorin 4, one of the four NO-carrying heme proteins from the salivary glands of Rhodnius prolixus, forms a homodimer at pH 5.0 with a Kd of ∼8 μM. This dimer begins to dissociate at pH 5.5 and is completely dissociated to monomer at pH 7.3, even at 3.7 mM. The dimer is significantly stabilized by binding NO to the heme and at pH 7.3 would require dilution to well below 0.2 mM to completely dissociate the NP4-NO homodimer. The primary techniques used for investigating the homodimer and the monomer-dimer equilibrium were size-exclusion fast protein liquid chromatography at pH 5.0 and (1)H{(15)N} heteronuclear single-quantum coherence spectroscopy as a function of pH and concentration. Preparation of site-directed mutants of NP4 (A1K, D30A, D30N, V36A/D129A/L130A, K38A, R39A, K125A, K125E, D132A, L133V, and K38Q/R39Q/K125Q) showed that the N-terminus, D30, D129, D132, at least one heme propionate, and, by association, likely also E32 and D35 are involved in the dimerization. The "closed loop" form of the A-B and G-H flexible loops of monomeric NP4, which predominates in crystal structures of the monomeric protein reported at pH 5.6 but not at pH 7.5 and which involves all of the residues listed above except D132, is required for dimer formation. Wild-type NP1 does not form a homodimer, but NP1(K1A) and native N-terminal NP1 form dimers in the presence of NO. The homodimer of NP1, however, is considerably less stable than that of NP4 in the absence of NO. This suggests that additional aspartate or glutamate residues present in the C-terminal region of NP4, but not NP1, are also involved in stabilizing the dimer.

Dimerization of Nitrophorin 4 at Low pH and Comparison to the K1A Mutant of Nitrophorin 1

PubMed Central

2015-01-01

Nitrophorin 4, one of the four NO-carrying heme proteins from the salivary glands of Rhodnius prolixus, forms a homodimer at pH 5.0 with a Kd of ∼8 μM. This dimer begins to dissociate at pH 5.5 and is completely dissociated to monomer at pH 7.3, even at 3.7 mM. The dimer is significantly stabilized by binding NO to the heme and at pH 7.3 would require dilution to well below 0.2 mM to completely dissociate the NP4-NO homodimer. The primary techniques used for investigating the homodimer and the monomer–dimer equilibrium were size-exclusion fast protein liquid chromatography at pH 5.0 and 1H{15N} heteronuclear single-quantum coherence spectroscopy as a function of pH and concentration. Preparation of site-directed mutants of NP4 (A1K, D30A, D30N, V36A/D129A/L130A, K38A, R39A, K125A, K125E, D132A, L133V, and K38Q/R39Q/K125Q) showed that the N-terminus, D30, D129, D132, at least one heme propionate, and, by association, likely also E32 and D35 are involved in the dimerization. The “closed loop” form of the A–B and G–H flexible loops of monomeric NP4, which predominates in crystal structures of the monomeric protein reported at pH 5.6 but not at pH 7.5 and which involves all of the residues listed above except D132, is required for dimer formation. Wild-type NP1 does not form a homodimer, but NP1(K1A) and native N-terminal NP1 form dimers in the presence of NO. The homodimer of NP1, however, is considerably less stable than that of NP4 in the absence of NO. This suggests that additional aspartate or glutamate residues present in the C-terminal region of NP4, but not NP1, are also involved in stabilizing the dimer. PMID:25489673

Dimerization of Nitrophorin 4 at Low pH and Comparison to the K1A Mutant of Nitrophorin 1

DOE PAGES

Berry, Robert E.; Yang, Fei; Shokhireva, Tatiana K.; ...

2014-12-09

Nitrophorin 4, one of the four NO-carrying heme proteins from the salivary glands of Rhodnius prolixus, forms a homodimer at pH 5.0 with a K d of ~8 μM. This dimer begins to dissociate at pH 5.5 and is completely dissociated to monomer at pH 7.3, even at 3.7 mM. The dimer is significantly stabilized by binding NO to the heme and at pH 7.3 would require dilution to well below 0.2 mM to completely dissociate the NP4-NO homodimer. The primary techniques used for investigating the homodimer and the monomer–dimer equilibrium were size-exclusion fast protein liquid chromatography at pH 5.0more » and 1H{ 15N} heteronuclear single-quantum coherence spectroscopy as a function of pH and concentration. Preparation of site-directed mutants of NP4 (A1K, D30A, D30N, V36A/D129A/L130A, K38A, R39A, K125A, K125E, D132A, L133V, and K38Q/R39Q/K125Q) showed that the N-terminus, D30, D129, D132, at least one heme propionate, and, by association, likely also E32 and D35 are involved in the dimerization. The “closed loop” form of the A–B and G–H flexible loops of monomeric NP4, which predominates in crystal structures of the monomeric protein reported at pH 5.6 but not at pH 7.5 and which involves all of the residues listed above except D132, is required for dimer formation. Wild-type NP1 does not form a homodimer, but NP1(K1A) and native N-terminal NP1 form dimers in the presence of NO. Lastly, the homodimer of NP1, however, is considerably less stable than that of NP4 in the absence of NO. This suggests that additional aspartate or glutamate residues present in the C-terminal region of NP4, but not NP1, are also involved in stabilizing the dimer.« less

Hydrolysis at One of the Two Nucleotide-binding Sites Drives the Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zoghbi, M. E.; Altenberg, G. A.

The functional unit of ATP-binding cassette (ABC) transporters consists of two transmembrane domains and two nucleotide-binding domains (NBDs). ATP binding elicits association of the two NBDs, forming a dimer in a head-to-tail arrangement, with two nucleotides “sandwiched” at the dimer interface. Each of the two nucleotide-binding sites is formed by residues from the two NBDs. We recently found that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii dimerizes in response to ATP binding and dissociates completely following ATP hydrolysis. However, it is still unknown whether dissociation of NBD dimers follows ATP hydrolysis at one or both nucleotide-binding sites. Here, we usedmore » luminescence resonance energy transfer to study heterodimers formed by one active (donor-labeled) and one catalytically defective (acceptor-labeled) NBD. Rapid mixing experiments in a stop-flow chamber showed that NBD heterodimers with one functional and one inactive site dissociated at a rate indistinguishable from that of dimers with two hydrolysis-competent sites. Comparison of the rates of NBD dimer dissociation and ATP hydrolysis indicated that dissociation followed hydrolysis of one ATP. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dimer dissociation.« less

SARS-CoV 3CL protease cleaves its C-terminal autoprocessing site by novel subsite cooperativity.

PubMed

Muramatsu, Tomonari; Takemoto, Chie; Kim, Yong-Tae; Wang, Hongfei; Nishii, Wataru; Terada, Takaho; Shirouzu, Mikako; Yokoyama, Shigeyuki

2016-11-15

The 3C-like protease (3CL pro ) of severe acute respiratory syndrome coronavirus (SARS-CoV) cleaves 11 sites in the polyproteins, including its own N- and C-terminal autoprocessing sites, by recognizing P4-P1 and P1'. In this study, we determined the crystal structure of 3CL pro with the C-terminal prosequence and the catalytic-site C145A mutation, in which the enzyme binds the C-terminal prosequence of another molecule. Surprisingly, Phe at the P3' position [Phe(P3')] is snugly accommodated in the S3' pocket. Mutations of Phe(P3') impaired the C-terminal autoprocessing, but did not affect N-terminal autoprocessing. This difference was ascribed to the P2 residue, Phe(P2) and Leu(P2), in the C- and N-terminal sites, as follows. The S3' subsite is formed by Phe(P2)-induced conformational changes of 3CL pro and the direct involvement of Phe(P2) itself. In contrast, the N-terminal prosequence with Leu(P2) does not cause such conformational changes for the S3' subsite formation. In fact, the mutation of Phe(P2) to Leu in the C-terminal autoprocessing site abolishes the dependence on Phe(P3'). These mechanisms explain why Phe is required at the P3' position when the P2 position is occupied by Phe rather than Leu, which reveals a type of subsite cooperativity. Moreover, the peptide consisting of P4-P1 with Leu(P2) inhibits protease activity, whereas that with Phe(P2) exhibits a much smaller inhibitory effect, because Phe(P3') is missing. Thus, this subsite cooperativity likely exists to avoid the autoinhibition of the enzyme by its mature C-terminal sequence, and to retain the efficient C-terminal autoprocessing by the use of Phe(P2).

Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hastings, Adam F.; Otting, Gottfried; Folmer, Rutger H.A.

2005-09-23

Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29 kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in {sup 1}H-{sup 15}N correlation spectra of a {sup 15}N-labelled RTP.C110S mutant upon the addition of a 21 base pair symmetrical DNA binding site. Assignment of the {sup 1}H-{sup 15}N correlations was achieved using a suite of triple resonance NMR experiments with {sup 15}N,{sup 13}C,70% {sup 2}H enriched protein recorded at 800 MHz and using TROSY pulse sequences. Perturbationsmore » to {sup 1}H-{sup 15}N spectra revealed that the N-termini, {alpha}3-helices and several loops are affected by the binding interaction. An analysis of this data in light of the crystallographically determined apo- and DNA-bound forms of RTP.C110S revealed that the NMR spectral perturbations correlate more closely to protein structural changes upon complex formation rather than to interactions at the protein-DNA interface.« less

Acetylation within the N- and C-Terminal Domains of Src Regulates Distinct Roles of STAT3-Mediated Tumorigenesis.

PubMed

Huang, Chao; Zhang, Zhe; Chen, Lihan; Lee, Hank W; Ayrapetov, Marina K; Zhao, Ting C; Hao, Yimei; Gao, Jinsong; Yang, Chunzhang; Mehta, Gautam U; Zhuang, Zhengping; Zhang, Xiaoren; Hu, Guohong; Chin, Y Eugene

2018-06-01

Posttranslational modifications of mammalian c-Src N-terminal and C-terminal domains regulate distinct functions. Myristoylation of G 2 controls its cell membrane association and phosphorylation of Y419/Y527 controls its activation or inactivation, respectively. We provide evidence that Src-cell membrane association-dissociation and catalytic activation-inactivation are both regulated by acetylation. In EGF-treated cells, CREB binding protein (CBP) acetylates an N-terminal lysine cluster (K5, K7, and K9) of c-Src to promote dissociation from the cell membrane. CBP also acetylates the C-terminal K401, K423, and K427 of c-Src to activate intrinsic kinase activity for STAT3 recruitment and activation. N-terminal domain phosphorylation (Y14, Y45, and Y68) of STAT3 by c-Src activates transcriptionally active dimers of STAT3. Moreover, acetyl-Src translocates into nuclei, where it forms the Src-STAT3 enhanceosome for gene regulation and cancer cell proliferation. Thus, c-Src acetylation in the N-terminal and C-terminal domains play distinct roles in Src activity and regulation. Significance: CBP-mediated acetylation of lysine clusters in both the N-terminal and C-terminal regions of c-Src provides additional levels of control over STAT3 transcriptional activity. Cancer Res; 78(11); 2825-38. ©2018 AACR . ©2018 American Association for Cancer Research.

Characterization of local polarity and hydrophobic binding sites of beta-lactoglobulin by using N-terminal specific fluorescence labeling.

PubMed

Dong, Su-Ying; Zhao, Zhen-Wen; Ma, Hui-Min

2006-01-01

Because of wide ligand-binding ability and significant industrial interest of beta-lactoglobulin (beta-LG), its binding properties have been extensively studied. However, there still exists a controversy as to where a ligand binds, since at least two potential hydrophobic binding sites in beta-LG have been postulated for ligand binding: an internal one (calyx) and an external one (near the N-terminus). In this work, the local polarity and hydrophobic binding sites of beta-LG have been characterized by using N-terminal specific fluorescence labeling combined with a polarity-sensitive fluorescent probe 3-(4-chloro-6-hydrazino- 1,3,5-triazinylamino)-7-(dimethylamino)-2-methylphenazine (CHTDP). The polarity within the calyx is found to be extremely low, which is explained in terms of superhydrophobicity possibly resulting from its nanostructure, and the polarity is increased with the destruction of the calyx by heat treatment. However, the polarity of the N-terminal domain in native beta-LG is decreased after thermal denaturation. This polarity trend toward decreasing instead of increasing shows that beta-LG may have no definite external hydrophobic binding site. The hydrophobic binding of a ligand such as CHTDP at the surface of the protein is probably achieved via appropriate assembling of corresponding hydrophobic residues rather than via a fixed external hydrophobic binding site. Also, the ligand-binding location in beta-LG is found to be relevant to not only experimental conditions (pH < or = 6.2 or pH > 7.1) but also binding mechanisms (hydrophobic affinity or electrostatic interaction).

A competent catalytic active site is necessary for substrate induced dimer assembly in triosephosphate isomerase.

PubMed

Jimenez-Sandoval, Pedro; Vique-Sanchez, Jose Luis; Hidalgo, Marisol López; Velazquez-Juarez, Gilberto; Diaz-Quezada, Corina; Arroyo-Navarro, Luis Fernando; Moran, Gabriela Montero; Fattori, Juliana; Jessica Diaz-Salazar, A; Rudiño-Pinera, Enrique; Sotelo-Mundo, Rogerio; Figueira, Ana Carolina Migliorini; Lara-Gonzalez, Samuel; Benítez-Cardoza, Claudia G; Brieba, Luis G

2017-11-01

The protozoan parasite Trichomonas vaginalis contains two nearly identical triosephosphate isomerases (TvTIMs) that dissociate into stable monomers and dimerize upon substrate binding. Herein, we compare the role of the "ball and socket" and loop 3 interactions in substrate assisted dimer assembly in both TvTIMs. We found that point mutants at the "ball" are only 39 and 29-fold less catalytically active than their corresponding wild-type counterparts, whereas Δloop 3 deletions are 1502 and 9400-fold less active. Point and deletion mutants dissociate into stable monomers. However, point mutants assemble as catalytic competent dimers upon binding of the transition state substrate analog PGH, whereas loop 3 deletions remain monomeric. A comparison between crystal structures of point and loop 3 deletion monomeric mutants illustrates that the catalytic residues in point mutants and wild-type TvTIMs are maintained in the same orientation, whereas the catalytic residues in deletion mutants show an increase in thermal mobility and present structural disorder that may hamper their catalytic role. The high enzymatic activity present in monomeric point mutants correlates with the formation of dimeric TvTIMs upon substrate binding. In contrast, the low activity and lack of dimer assembly in deletion mutants suggests a role of loop 3 in promoting the formation of the active site as well as dimer assembly. Our results suggest that in TvTIMs the active site is assembled during dimerization and that the integrity of loop 3 and ball and socket residues is crucial to stabilize the dimer. Copyright © 2017 Elsevier B.V. All rights reserved.

Crystal structure, biochemical and genetic characterization of yeast and E. cuniculi TAF(II)5 N-terminal domain: implications for TFIID assembly.

PubMed

Romier, Christophe; James, Nicole; Birck, Catherine; Cavarelli, Jean; Vivarès, Christian; Collart, Martine A; Moras, Dino

2007-05-18

General transcription factor TFIID plays an essential role in transcription initiation by RNA polymerase II at numerous promoters. However, understanding of the assembly and a full structural characterization of this large 15 subunit complex is lacking. TFIID subunit TAF(II)5 has been shown to be present twice in this complex and to be critical for the function and assembly of TFIID. Especially, the TAF(II)5 N-terminal domain is required for its incorporation within TFIID and immuno-labelling experiments carried out by electron microscopy at low resolution have suggested that this domain might homodimerize, possibly explaining the three-lobed architecture of TFIID. However, the resolution at which the electron microscopy (EM) analyses were conducted is not sufficient to determine whether homodimerization occurs or whether a more intricate assembly implying other subunits is required. Here we report the X-ray structures of the fully evolutionary conserved C-terminal sub-domain of the TAF(II)5 N terminus, from yeast and the mammalian parasite Encephalitozoon cuniculi. This sub-domain displays a novel fold with specific surfaces having conserved physico-chemical properties that can form protein-protein interactions. Although a crystallographic dimer implying one of these surfaces is present in one of the crystal forms, several biochemical analyses show that this sub-domain is monomeric in solution, even at various salt conditions and in presence of different divalent cations. Consequently, the N-terminal sub-domain of the TAF(II)5 N terminus, which is homologous to a dimerization motif but has not been fully conserved during evolution, was studied by analytical ultracentrifugation and yeast genetics. Our results show that this sub-domain dimerizes at very high concentration but is neither required for yeast viability, nor for incorporation of two TAF(II)5 molecules within TFIID and for the assembly of this complex. Altogether, although our results do not argue in

The N-terminal Part of Arabidopsis thaliana Starch Synthase 4 Determines the Localization and Activity of the Enzyme.

PubMed

Raynaud, Sandy; Ragel, Paula; Rojas, Tomás; Mérida, Ángel

2016-05-13

Starch synthase 4 (SS4) plays a specific role in starch synthesis because it controls the number of starch granules synthesized in the chloroplast and is involved in the initiation of the starch granule. We showed previously that SS4 interacts with fibrillins 1 and is associated with plastoglobules, suborganelle compartments physically attached to the thylakoid membrane in chloroplasts. Both SS4 localization and its interaction with fibrillins 1 were mediated by the N-terminal part of SS4. Here we show that the coiled-coil region within the N-terminal portion of SS4 is involved in both processes. Elimination of this region prevents SS4 from binding to fibrillins 1 and alters SS4 localization in the chloroplast. We also show that SS4 forms dimers, which depends on a region located between the coiled-coil region and the glycosyltransferase domain of SS4. This region is highly conserved between all SS4 enzymes sequenced to date. We show that the dimerization seems to be necessary for the activity of the enzyme. Both dimerization and the functionality of the coiled-coil region are conserved among SS4 proteins from phylogenetically distant species, such as Arabidopsis and Brachypodium This finding suggests that the mechanism of action of SS4 is conserved among different plant species. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A Phytophthora sojae effector PsCRN63 forms homo-/hetero-dimers to suppress plant immunity via an inverted association manner.

PubMed

Li, Qi; Zhang, Meixiang; Shen, Danyu; Liu, Tingli; Chen, Yanyu; Zhou, Jian-Min; Dou, Daolong

2016-05-31

Oomycete pathogens produce a large number of effectors to promote infection. Their mode of action are largely unknown. Here we show that a Phytophthora sojae effector, PsCRN63, suppresses flg22-induced expression of FRK1 gene, a molecular marker in pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI). However, PsCRN63 does not suppress upstream signaling events including flg22-induced MAPK activation and BIK1 phosphorylation, indicating that it acts downstream of MAPK cascades. The PsCRN63-transgenic Arabidopsis plants showed increased susceptibility to bacterial pathogen Pseudomonas syringae pathovar tomato (Pst) DC3000 and oomycete pathogen Phytophthora capsici. The callose deposition were suppressed in PsCRN63-transgenic plants compared with the wild-type control plants. Genes involved in PTI were also down-regulated in PsCRN63-transgenic plants. Interestingly, we found that PsCRN63 forms an dimer that is mediated by inter-molecular interactions between N-terminal and C-terminal domains in an inverted association manner. Furthermore, the N-terminal and C-terminal domains required for the dimerization are widely conserved among CRN effectors, suggesting that homo-/hetero-dimerization of Phytophthora CRN effectors is required to exert biological functions. Indeed, the dimerization was required for PTI suppression and cell death-induction activities of PsCRN63.

Conformation changes, N-terminal involvement and cGMP signal relay in phosphodiesterase-5 GAF domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, H.; Robinson, H.; Ke, H.

2010-12-03

The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, whichmore » may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98-147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes.« less

Molecular evidence of stereo-specific lactoferrin dimers in solution.

PubMed

Persson, Björn A; Lund, Mikael; Forsman, Jan; Chatterton, Dereck E W; Akesson, Torbjörn

2010-10-01

Gathering experimental evidence suggests that bovine as well as human lactoferrin self-associate in aqueous solution. Still, a molecular level explanation is unavailable. Using force field based molecular modeling of the protein-protein interaction free energy we demonstrate (1) that lactoferrin forms highly stereo-specific dimers at neutral pH and (2) that the self-association is driven by a high charge complementarity across the contact surface of the proteins. Our theoretical predictions of dimer formation are verified by electrophoretic mobility and N-terminal sequence analysis on bovine lactoferrin. 2010 Elsevier B.V. All rights reserved.

Involvement of the N-terminal part of cyclophilin B in the interaction with specific Jurkat T-cell binding sites.

PubMed

Mariller, C; Haendler, B; Allain, F; Denys, A; Spik, G

1996-07-15

Cyclophilin B (CyPB) is secreted in biological fluids such as blood or milk and binds to a specific receptor present on the human lymphoblastic cell line Jurkat and on human peripheral blood lymphocytes. This study was intended to specify the areas of CyPB that are involved in the interaction with the receptor. A synthetic peptide corresponding to the first 24 N-terminal amino acid residues of CyPB was shown to specifically recognize the receptor. Moreover, modification of Arg18 of CyPB by p-hydroxyphenlglyoxal led to a dramatic loss of affinity for the receptor. However, when this residue was replaced by an alanine residue using site-directed mutagenesis, no modification of the binding properties was found, suggesting that Arg18 is not directly involved but is sufficiently close to the interaction site to interfere with the binding when modified. Competitive binding experiments using a chimaeric protein made up of the 24 N-terminal amino acid residues of CyPB fused to the cyclophilin A core sequence confirmed the involvement of this region of CyPB in receptor binding.

Glycosylation Alters Dimerization Properties of a Cell-surface Signaling Protein, Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1)*

PubMed Central

Zhuo, You; Yang, Jeong-Yeh; Moremen, Kelley W.; Prestegard, James H.

2016-01-01

Human carcinoembryonic antigen-related cell adhesion molecule 1 (C?/Au: EACAM1) is a cell-surface signaling molecule involved in cell adhesion, proliferation, and immune response. It is also implicated in cancer angiogenesis, progression, and metastasis. This diverse set of effects likely arises as a result of the numerous homophilic and heterophilic interactions that CEACAM1 can have with itself and other molecules. Its N-terminal Ig variable (IgV) domain has been suggested to be a principal player in these interactions. Previous crystal structures of the β-sandwich-like IgV domain have been produced using Escherichia coli-expressed material, which lacks native glycosylation. These have led to distinctly different proposals for dimer interfaces, one involving interactions of ABED β-strands and the other involving GFCC′C″ β-strands, with the former burying one prominent glycosylation site. These structures raise questions as to which form may exist in solution and what the effect of glycosylation may have on this form. Here, we use NMR cross-correlation measurements to examine the effect of glycosylation on CEACAM1-IgV dimerization and use residual dipolar coupling (RDC) measurements to characterize the solution structure of the non-glycosylated form. Our findings demonstrate that even addition of a single N-linked GlcNAc at potential glycosylation sites inhibits dimer formation. Surprisingly, RDC data collected on E. coli expressed material in solution indicate that a dimer using the non-glycosylated GFCC′C″ interface is preferred even in the absence of glycosylation. The results open new questions about what other factors may facilitate dimerization of CEACAM1 in vivo, and what roles glycosylation may play in heterophylic interactions. PMID:27471271

Protein sequence analysis, cloning, and expression of flammutoxin, a pore-forming cytolysin from Flammulina velutipes. Maturation of dimeric precursor to monomeric active form by carboxyl-terminal truncation.

PubMed

Tomita, Toshio; Mizumachi, Yoshihiro; Chong, Kang; Ogawa, Kanako; Konishi, Norihide; Sugawara-Tomita, Noriko; Dohmae, Naoshi; Hashimoto, Yohichi; Takio, Koji

2004-12-24

Flammutoxin (FTX), a 31-kDa pore-forming cytolysin from Flammulina velutipes, is specifically expressed during the fruiting body formation. We cloned and expressed the cDNA encoding a 272-residue protein with an identical N-terminal sequence with that of FTX but failed to obtain hemolytically active protein. This, together with the presence of multiple FTX family proteins in the mushroom, prompted us to determine the complete primary structure of FTX by protein sequence analysis. The N-terminal 72 and C-terminal 107 residues were sequenced by Edman degradation of the fragments generated from the alkylated FTX by enzymatic digestions with Achromobacter protease I or Staphylococcus aureus V8 protease and by chemical cleavages with CNBr, hydroxylamine, or 1% formic acid. The central part of FTX was sequenced with a surface-adhesive 7-kDa fragment, which was generated by a tryptic digestion of FTX and recovered by rinsing the wall of a test tube with 6 M guanidine HCl. The 7-kDa peptide was cleaved with 12 M HCl, thermolysin, or S. aureus V8 protease to produce smaller peptides for sequence analysis. As a result, FTX consisted of 251 residues, and protein and nucleotide sequences were in accord except for the lack of the initial Met and the C-terminal 20 residues in protein. Recombinant FTX (rFTX) with or without the C-terminal 20 residues (rFTX271 or rFTX251, respectively) was prepared to study the maturation process of FTX. Like natural FTX, rFTX251 existed as a monomer in solution and assembled into an SDS-stable, ring-shaped pore complex on human erythrocytes, causing hemolysis. In contrast, rFTX271, existing as a dimer in solution, bound to the cells but failed to form pore complex. The dimeric rFTX271 was converted to hemolytically active monomers upon the cleavage between Lys(251) and Met(252) by trypsin.

Identification of C-terminal phosphorylation sites of N-formyl peptide receptor-1 (FPR1) in human blood neutrophils.

PubMed

Maaty, Walid S; Lord, Connie I; Gripentrog, Jeannie M; Riesselman, Marcia; Keren-Aviram, Gal; Liu, Ting; Dratz, Edward A; Bothner, Brian; Jesaitis, Algirdas J

2013-09-20

Accumulation, activation, and control of neutrophils at inflammation sites is partly driven by N-formyl peptide chemoattractant receptors (FPRs). Occupancy of these G-protein-coupled receptors by formyl peptides has been shown to induce regulatory phosphorylation of cytoplasmic serine/threonine amino acid residues in heterologously expressed recombinant receptors, but the biochemistry of these modifications in primary human neutrophils remains relatively unstudied. FPR1 and FPR2 were partially immunopurified using antibodies that recognize both receptors (NFPRa) or unphosphorylated FPR1 (NFPRb) in dodecylmaltoside extracts of unstimulated and N-formyl-Met-Leu-Phe (fMLF) + cytochalasin B-stimulated neutrophils or their membrane fractions. After deglycosylation and separation by SDS-PAGE, excised Coomassie Blue-staining bands (∼34,000 Mr) were tryptically digested, and FPR1, phospho-FPR1, and FPR2 content was confirmed by peptide mass spectrometry. C-terminal FPR1 peptides (Leu(312)-Arg(322) and Arg(323)-Lys(350)) and extracellular FPR1 peptide (Ile(191)-Arg(201)) as well as three similarly placed FPR2 peptides were identified in unstimulated and fMLF + cytochalasin B-stimulated samples. LC/MS/MS identified seven isoforms of Ala(323)-Lys(350) only in the fMLF + cytochalasin B-stimulated sample. These were individually phosphorylated at Thr(325), Ser(328), Thr(329), Thr(331), Ser(332), Thr(334), and Thr(339). No phospho-FPR2 peptides were detected. Cytochalasin B treatment of neutrophils decreased the sensitivity of fMLF-dependent NFPRb recognition 2-fold, from EC50 = 33 ± 8 to 74 ± 21 nM. Our results suggest that 1) partial immunopurification, deglycosylation, and SDS-PAGE separation of FPRs is sufficient to identify C-terminal FPR1 Ser/Thr phosphorylations by LC/MS/MS; 2) kinases/phosphatases activated in fMLF/cytochalasin B-stimulated neutrophils produce multiple C-terminal tail FPR1 Ser/Thr phosphorylations but have little effect on corresponding FPR2 sites

Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily

PubMed Central

Ohnishi, Satoshi; Tochio, Naoya; Tomizawa, Tadashi; Akasaka, Ryogo; Harada, Takushi; Seki, Eiko; Sato, Manami; Watanabe, Satoru; Fujikura, Yukiko; Koshiba, Seizo; Terada, Takaho; Shirouzu, Mikako; Tanaka, Akiko; Kigawa, Takanori; Yokoyama, Shigeyuki

2008-01-01

The second WW domain in mammalian Salvador protein (SAV1 WW2) is quite atypical, as it forms a β-clam-like homodimer. The second WW domain in human MAGI1 (membrane associated guanylate kinase, WW and PDZ domain containing 1) (MAGI1 WW2) shares high sequence similarity with SAV1 WW2, suggesting comparable dimerization. However, an analytical ultracentrifugation study revealed that MAGI1 WW2 (Leu355–Pro390) chiefly exists as a monomer at low protein concentrations, with an association constant of 1.3 × 102 M−1. We determined its solution structure, and a structural comparison with the dimeric SAV1 WW2 suggested that an Asp residue is crucial for the inhibition of the dimerization. The substitution of this acidic residue with Ser resulted in the dimerization of MAGI1 WW2. The spin-relaxation data suggested that the MAGI1 WW2 undergoes a dynamic process of transient dimerization that is limited by the charge repulsion. Additionally, we characterized a longer construct of this WW domain with a C-terminal extension (Leu355–Glu401), as the formation of an extra α-helix was predicted. An NMR structural determination confirmed the formation of an α-helix in the extended C-terminal region, which appears to be independent from the dimerization regulation. A thermal denaturation study revealed that the dimerized MAGI1 WW2 with the Asp-to-Ser mutation gained apparent stability in a protein concentration-dependent manner. A structural comparison between the two constructs with different lengths suggested that the formation of the C-terminal α-helix stabilized the global fold by facilitating contacts between the N-terminal linker region and the main body of the WW domain. PMID:18562638

Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily.

PubMed

Ohnishi, Satoshi; Tochio, Naoya; Tomizawa, Tadashi; Akasaka, Ryogo; Harada, Takushi; Seki, Eiko; Sato, Manami; Watanabe, Satoru; Fujikura, Yukiko; Koshiba, Seizo; Terada, Takaho; Shirouzu, Mikako; Tanaka, Akiko; Kigawa, Takanori; Yokoyama, Shigeyuki

2008-09-01

The second WW domain in mammalian Salvador protein (SAV1 WW2) is quite atypical, as it forms a beta-clam-like homodimer. The second WW domain in human MAGI1 (membrane associated guanylate kinase, WW and PDZ domain containing 1) (MAGI1 WW2) shares high sequence similarity with SAV1 WW2, suggesting comparable dimerization. However, an analytical ultracentrifugation study revealed that MAGI1 WW2 (Leu355-Pro390) chiefly exists as a monomer at low protein concentrations, with an association constant of 1.3 x 10(2) M(-1). We determined its solution structure, and a structural comparison with the dimeric SAV1 WW2 suggested that an Asp residue is crucial for the inhibition of the dimerization. The substitution of this acidic residue with Ser resulted in the dimerization of MAGI1 WW2. The spin-relaxation data suggested that the MAGI1 WW2 undergoes a dynamic process of transient dimerization that is limited by the charge repulsion. Additionally, we characterized a longer construct of this WW domain with a C-terminal extension (Leu355-Glu401), as the formation of an extra alpha-helix was predicted. An NMR structural determination confirmed the formation of an alpha-helix in the extended C-terminal region, which appears to be independent from the dimerization regulation. A thermal denaturation study revealed that the dimerized MAGI1 WW2 with the Asp-to-Ser mutation gained apparent stability in a protein concentration-dependent manner. A structural comparison between the two constructs with different lengths suggested that the formation of the C-terminal alpha-helix stabilized the global fold by facilitating contacts between the N-terminal linker region and the main body of the WW domain.

Conformation Changes, N-terminal Involvement, and cGMP Signal Relay in the Phosphodiesterase-5 GAF Domain*

PubMed Central

Wang, Huanchen; Robinson, Howard; Ke, Hengming

2010-01-01

The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, which may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98–147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes. PMID:20861010

Conformation Changes N-terminal Involvement and cGMP Signal Relay in the Phosphodiesterase-5 GAF Domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

H Wang; H Robinson; H Ke

2011-12-31

The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, whichmore » may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98-147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes.« less

Consequences of Energetic Frustration on the Ligand-Coupled Folding/Dimerization Dynamics of Allosteric Protein S100A12.

PubMed

Ren, Weitong; Li, Wenfei; Wang, Jun; Zhang, Jian; Wang, Wei

2017-10-26

Allosteric proteins are featured by energetic degeneracy of two (or more) functionally relevant conformations, therefore their energy landscapes are often locally frustrated. How such frustration affects the protein folding/binding dynamics is not well understood. Here, by using molecular simulations we study the consequences of local frustration in the dimerization dynamics of allosteric proteins based on a homodimer protein S100A12. Despite of the structural symmetry of the two EF-hand motifs in the three-dimensional structures, the S100A12 homodimer shows allosteric behaviors and local frustration only in half of its structural elements, i.e., the C-terminal EF-hand. We showed that such spatially asymmetric location of frustration leads to asymmetric dimerization pathways, in which the dimerization is dominantly initiated by the interchain binding of the minimally frustrated N-terminal EF-hands, achieving optimal balance between the requirements of rapid conformational switching and interchain assembling to the energy landscapes. We also showed that the local frustration, as represented by the double-basin topography of the energy landscape, gives rise to multiple cross-linked dimerization pathways, in which the dimerization is coupled with the allosteric motions of the C-terminal EF-hands. Binding of metal ions tends to reshape the energy landscape and modulate the dimerization pathways. In addition, by employing the frustratometer method, we showed that the highly frustrated residue-pairs in the C-terminal EF-hand are partially unfolded during the conformational transitions of the native homodimer, leading to lowing of free energy barrier. Our results revealed tight interplay between the local frustration of the energy landscape and the dimerization dynamics for allosteric proteins.

Resolving Hot Spots in the C-Terminal Dimerization Domain that Determine the Stability of the Molecular Chaperone Hsp90

PubMed Central

Reimann, Sven; Smits, Sander H. J.; Schmitt, Lutz; Groth, Georg; Gohlke, Holger

2014-01-01

Human heat shock protein of 90 kDa (hHsp90) is a homodimer that has an essential role in facilitating malignant transformation at the molecular level. Inhibiting hHsp90 function is a validated approach for treating different types of tumors. Inhibiting the dimerization of hHsp90 via its C-terminal domain (CTD) should provide a novel way to therapeutically interfere with hHsp90 function. Here, we predicted hot spot residues that cluster in the CTD dimerization interface by a structural decomposition of the effective energy of binding computed by the MM-GBSA approach and confirmed these predictions using in silico alanine scanning with DrugScorePPI. Mutation of these residues to alanine caused a significant decrease in the melting temperature according to differential scanning fluorimetry experiments, indicating a reduced stability of the mutant hHsp90 complexes. Size exclusion chromatography and multi-angle light scattering studies demonstrate that the reduced stability of the mutant hHsp90 correlates with a lower complex stoichiometry due to the disruption of the dimerization interface. These results suggest that the identified hot spot residues can be used as a pharmacophoric template for identifying and designing small-molecule inhibitors of hHsp90 dimerization. PMID:24760083

N-terminal Proteomics Assisted Profiling of the Unexplored Translation Initiation Landscape in Arabidopsis thaliana *

PubMed Central

Ndah, Elvis; Jonckheere, Veronique

2017-01-01

Proteogenomics is an emerging research field yet lacking a uniform method of analysis. Proteogenomic studies in which N-terminal proteomics and ribosome profiling are combined, suggest that a high number of protein start sites are currently missing in genome annotations. We constructed a proteogenomic pipeline specific for the analysis of N-terminal proteomics data, with the aim of discovering novel translational start sites outside annotated protein coding regions. In summary, unidentified MS/MS spectra were matched to a specific N-terminal peptide library encompassing protein N termini encoded in the Arabidopsis thaliana genome. After a stringent false discovery rate filtering, 117 protein N termini compliant with N-terminal methionine excision specificity and indicative of translation initiation were found. These include N-terminal protein extensions and translation from transposable elements and pseudogenes. Gene prediction provided supporting protein-coding models for approximately half of the protein N termini. Besides the prediction of functional domains (partially) contained within the newly predicted ORFs, further supporting evidence of translation was found in the recently released Araport11 genome re-annotation of Arabidopsis and computational translations of sequences stored in public repositories. Most interestingly, complementary evidence by ribosome profiling was found for 23 protein N termini. Finally, by analyzing protein N-terminal peptides, an in silico analysis demonstrates the applicability of our N-terminal proteogenomics strategy in revealing protein-coding potential in species with well- and poorly-annotated genomes. PMID:28432195

N-terminal Proteomics Assisted Profiling of the Unexplored Translation Initiation Landscape in Arabidopsis thaliana.

PubMed

Willems, Patrick; Ndah, Elvis; Jonckheere, Veronique; Stael, Simon; Sticker, Adriaan; Martens, Lennart; Van Breusegem, Frank; Gevaert, Kris; Van Damme, Petra

2017-06-01

Proteogenomics is an emerging research field yet lacking a uniform method of analysis. Proteogenomic studies in which N-terminal proteomics and ribosome profiling are combined, suggest that a high number of protein start sites are currently missing in genome annotations. We constructed a proteogenomic pipeline specific for the analysis of N-terminal proteomics data, with the aim of discovering novel translational start sites outside annotated protein coding regions. In summary, unidentified MS/MS spectra were matched to a specific N-terminal peptide library encompassing protein N termini encoded in the Arabidopsis thaliana genome. After a stringent false discovery rate filtering, 117 protein N termini compliant with N-terminal methionine excision specificity and indicative of translation initiation were found. These include N-terminal protein extensions and translation from transposable elements and pseudogenes. Gene prediction provided supporting protein-coding models for approximately half of the protein N termini. Besides the prediction of functional domains (partially) contained within the newly predicted ORFs, further supporting evidence of translation was found in the recently released Araport11 genome re-annotation of Arabidopsis and computational translations of sequences stored in public repositories. Most interestingly, complementary evidence by ribosome profiling was found for 23 protein N termini. Finally, by analyzing protein N-terminal peptides, an in silico analysis demonstrates the applicability of our N-terminal proteogenomics strategy in revealing protein-coding potential in species with well- and poorly-annotated genomes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Dimerization of the EphA1 Receptor Tyrosine Kinase Transmembrane Domain: Insights into the Mechanism of Receptor Activation

PubMed Central

2014-01-01

EphA1 is a receptor tyrosine kinase (RTK) that plays a key role in developmental processes, including guidance of the migration of axons and cells in the nervous system. EphA1, in common with other RTKs, contains an N-terminal extracellular domain, a single transmembrane (TM) α-helix, and a C-terminal intracellular kinase domain. The TM helix forms a dimer, as seen in recent NMR studies. We have modeled the EphA1 TM dimer using a multiscale approach combining coarse-grain (CG) and atomistic molecular dynamics (MD) simulations. The one-dimensional potential of mean force (PMF) for this system, based on interhelix separation, has been calculated using CG MD simulations. This provides a view of the free energy landscape for helix–helix interactions of the TM dimer in a lipid bilayer. The resulting PMF profiles suggest two states, consistent with a rotation-coupled activation mechanism. The more stable state corresponds to a right-handed helix dimer interacting via an N-terminal glycine zipper motif, consistent with a recent NMR structure (2K1K). A second metastable state corresponds to a structure in which the glycine zipper motif is not involved. Analysis of unrestrained CG MD simulations based on representative models from the PMF calculations or on the NMR structure reveals possible pathways of interconversion between these two states, involving helix rotations about their long axes. This suggests that the interaction of TM helices in EphA1 dimers may be intrinsically dynamic. This provides a potential mechanism for signaling whereby extracellular events drive a shift in the repopulation of the underlying TM helix dimer energy landscape. PMID:25286141

Design, Synthesis, and Evaluation of N- and C-Terminal Protein Bioconjugates as G Protein-Coupled Receptor Agonists.

PubMed

Healey, Robert D; Wojciechowski, Jonathan P; Monserrat-Martinez, Ana; Tan, Susan L; Marquis, Christopher P; Sierecki, Emma; Gambin, Yann; Finch, Angela M; Thordarson, Pall

2018-02-21

A G protein-coupled receptor (GPCR) agonist protein, thaumatin, was site-specifically conjugated at the N- or C-terminus with a fluorophore for visualization of GPCR:agonist interactions. The N-terminus was specifically conjugated using a synthetic 2-pyridinecarboxyaldehyde reagent. The interaction profiles observed for N- and C-terminal conjugates were varied; N-terminal conjugates interacted very weakly with the GPCR of interest, whereas C-terminal conjugates bound to the receptor. These chemical biology tools allow interactions of therapeutic proteins:GPCR to be monitored and visualized. The methodology used for site-specific bioconjugation represents an advance in application of 2-pyridinecarboxyaldehydes for N-terminal specific bioconjugations.

Glycosylation Alters Dimerization Properties of a Cell-surface Signaling Protein, Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1).

PubMed

Zhuo, You; Yang, Jeong-Yeh; Moremen, Kelley W; Prestegard, James H

2016-09-16

Human carcinoembryonic antigen-related cell adhesion molecule 1 (C?/Au: EACAM1) is a cell-surface signaling molecule involved in cell adhesion, proliferation, and immune response. It is also implicated in cancer angiogenesis, progression, and metastasis. This diverse set of effects likely arises as a result of the numerous homophilic and heterophilic interactions that CEACAM1 can have with itself and other molecules. Its N-terminal Ig variable (IgV) domain has been suggested to be a principal player in these interactions. Previous crystal structures of the β-sandwich-like IgV domain have been produced using Escherichia coli-expressed material, which lacks native glycosylation. These have led to distinctly different proposals for dimer interfaces, one involving interactions of ABED β-strands and the other involving GFCC'C″ β-strands, with the former burying one prominent glycosylation site. These structures raise questions as to which form may exist in solution and what the effect of glycosylation may have on this form. Here, we use NMR cross-correlation measurements to examine the effect of glycosylation on CEACAM1-IgV dimerization and use residual dipolar coupling (RDC) measurements to characterize the solution structure of the non-glycosylated form. Our findings demonstrate that even addition of a single N-linked GlcNAc at potential glycosylation sites inhibits dimer formation. Surprisingly, RDC data collected on E. coli expressed material in solution indicate that a dimer using the non-glycosylated GFCC'C″ interface is preferred even in the absence of glycosylation. The results open new questions about what other factors may facilitate dimerization of CEACAM1 in vivo, and what roles glycosylation may play in heterophylic interactions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of two conformationally trapped n-propanol-water dimers in a supersonic expansion

NASA Astrophysics Data System (ADS)

Mead, Griffin J.; Alonso, Elena R.; Finneran, Ian A.; Carroll, P. Brandon; Blake, Geoffrey A.

2017-05-01

Two conformers of the n-propanol-water dimer have been observed in a supersonic expansion using chirped-pulse Fourier-transform microwave (CPFTMW) spectroscopy. Structural assignments reveal the n-propanol sub-unit is conformationally trapped, with its methyl group in both Gauche and Trans orientations. Despite different carbon backbone conformations, both dimers display the same water-donor/alcohol-acceptor hydrogen bonding motif. This work builds upon other reported alcohol-water dimers and upon previous work detailing the trapping of small molecules into multiple structural minima in rare gas supersonic expansions.

Sequential cancer immunotherapy: targeted activity of dimeric TNF and IL-8

PubMed Central

Adrian, Nicole; Siebenborn, Uta; Fadle, Natalie; Plesko, Margarita; Fischer, Eliane; Wüest, Thomas; Stenner, Frank; Mertens, Joachim C.; Knuth, Alexander; Ritter, Gerd; Old, Lloyd J.; Renner, Christoph

2009-01-01

Polymorphonuclear neutrophils (PMNs) are potent effectors of inflammation and their attempts to respond to cancer are suggested by their systemic, regional and intratumoral activation. We previously reported on the recruitment of CD11b+ leukocytes due to tumor site-specific enrichment of TNF activity after intravenous administration of a dimeric TNF immunokine with specificity for fibroblast activation protein (FAP). However, TNF-induced chemo-attraction and extravasation of PMNs from blood into the tumor is a multistep process essentially mediated by interleukin 8. With the aim to amplify the TNF-induced and IL-8-mediated chemotactic response, we generated immunocytokines by N-terminal fusion of a human anti-FAP scFv fragment with human IL-8 (IL-872) and its N-terminally truncated form IL-83-72. Due to the dramatic difference in chemotaxis induction in vitro, we favored the mature chemokine fused to the anti-FAP scFv for further investigation in vivo. BALB/c nu/nu mice were simultaneously xenografted with FAP-positive or -negative tumors and extended chemo-attraction of PMNs was only detectable in FAP-expressing tissue after intravenous administration of the anti-FAP scFv-IL-872 construct. As TNF-activated PMNs are likewise producers and primary targets for IL-8, we investigated the therapeutic efficacy of co-administration of both effectors: Sequential application of scFv-IL-872 and dimeric IgG1-TNF fusion proteins significantly enhanced anti-tumor activity when compared either to a single effector treatment regimen or sequential application of non-targeted cytokines, indicating that the tumor-restricted sequential application of IL-872 and TNF is a promising approach for cancer therapy. PMID:19267427

Coarse-Grained Molecular Simulation of Epidermal Growth Factor Receptor Protein Tyrosine Kinase Multi-Site Self-Phosphorylation

PubMed Central

Koland, John G.

2014-01-01

Upon the ligand-dependent dimerization of the epidermal growth factor receptor (EGFR), the intrinsic protein tyrosine kinase (PTK) activity of one receptor monomer is activated, and the dimeric receptor undergoes self-phosphorylation at any of eight candidate phosphorylation sites (P-sites) in either of the two C-terminal (CT) domains. While the structures of the extracellular ligand binding and intracellular PTK domains are known, that of the ∼225-amino acid CT domain is not, presumably because it is disordered. Receptor phosphorylation on CT domain P-sites is critical in signaling because of the binding of specific signaling effector molecules to individual phosphorylated P-sites. To investigate how the combination of conventional substrate recognition and the unique topological factors involved in the CT domain self-phosphorylation reaction lead to selectivity in P-site phosphorylation, we performed coarse-grained molecular simulations of the P-site/catalytic site binding reactions that precede EGFR self-phosphorylation events. Our results indicate that self-phosphorylation of the dimeric EGFR, although generally believed to occur in trans, may well occur with a similar efficiency in cis, with the P-sites of both receptor monomers being phosphorylated to a similar extent. An exception was the case of the most kinase-proximal P-site-992, the catalytic site binding of which occurred exclusively in cis via an intramolecular reaction. We discovered that the in cis interaction of P-site-992 with the catalytic site was facilitated by a cleft between the N-terminal and C-terminal lobes of the PTK domain that allows the short CT domain sequence tethering P-site-992 to the PTK core to reach the catalytic site. Our work provides several new mechanistic insights into the EGFR self-phosphorylation reaction, and demonstrates the potential of coarse-grained molecular simulation approaches for investigating the complexities of self-phosphorylation in molecules such as EGFR

Identification of an N-terminal glycogen synthase kinase 3 phosphorylation site which regulates the functional localisation of polycystin-2 in vivo and in vitro

PubMed Central

Streets, Andrew J; Moon, David J; Kane, Michelle E; Obara, Tomoko; Ong, Albert CM

2008-01-01

PKD2 is mutated in 15% of patients with autosomal dominant polycystic kidney disease (ADPKD). Polycystin-2 (PC2), the PKD2 protein, is a nonselective Ca2+-permeable cation channel which may function at the cell surface and ER. Nevertheless, the factors that regulate the dynamic translocation of PC2 between the ER and other compartments are not well understood. Constitutive phosphorylation of PC2 at a single C-terminal site (Ser812) has been previously reported. Since we were unable to abolish phospholabelling of PC2 in HEK293 cells by site-directed mutagenesis of Ser812 or all 5 predicted phosphorylation sites in the C-terminus, we hypothesised that PC2 could also be phosphorylated at the N-terminus. In this paper, we report the identification of a new phosphorylation site for PC2 within its N-terminal domain (Ser76) and demonstrate that this residue is phosphorylated by glycogen synthase kinase 3 (GSK-3). The consensus recognition sequence for GSK-3 (Ser76/Ser80) is evolutionarily conserved down to lower vertebrates. In the presence of specific GSK-3 inhibitors, the lateral plasma membrane pool of endogenous PC2 redistributes into an intracellular compartment in MDCK cells without a change in primary cilia localization. Finally, co-injection of wild-type but not a S76A/S80A mutant PKD2 capped mRNA could rescue the cystic phenotype induced by an antisense morpholino oligonucleotide to pkd2 in zebrafish pronephric kidney. We conclude that surface localization of PC2 is regulated by phosphorylation at a unique GSK-3 site in its N-terminal domain in vivo and in vitro. This site is functionally significant for the maintenance of normal glomerular and tubular morphology. PMID:16551655

Structural insight into arginine methylation by the mouse protein arginine methyltransferase 7: a zinc finger freezes the mimic of the dimeric state into a single active site.

PubMed

Cura, Vincent; Troffer-Charlier, Nathalie; Wurtz, Jean Marie; Bonnefond, Luc; Cavarelli, Jean

2014-09-01

Protein arginine methyltransferase 7 (PRMT7) is a type III arginine methyltransferase which has been implicated in several biological processes such as transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation and metastasis. PRMT7 is a unique but less characterized member of the family of PRMTs. The crystal structure of full-length PRMT7 from Mus musculus refined at 1.7 Å resolution is described. The PRMT7 structure is composed of two catalytic modules in tandem forming a pseudo-dimer and contains only one AdoHcy molecule bound to the N-terminal module. The high-resolution crystal structure presented here revealed several structural features showing that the second active site is frozen in an inactive state by a conserved zinc finger located at the junction between the two PRMT modules and by the collapse of two degenerated AdoMet-binding loops.

The two-state dimer receptor model: a general model for receptor dimers.

PubMed

Franco, Rafael; Casadó, Vicent; Mallol, Josefa; Ferrada, Carla; Ferré, Sergi; Fuxe, Kjell; Cortés, Antoni; Ciruela, Francisco; Lluis, Carmen; Canela, Enric I

2006-06-01

Nonlinear Scatchard plots are often found for agonist binding to G-protein-coupled receptors. Because there is clear evidence of receptor dimerization, these nonlinear Scatchard plots can reflect cooperativity on agonist binding to the two binding sites in the dimer. According to this, the "two-state dimer receptor model" has been recently derived. In this article, the performance of the model has been analyzed in fitting data of agonist binding to A(1) adenosine receptors, which are an example of receptor displaying concave downward Scatchard plots. Analysis of agonist/antagonist competition data for dopamine D(1) receptors using the two-state dimer receptor model has also been performed. Although fitting to the two-state dimer receptor model was similar to the fitting to the "two-independent-site receptor model", the former is simpler, and a discrimination test selects the two-state dimer receptor model as the best. This model was also very robust in fitting data of estrogen binding to the estrogen receptor, for which Scatchard plots are concave upward. On the one hand, the model would predict the already demonstrated existence of estrogen receptor dimers. On the other hand, the model would predict that concave upward Scatchard plots reflect positive cooperativity, which can be neither predicted nor explained by assuming the existence of two different affinity states. In summary, the two-state dimer receptor model is good for fitting data of binding to dimeric receptors displaying either linear, concave upward, or concave downward Scatchard plots.

A structural study of the K adsorption site on a Si(001)2 × 1 surface: Dimer, caves or both

NASA Astrophysics Data System (ADS)

Asensio, M. C.; Michel, E. G.; Alvarez, J.; Ocal, C.; Miranda, R.; Ferrer, S.

1989-04-01

The atomic structure of the clean Si(100) and K covered surfaces has been investigated by Auger electron diffraction (AED) monitoring the intensities along polar scans. This technique is sensitive to the asymmetric-dimer nature of the 2 × 1 reconstruction of the Si(001) surface. Data taken at room temperature for submonolayer coverages are consistent with adsorption of K on the troughs (cave position) existing between two consecutive dimer chains along the [110] direction. At 110 K both dimer and cave sites are occupied. A mild annealing to 300 K produces an overlayer redistribution in favor of the "cave" site further indicating that this site is energetically favoured as found in some recent calculations.

The N-Terminal CCHC Zinc Finger Motif Mediates Homodimerization of Transcription Factor BCL11B.

PubMed

Grabarczyk, Piotr; Winkler, Passorn; Delin, Martin; Sappa, Praveen K; Bekeschus, Sander; Hildebrandt, Petra; Przybylski, Grzegorz K; Völker, Uwe; Hammer, Elke; Schmidt, Christian A

2018-03-01

The BCL11B gene encodes a Krüppel-like, sequence-specific zinc finger (ZF) transcription factor that acts as either a repressor or an activator, depending on its posttranslational modifications. The importance of BCL11B in numerous biological processes in multiple organs has been well established in mouse knockout models. The phenotype of the first de novo monoallelic germ line missense mutation in the BCL11B gene (encoding N441K) strongly implies that the mutant protein acts in a dominant-negative manner by neutralizing the unaffected protein through the formation of a nonfunctional dimer. Using a Förster resonance energy transfer-assisted fluorescence-activated cell sorting (FACS-FRET) assay and affinity purification followed by mass spectrometry (AP-MS), we show that the N-terminal CCHC zinc finger motif is necessary and sufficient for the formation of the BCL11B dimer. Mutation of the CCHC ZF in BCL11B abolishes its transcription-regulatory activity. In addition, unlike wild-type BCL11B, this mutant is incapable of inducing cell cycle arrest and protecting against DNA damage-driven apoptosis. Our results confirm the BCL11B dimerization hypothesis and prove its importance for BCL11B function. By mapping the relevant regions to the CCHC domain, we describe a previously unidentified mechanism of transcription factor homodimerization. Copyright © 2018 American Society for Microbiology.

The Reach of Linear Protein-DNA Dimerizers

PubMed Central

Stafford, Ryan L.; Dervan, Peter B.

2008-01-01

A protein-DNA dimerizer constructed from a DNA-binding pyrrole-imidazole polyamide and the peptide FYPWMK facilitates binding of the natural transcription factor Exd to an adjacent DNA site. Previous dimerizers have been constructed with the peptide attached to an internal pyrrole monomer in an overall branched oligomer. Linear oligomers constructed by attaching the peptide to the polyamide C-terminus expand the range of protein-DNA dimerization to six additional DNA sites. Replacing the FYPWMK hexapeptide with a WM dipeptide, which was previously functional in branched compounds, does not lead to a functional linear dimerizer. Instead, inserting an additional lysine generates a minimal, linear WMK tripeptide conjugate that maintains the activity of the larger FYPWMK dimerizers in a single DNA-binding site orientation. These studies provide insight into the importance of linker length and composition, binding site spacing and orientation, and the protein-binding domain content that are important for the optimization of protein DNA-dimerizers suitable for biological experiments. PMID:17949089

Genetically encoded photocross-linkers determine the biological binding site of exendin-4 peptide in the N-terminal domain of the intact human glucagon-like peptide-1 receptor (GLP-1R)

PubMed Central

Koole, Cassandra; Reynolds, Christopher A.; Mobarec, Juan C.; Hick, Caroline; Sexton, Patrick M.; Sakmar, Thomas P.

2017-01-01

The glucagon-like peptide-1 receptor (GLP-1R) is a key therapeutic target in the management of type II diabetes mellitus, with actions including regulation of insulin biosynthesis and secretion, promotion of satiety, and preservation of β-cell mass. Like most class B G protein-coupled receptors (GPCRs), there is limited knowledge linking biological activity of the GLP-1R with the molecular structure of an intact, full-length, and functional receptor·ligand complex. In this study, we have utilized genetic code expansion to site-specifically incorporate the photoactive amino acid p-azido-l-phenylalanine (azF) into N-terminal residues of a full-length functional human GLP-1R in mammalian cells. UV-mediated photolysis of azF was then carried out to induce targeted photocross-linking to determine the proximity of the azido group in the mutant receptor with the peptide exendin-4. Cross-linking data were compared directly with the crystal structure of the isolated N-terminal extracellular domain of the GLP-1R in complex with exendin(9–39), revealing both similarities as well as distinct differences in the mode of interaction. Generation of a molecular model to accommodate the photocross-linking constraints highlights the potential influence of environmental conditions on the conformation of the receptor·peptide complex, including folding dynamics of the peptide and formation of dimeric and higher order oligomeric receptor multimers. These data demonstrate that crystal structures of isolated receptor regions may not give a complete reflection of peptide/receptor interactions and should be combined with additional experimental constraints to reveal peptide/receptor interactions occurring in the dynamic, native, and full-length receptor state. PMID:28283573

Structural bases of dimerization of yeast telomere protein Cdc13 and its interaction with the catalytic subunit of DNA polymerase [alpha

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Jia; Yang, Yuting; Wan, Ke

Budding yeast Cdc13-Stn1-Ten1 (CST) complex plays an essential role in telomere protection and maintenance, and has been proposed to be a telomere-specific replication protein A (RPA)-like complex. Previous genetic and structural studies revealed a close resemblance between Stn1-Ten1 and RPA32-RPA14. However, the relationship between Cdc13 and RPA70, the largest subunit of RPA, has remained unclear. Here, we report the crystal structure of the N-terminal OB (oligonucleotide/oligosaccharide binding) fold of Cdc13. Although Cdc13 has an RPA70-like domain organization, the structures of Cdc13 OB folds are significantly different from their counterparts in RPA70, suggesting that they have distinct evolutionary origins. Furthermore, ourmore » structural and biochemical analyses revealed unexpected dimerization by the N-terminal OB fold and showed that homodimerization is probably a conserved feature of all Cdc13 proteins. We also uncovered the structural basis of the interaction between the Cdc13 N-terminal OB fold and the catalytic subunit of DNA polymerase {alpha} (Pol1), and demonstrated a role for Cdc13 dimerization in Pol1 binding. Analysis of the phenotypes of mutants defective in Cdc13 dimerization and Cdc13-Pol1 interaction revealed multiple mechanisms by which dimerization regulates telomere lengths in vivo. Collectively, our findings provide novel insights into the mechanisms and evolution of Cdc13.« less

Crystal Structure of the Full-Length Feline Immunodeficiency Virus Capsid Protein Shows an N-Terminal β-Hairpin in the Absence of N-Terminal Proline

PubMed Central

Folio, Christelle; Sierra, Natalia; Dujardin, Marie; Alvarez, Guzman

2017-01-01

Feline immunodeficiency virus (FIV) is a member of the Retroviridae family. It is the causative agent of an acquired immunodeficiency syndrome (AIDS) in cats and wild felines. Its capsid protein (CA) drives the assembly of the viral particle, which is a critical step in the viral replication cycle. Here, the first atomic structure of full-length FIV CA to 1.67 Å resolution is determined. The crystallized protein exhibits an original tetrameric assembly, composed of dimers which are stabilized by an intermolecular disulfide bridge induced by the crystallogenesis conditions. The FIV CA displays a standard α-helical CA topology with two domains, separated by a linker shorter than other retroviral CAs. The β-hairpin motif at its amino terminal end, which interacts with nucleotides in HIV-1, is unusually long in FIV CA. Interestingly, this functional β-motif is formed in this construct in the absence of the conserved N-terminal proline. The FIV CA exhibits a cis Arg–Pro bond in the CypA-binding loop, which is absent in known structures of lentiviral CAs. This structure represents the first tri-dimensional structure of a functional, full-length FIV CA. PMID:29120364

Complementation analysis of mutants of nitric oxide synthase reveals that the active site requires two hemes.

PubMed Central

Xie, Q W; Leung, M; Fuortes, M; Sassa, S; Nathan, C

1996-01-01

For catalytic activity, nitric oxide synthases (NOSs) must be dimeric. Previous work revealed that the requirements for stable dimerization included binding of tetrahydrobiopterin (BH4), arginine, and heme. Here we asked what function is served by dimerization. We assessed the ability of individually inactive mutants of mouse inducible NOS (iNOS; NOS2), each deficient in binding a particular cofactor or cosubstrate, to complement each other by generating NO upon cotransfection into human epithelial cells. The ability of the mutants to homodimerize was gauged by gel filtration and/or PAGE under partially denaturing conditions, both followed by immunoblot. Their ability to heterodimerize was assessed by coimmunoprecipitation. Heterodimers that contained only one COOH-terminal hemimer and only one BH4-binding site could both form and function, even though the NADPH-, FAD-, and FMN-binding domains (in the COOH-terminal hemimer) and the BH4-binding sites (in the NH2-terminal hemimer) were contributed by opposite chains. Heterodimers that contained only one heme-binding site (Cys-194) could also form, either in cis or in trans to the nucleotide-binding domains. However, for NO production, both chains had to bind heme. Thus, NO production by iNOS requires dimerization because the active site requires two hemes. Images Fig. 2 Fig. 3 Fig. 4 Fig. 7 PMID:8643499

Analysis of hepatitis C virus RNA dimerization and core–RNA interactions

PubMed Central

Ivanyi-Nagy, Roland; Kanevsky, Igor; Gabus, Caroline; Lavergne, Jean-Pierre; Ficheux, Damien; Penin, François; Fossé, Philippe; Darlix, Jean-Luc

2006-01-01

The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3′-untranslated region (3′-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623–2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3′-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus. PMID:16707664

Analysis of hepatitis C virus RNA dimerization and core-RNA interactions.

PubMed

Ivanyi-Nagy, Roland; Kanevsky, Igor; Gabus, Caroline; Lavergne, Jean-Pierre; Ficheux, Damien; Penin, François; Fossé, Philippe; Darlix, Jean-Luc

2006-01-01

The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3'-untranslated region (3'-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623-2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3'-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus.

Multiple C-terminal tail Ca2+/CaMs regulate CaV1.2 function but do not mediate channel dimerization

PubMed Central

Kim, Eun Young; Rumpf, Christine H; Van Petegem, Filip; Arant, Ryan J; Findeisen, Felix; Cooley, Elizabeth S; Isacoff, Ehud Y; Minor, Daniel L

2010-01-01

Interactions between voltage-gated calcium channels (CaVs) and calmodulin (CaM) modulate CaV function. In this study, we report the structure of a Ca2+/CaM CaV1.2 C-terminal tail complex that contains two PreIQ helices bridged by two Ca2+/CaMs and two Ca2+/CaM–IQ domain complexes. Sedimentation equilibrium experiments establish that the complex has a 2:1 Ca2+/CaM:C-terminal tail stoichiometry and does not form higher order assemblies. Moreover, subunit-counting experiments demonstrate that in live cell membranes CaV1.2s are monomers. Thus, contrary to previous proposals, the crystallographic dimer lacks physiological relevance. Isothermal titration calorimetry and biochemical experiments show that the two Ca2+/CaMs in the complex have different properties. Ca2+/CaM bound to the PreIQ C-region is labile, whereas Ca2+/CaM bound to the IQ domain is not. Furthermore, neither of lobes of apo-CaM interacts strongly with the PreIQ domain. Electrophysiological studies indicate that the PreIQ C-region has a role in calcium-dependent facilitation. Together, the data show that two Ca2+/CaMs can bind the CaV1.2 tail simultaneously and indicate a functional role for Ca2+/CaM at the C-region site. PMID:20953164

Multiple C-terminal tail Ca(2+)/CaMs regulate Ca(V)1.2 function but do not mediate channel dimerization.

PubMed

Kim, Eun Young; Rumpf, Christine H; Van Petegem, Filip; Arant, Ryan J; Findeisen, Felix; Cooley, Elizabeth S; Isacoff, Ehud Y; Minor, Daniel L

2010-12-01

Interactions between voltage-gated calcium channels (Ca(V)s) and calmodulin (CaM) modulate Ca(V) function. In this study, we report the structure of a Ca(2+)/CaM Ca(V)1.2 C-terminal tail complex that contains two PreIQ helices bridged by two Ca(2+)/CaMs and two Ca(2+)/CaM-IQ domain complexes. Sedimentation equilibrium experiments establish that the complex has a 2:1 Ca(2+)/CaM:C-terminal tail stoichiometry and does not form higher order assemblies. Moreover, subunit-counting experiments demonstrate that in live cell membranes Ca(V)1.2s are monomers. Thus, contrary to previous proposals, the crystallographic dimer lacks physiological relevance. Isothermal titration calorimetry and biochemical experiments show that the two Ca(2+)/CaMs in the complex have different properties. Ca(2+)/CaM bound to the PreIQ C-region is labile, whereas Ca(2+)/CaM bound to the IQ domain is not. Furthermore, neither of lobes of apo-CaM interacts strongly with the PreIQ domain. Electrophysiological studies indicate that the PreIQ C-region has a role in calcium-dependent facilitation. Together, the data show that two Ca(2+)/CaMs can bind the Ca(V)1.2 tail simultaneously and indicate a functional role for Ca(2+)/CaM at the C-region site.

Site-Selective Benzannulation of N-Heterocycles in Bidentate Ligands Leads to Blue-Shifted Emission from [( P^N)Cu]2(μ-X)2 Dimers.

PubMed

Mondal, Rajarshi; Lozada, Issiah B; Davis, Rebecca L; Williams, J A Gareth; Herbert, David E

2018-05-07

Benzannulated bidentate pyridine/phosphine ( P^N) ligands bearing quinoline or phenanthridine (3,4-benzoquinoline) units have been prepared, along with their halide-bridged, dimeric Cu(I) complexes of the form [( P^N)Cu] 2 (μ-X) 2 . The copper complexes are phosphorescent in the orange-red region of the spectrum in the solid-state under ambient conditions. Structural characterization in solution and the solid-state reveals a flexible conformational landscape, with both diamond-like and butterfly motifs available to the Cu 2 X 2 cores. Comparing the photophysical properties of complexes of (quinolinyl)phosphine ligands with those of π-extended (phenanthridinyl)phosphines has revealed a counterintuitive impact of site-selective benzannulation. Contrary to conventional assumptions regarding π-extension and a bathochromic shift in the lowest energy absorption maxima, a blue shift of nearly 40 nm in the emission wavelength is observed for the complexes with larger ligand π-systems, which is assigned as phosphorescence on the basis of emission energies and lifetimes. Comparison of the ground-state and triplet excited state structures optimized from DFT and TD-DFT calculations allows attribution of this effect to a greater rigidity for the benzannulated complexes resulting in a higher energy emissive triplet state, rather than significant perturbation of orbital energies. This study reveals that ligand structure can impact photophysical properties for emissive molecules by influencing their structural rigidity, in addition to their electronic structure.

Electron-temperature dependence of dissociative recombination of electrons with N2/+/.N2 dimer ions

NASA Technical Reports Server (NTRS)

Whitaker, M.; Biondi, M. A.; Johnsen, R.

1981-01-01

The variation with electron temperature of the dissociative recombination of electrons with N2(+).N2 dimer ions is investigated in light of the importance of such ions in the lower ionosphere and in laser plasmas. Dissociative recombination coefficients were determined by means of a microwave afterglow mass spectrometer technique for electron temperatures from 300-5600 K and an ion and neutral temperature of 300 K. The recombination coefficient is found to be proportional to the -0.41 power of the electron temperature in this range, similar to that observed for the CO(+).CO dimer ion and consistent with the expected energy dependence for a fast dissociative process.

Neurospora tryptophan synthase: N-terminal analysis and the sequence of the pyridoxal phosphate active site peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pratt, M.L.; Hsu, P.Y.; DeMoss, J.A.

1986-05-01

Tryptophan synthase (TS), which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein requiring pyridoxal phosphate (B6P) for two of its three distinct enzyme activities. TS from Neurospora has a blocked N-terminal, is a homodimer of 150 KDa and binds one mole of B6P per mole of subunit. The authors shown the N-terminal residue to be acyl-serine. The B6P-active site of holoenzyme was labelled by reduction of the B6P-Schiff base with (/sup 3/H)-NaBH/sub 4/, and resulted in a proportionate loss of activity in the two B6P-requiring reactions. SDS-polyacrylamide gel electrophoresis of CNBr-generated peptides showed the labelled, active sitemore » peptide to be 6 KDa. The sequence of this peptide, purified to apparent homogeneity by a combination of C-18 reversed phase and TSK gel filtration HPLC is: gly-arg-pro-gly-gln-leu-his-lys-ala-glu-arg-leu-thr-glu-tyr-ala-gly-gly-ala-gln-ile-xxx-leu-lys-arg-glu-asp-leu-asn-his-xxx-gly-xxx-his-/sub ***/-ile-asn-asn-ala-leu. Although four residues (xxx, /sub ***/) are unidentified, this peptide is minimally 78% homologous with the corresponding peptide from yeast TS, in which residue (/sub ***/) is the lysine that binds B6P.« less

Study of structural stability and damaging effect on membrane for four Aβ42 dimers

PubMed Central

Feng, Wei; Lei, Huimin; Si, Jiarui; Zhang, Tao

2017-01-01

Increasing evidence shows that Aβ oligomers are key pathogenic molecules in Alzheimer’s disease. Among Aβ oligomers, dimer is the smallest aggregate and toxic unit. Therefore, understanding its structural and dynamic properties is quite useful to prevent the formation and toxicity of the Aβ oligomers. In this study, we performed molecular dynamic simulations on four Aβ42 dimers, 2NCb, CNNC, NCNC and NCCN, within the hydrated DPPC membrane. Four Aβ42 dimers differ in the arrangements of two Aβ42 peptides. This study aims to investigate the impact of aggregation pattern of two Aβ peptides on the structural stability of the Aβ42 dimer and its disruption to the biological membrane. The MD results demonstrate that the NCCN, CNNC and NCNC have the larger structural fluctuation at the N-terminus of Aβ42 peptide, where the β-strand structure converts into the coil structure. The loss of the N-terminal β-strand further impairs the aggregate ability of Aβ42 dimer. In addition, inserting Aβ42 dimer into the membrane can considerably decrease the average APL of DPPC membrane. Moreover this decrease effect is largely dependent on the distance to the location of Aβ42 dimer and its secondary structure forms. Based on the results, the 2NCb is considered as a stable dimeric unit for aggregating the larger Aβ42 oligomer, and has a potent ability to disrupt the membrane. PMID:28594887

Polyhydroxylated [60]fullerene binds specifically to functional recognition sites on a monomeric and a dimeric ubiquitin

NASA Astrophysics Data System (ADS)

Zanzoni, Serena; Ceccon, Alberto; Assfalg, Michael; Singh, Rajesh K.; Fushman, David; D'Onofrio, Mariapina

2015-04-01

The use of nanoparticles (NPs) in biomedical applications requires an in-depth understanding of the mechanisms by which NPs interact with biomolecules. NPs associating with proteins may interfere with protein-protein interactions and affect cellular communication pathways, however the impact of NPs on biomolecular recognition remains poorly characterized. In this respect, particularly relevant is the study of NP-induced functional perturbations of proteins implicated in the regulation of key biochemical pathways. Ubiquitin (Ub) is a prototypical protein post-translational modifier playing a central role in numerous essential biological processes. To contribute to the understanding of the interactions between this universally distributed biomacromolecule and NPs, we investigated the adsorption of polyhydroxylated [60]fullerene on monomeric Ub and on a minimal polyubiquitin chain in vitro at atomic resolution. Site-resolved chemical shift and intensity perturbations of Ub's NMR signals, together with 15N spin relaxation rate changes, exchange saturation transfer effects, and fluorescence quenching data were consistent with the reversible formation of soluble aggregates incorporating fullerenol clusters. The specific interaction epitopes were identified, coincident with functional recognition sites in a monomeric and lysine48-linked dimeric Ub. Fullerenol appeared to target the open state of the dynamic structure of a dimeric Ub according to a conformational selection mechanism. Importantly, the protein-NP association prevented the enzyme-catalyzed synthesis of polyubiquitin chains. Our findings provide an experiment-based insight into protein/fullerenol recognition, with implications in functional biomolecular communication, including regulatory protein turnover, and for the opportunity of therapeutic intervention in Ub-dependent cellular pathways.The use of nanoparticles (NPs) in biomedical applications requires an in-depth understanding of the mechanisms by which

Deletion of the N-terminal Domain (NTD) Alters the Ethanol Inhibition of NMDA Receptors in a Subunit-Dependent Manner

PubMed Central

Smothers, C. Thetford; Jin, Chun; Woodward, John J.

2013-01-01

Background Ethanol inhibition of NMDA receptors is poorly understood due in part to the organizational complexity of the receptor that provides ample locations for sites of action. Among these the N-terminal domain of NMDA receptor subunits contains binding sites for a variety of modulatory agents including zinc, protons and GluN2B selective antagonists such as ifenprodil or Ro-25–6981. Ethanol inhibition of neuronal NMDA receptors expressed in some brain areas has been reported to be occluded by the presence of ifenprodil or similar compounds suggesting that the N-terminal domain may be important in regulating the ethanol sensitivity of NMDA receptors. Methods Wild-type GluN1 and GluN2 subunits and those in which the coding sequence for the N-terminal domain was deleted were expressed in HEK293 cells. Whole-cell voltage-clamp recording was used to assess ethanol inhibition of wild-type and mutant receptors lacking the N-terminal domain. Results As compared to wild-type GluN1/GluN2A receptors, ethanol inhibition was slightly greater in cells expressing GluN2A subunits lacking the N-terminal domain. In contrast, GluN2B N-terminal deletion mutants showed normal ethanol inhibition while those lacking the N-terminal domain in both GluN1 and GluN2B subunits had decreased ethanol inhibition as compared to wild-type receptors. N-terminal domain lacking GluN2B receptors were insensitive to ifenprodil but retained normal sensitivity to ethanol. Conclusions These findings indicate that the N-terminal domain modestly influences the ethanol sensitivity of NMDA receptors in a subunit-dependent manner. They also show that ifenprodil’s actions on GluN2B containing receptors can be dissociated from those of ethanol. These results suggest that while the N-terminal domain is not a primary site of action for ethanol on NMDA receptors, it likely affects sensitivity via actions on intrinsic channel properties. PMID:23905549

1 / n Expansion for the Number of Matchings on Regular Graphs and Monomer-Dimer Entropy

NASA Astrophysics Data System (ADS)

Pernici, Mario

2017-08-01

Using a 1 / n expansion, that is an expansion in descending powers of n, for the number of matchings in regular graphs with 2 n vertices, we study the monomer-dimer entropy for two classes of graphs. We study the difference between the extensive monomer-dimer entropy of a random r-regular graph G (bipartite or not) with 2 n vertices and the average extensive entropy of r-regular graphs with 2 n vertices, in the limit n → ∞. We find a series expansion for it in the numbers of cycles; with probability 1 it converges for dimer density p < 1 and, for G bipartite, it diverges as |ln(1-p)| for p → 1. In the case of regular lattices, we similarly expand the difference between the specific monomer-dimer entropy on a lattice and the one on the Bethe lattice; we write down its Taylor expansion in powers of p through the order 10, expressed in terms of the number of totally reducible walks which are not tree-like. We prove through order 6 that its expansion coefficients in powers of p are non-negative.

Structure of the Response Regulator PhoP from Mycobacterium tuberculosis Reveals a Dimer Through the Receiver Domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

S Menon; S Wang

The PhoP protein from Mycobacterium tuberculosis is a response regulator of the OmpR/PhoB subfamily, whose structure consists of an N-terminal receiver domain and a C-terminal DNA-binding domain. How the DNA-binding activities are regulated by phosphorylation of the receiver domain remains unclear due to a lack of structural information on the full-length proteins. Here we report the crystal structure of the full-length PhoP of M. tuberculosis. Unlike other known structures of full-length proteins of the same subfamily, PhoP forms a dimer through its receiver domain with the dimer interface involving {alpha}4-{beta}5-{alpha}5, a common interface for activated receiver domain dimers. However, themore » switch residues, Thr99 and Tyr118, are in a conformation resembling those of nonactivated receiver domains. The Tyr118 side chain is involved in the dimer interface interactions. The receiver domain is tethered to the DNA-binding domain through a flexible linker and does not impose structural constraints on the DNA-binding domain. This structure suggests that phosphorylation likely facilitates/stabilizes receiver domain dimerization, bringing the DNA-binding domains to close proximity, thereby increasing their binding affinity for direct repeat DNA sequences.« less

Releasing N-glycan from peptide N-terminus by N-terminal succinylation assisted enzymatic deglycosylation.

PubMed

Weng, Yejing; Sui, Zhigang; Jiang, Hao; Shan, Yichu; Chen, Lingfan; Zhang, Shen; Zhang, Lihua; Zhang, Yukui

2015-04-22

Due to the important roles of N-glycoproteins in various biological processes, the global N-glycoproteome analysis has been paid much attention. However, by current strategies for N-glycoproteome profiling, peptides with glycosylated Asn at N-terminus (PGANs), generated by protease digestion, could hardly be identified, due to the poor deglycosylation capacity by enzymes. However, theoretically, PGANs occupy 10% of N-glycopeptides in the typical tryptic digests. Therefore, in this study, we developed a novel strategy to identify PGANs by releasing N-glycans through the N-terminal site-selective succinylation assisted enzymatic deglycosylation. The obtained PGANs information is beneficial to not only achieve the deep coverage analysis of glycoproteomes, but also discover the new biological functions of such modification.

Crystal structures of the F and pSLT plasmid TraJ N-terminal regions reveal similar homodimeric PAS folds with functional interchangeability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Jun; Wu, Ruiying; Adkins, Joshua N.

2014-09-16

In the F-family of conjugative plasmids, TraJ is an essential transcriptional activator of the tra operon that encodes most of the proteins required for conjugation. Here we report for the first time the X-ray crystal structures of the TraJ N-terminal regions from the prototypic F plasmid (TraJF11-130) and from the Salmonella virulence plasmid pSLT (TraJpSLT 1-128). Both proteins form similar homodimeric Per-ARNT-Sim (PAS) fold structures. Mutational analysis reveals that the observed dimeric interface is critical for TraJF transcriptional activation, indicating that dimerization of TraJ is required for its in vivo function. An artificial ligand (oxidized dithiothreitol) occupies a cavity inmore » the TraJF dimer interface, while a smaller cavity in corresponding region of the TraJpSLT structure lacks a ligand. Gas chromatography/mass spectrometry-electron ionization analysis of dithiothreitol-free TraJF suggests indole may be the natural TraJ ligand; however, disruption of the indole biosynthetic pathway does not affect TraJF function. Heterologous PAS domains from pSLT and R100 TraJ can functionally replace the TraJF PAS domain, suggesting that TraJ allelic specificity is mediated by the region C-terminal to the PAS domain.« less

Energy landscapes of the monomer and dimer of the Alzheimer's peptide A β (1 -28 )

NASA Astrophysics Data System (ADS)

Dong, Xiao; Chen, Wei; Mousseau, Normand; Derreumaux, Philippe

2008-03-01

The cytoxicity of Alzheimer's disease has been linked to the self-assembly of the 40 /42 amino acid of the amyloid-β (A β ) peptide into oligomers. To understand the assembly process, it is important to characterize the very first steps of aggregation at an atomic level of detail. Here, we focus on the N-terminal fragment 1-28, known to form fibrils in vitro. Circular dichroism and NMR experiments indicate that the monomer of A β (1 -28 ) is α -helical in a membranelike environment and random coil in aqueous solution. Using the activation-relaxation technique coupled with the OPEP coarse grained force field, we determine the structures of the monomer and of the dimer of A β (1 -28 ) . In agreement with experiments, we find that the monomer is predominantly random coil in character, but displays a non-negligible β -strand probability in the N-terminal region. Dimerization impacts the structure of each chain and leads to an ensemble of intertwined conformations with little β -strand content in the region Leu17-Ala21. All these structural characteristics are inconsistent with the amyloid fibril structure and indicate that the dimer has to undergo significant rearrangement en route to fibril formation.

Dimer formation and transcription activation in the sporulation response regulator Spo0A.

PubMed

Lewis, Richard J; Scott, David J; Brannigan, James A; Ladds, Joanne C; Cervin, Marguerite A; Spiegelman, George B; Hoggett, James G; Barák, Imrich; Wilkinson, Anthony J

2002-02-15

The response regulator Spo0A is the master control element in the initiation of sporulation in Bacillus subtilis. Like many other multi-domain response regulators, the latent activity of the effector, C-terminal domain is stimulated by phosphorylation on a conserved aspartic acid residue in the regulatory, N-terminal domain. If a threshold concentration of phosphorylated Spo0A is achieved, the transcription of genes required for sporulation is activated, whereas the genes encoding stationary phase sentinels are repressed, and sporulation proceeds. Despite detailed genetic, biochemical and structural characterisation, it is not understood how the phosphorylation signal in the receiver domain is transduced into DNA binding and transcription activation in the distal effector domain. An obstacle to our understanding of Spo0A function is the uncertainty concerning changes in quaternary structure that accompany phosphorylation. Here we have revisited this question and shown unequivocally that Spo0A forms dimers upon phosphorylation and that the subunit interactions in the dimer are mediated principally by the receiver domain. Purified dimers of two mutants of Spo0A, in which the phosphorylatable aspartic acid residue has been substituted, activate transcription from the spoIIG promoter in vitro, whereas monomers do not. This suggests that dimers represent the activated form of Spo0A. Copyright 2002 Elsevier Science Ltd.

Blocking an N-terminal acetylation–dependent protein interaction inhibits an E3 ligase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scott, Daniel C.; Hammill, Jared T.; Min, Jaeki

N-terminal acetylation is an abundant modification influencing protein functions. Because ~80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation–dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors aremore » highly selective with respect to other protein acetyl-amide–binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2–E3 ligases.« less

A multipronged strategy of an anti-terminator protein to overcome Rho-dependent transcription termination

PubMed Central

Muteeb, Ghazala; Dey, Debashish; Mishra, Saurabh; Sen, Ranjan

2012-01-01

One of the important role of Rho-dependent transcription termination in bacteria is to prevent gene expressions from the bacteriophage DNA. The transcription anti-termination systems of the lambdoid phages have been designed to overcome this Rho action. The anti-terminator protein N has three interacting regions, which interact with the mRNA, with the NusA and with the RNA polymerase. Here, we show that N uses all these interaction modules to overcome the Rho action. N and Rho co-occupy their overlapping binding sites on the nascent RNA (the nutR/tR1 site), and this configuration slows down the rate of ATP hydrolysis and the rate of RNA release by Rho from the elongation complex. N-RNA polymerase interaction is not too important for this Rho inactivation process near/at the nutR site. This interaction becomes essential when the elongation complex moves away from the nutR site. From the unusual NusA-dependence property of a Rho mutant E134K, a suppressor of N, we deduced that the N-NusA complex in the anti-termination machinery reduces the efficiency of Rho by removing NusA from the termination pathway. We propose that NusA-remodelling is also one of the mechanisms used by N to overcome the termination signals. PMID:23024214

A multipronged strategy of an anti-terminator protein to overcome Rho-dependent transcription termination.

PubMed

Muteeb, Ghazala; Dey, Debashish; Mishra, Saurabh; Sen, Ranjan

2012-12-01

One of the important role of Rho-dependent transcription termination in bacteria is to prevent gene expressions from the bacteriophage DNA. The transcription anti-termination systems of the lambdoid phages have been designed to overcome this Rho action. The anti-terminator protein N has three interacting regions, which interact with the mRNA, with the NusA and with the RNA polymerase. Here, we show that N uses all these interaction modules to overcome the Rho action. N and Rho co-occupy their overlapping binding sites on the nascent RNA (the nutR/tR1 site), and this configuration slows down the rate of ATP hydrolysis and the rate of RNA release by Rho from the elongation complex. N-RNA polymerase interaction is not too important for this Rho inactivation process near/at the nutR site. This interaction becomes essential when the elongation complex moves away from the nutR site. From the unusual NusA-dependence property of a Rho mutant E134K, a suppressor of N, we deduced that the N-NusA complex in the anti-termination machinery reduces the efficiency of Rho by removing NusA from the termination pathway. We propose that NusA-remodelling is also one of the mechanisms used by N to overcome the termination signals.

n-Dopants Based on Dimers of Benzimidazoline Radicals: Structures and Mechanism of Redox Reactions

PubMed Central

Zhang, Siyuan; Naab, Benjamin D.; Jucov, Evgheni V.; Parkin, Sean; Evans, Eric G. B.; Millhauser, Glenn L.; Timofeeva, Tatiana V.; Risko, Chad; Brédas, Jean-Luc; Bao, Zhenan; Barlow, Stephen; Marder, Seth R.

2015-01-01

Dimers of 2-substituted N,N'-dimethylbenzimidazoline radicals, (2-Y-DMBI)2 {Y = cyclohexyl (Cyc), ferrocenyl (Fc), ruthenocenyl (Rc)} have recently been reported as n-dopants for organic semiconductors. Here their structural and energetic characteristics are reported, along with the mechanisms by which they react with acceptors, A (PCBM, TIPS-pentacene), in solution. X-ray data and DFT both indicate a longer C—C bond for (2-Cyc-DMBI)2 than (2-Fc-DMBI)2, yet DFT and ESR data show that the latter dissociates more readily due to stabilization of the radical by Fc. Depending on the energetics of dimer (D2) dissociation and of D2-to-A electron transfer, D2 reacts with A to form D+ and A•– by either of two mechanisms, differing in whether the first step is endergonic dissociation or endergonic electron transfer. However, the D+/0.5D2 redox potentials – the effective reducing strengths of the dimers – vary little within the series (ca. –1.9 V vs. FeCp2+/0) due to cancelation of trends in the D+/0 potential and D2 dissociation energy. The implications of these findings for use of these dimers as n-dopants, and for future dopant design, are discussed. PMID:26088609

Mitochondrial Hsp90 is a ligand-activated molecular chaperone coupling ATP binding to dimer closure through a coiled-coil intermediate

PubMed Central

Sung, Nuri; Lee, Jungsoon; Kim, Ji-Hyun; Chang, Changsoo; Joachimiak, Andrzej; Lee, Sukyeong; Tsai, Francis T. F.

2016-01-01

Heat-shock protein of 90 kDa (Hsp90) is an essential molecular chaperone that adopts different 3D structures associated with distinct nucleotide states: a wide-open, V-shaped dimer in the apo state and a twisted, N-terminally closed dimer with ATP. Although the N domain is known to mediate ATP binding, how Hsp90 senses the bound nucleotide and facilitates dimer closure remains unclear. Here we present atomic structures of human mitochondrial Hsp90N (TRAP1N) and a composite model of intact TRAP1 revealing a previously unobserved coiled-coil dimer conformation that may precede dimer closure and is conserved in intact TRAP1 in solution. Our structure suggests that TRAP1 normally exists in an autoinhibited state with the ATP lid bound to the nucleotide-binding pocket. ATP binding displaces the ATP lid that signals the cis-bound ATP status to the neighboring subunit in a highly cooperative manner compatible with the coiled-coil intermediate state. We propose that TRAP1 is a ligand-activated molecular chaperone, which couples ATP binding to dramatic changes in local structure required for protein folding. PMID:26929380

DNA sequence-specific dimeric bisbenzimidazoles DBP(n) and DBPA(n) as inhibitors of H-NS silencing in bacterial cells.

PubMed

Melkina, Olga E; Koval, Vasilii S; Ivanov, Alexander A; Zhuze, Alexei L; Zavilgelsky, Gennadii B

2018-03-01

DNA sequence-specific fluorescent dimeric bisbenzimidazoles DBP(n) and DBPA(n), noncovalently interacting with A-T pairs in the minor groove of double-stranded DNA were used for studying and monitoring the expression of histone-like H-NS-dependent promoters. Histone-like H-NS selectively binds to AT-rich segments of DNA and silences a large number of genes in bacterial chromosomes. The H-NS-dependent promoters of Quorum Sensing (QS)-regulated lux operons of the marine bacteria mesophilic Aliivibrio fischeri, psychrophilic Aliivibrio logei were used. Escherichia coli lux biosensors were constructed by cloning fragments bearing QS-regulated promoters into the vector, thereby placing each fragment upstream of the promoterless Photorhabdus luminescens luxCDABE genes. It was shown that the dimeric bisbenzimidazoles DBP(n) and DBPA(n) counteract the H-NS silencing activity. Thus, the presence of DBP(n) or DBPA(n) in the medium leads to an approximately 10-100-fold increase in the level of transcription of QS promoters in E. coli hns + . The largest decrease in the level of H-NS repression was observed using ligands containing a linker with a length of ca. 18Å, such as DBP(2) and DBPA(2). Ligands containing linkers with n=1 and 3 are an order of magnitude less active; ligands with n=4 are inactive. DBPA(2) exhibits activity starting with a concentration of 0.5μM; the minimum concentration of DBP(2) is 5-7 times higher. It is suggested that A-T pairs located at five nucleotide pair intervals, which correspond to the linker length in highly active ligands with n=2, play a key role in the structure of H-NS-binding sites in QS-regulated promoters. Copyright © 2017 Elsevier GmbH. All rights reserved.

Targeting cysteine-mediated dimerization of the MUC1-C oncoprotein in human cancer cells

PubMed Central

RAINA, DEEPAK; AHMAD, REHAN; RAJABI, HASAN; PANCHAMOORTHY, GOVIND; KHARBANDA, SURENDER; KUFE, DONALD

2012-01-01

The MUC1 heterodimeric protein is aberrantly overexpressed in diverse human carcinomas and contributes to the malignant phenotype. The MUC1-C transmembrane subunit contains a CQC motif in the cytoplasmic domain that has been implicated in the formation of dimers and in its oncogenic function. The present study demonstrates that MUC1-C forms dimers in human breast and lung cancer cells. MUC1-C dimerization was detectable in the cytoplasm and was independent of MUC1-N, the N-terminal mucin subunit that extends outside the cell. We show that the MUC1-C cytoplasmic domain forms dimers in vitro that are disrupted by reducing agents. Moreover, dimerization of the MUC1-C subunit in cancer cells was blocked by reducing agents and increased by oxidative stress, supporting involvement of the CQC motif in forming disulfide bonds. In support of these observations, mutation of the MUC1-C CQC motif to AQA completely blocked MUC1-C dimerization. Importantly, this study was performed with MUC1-C devoid of fluorescent proteins, such as GFP, CFP and YFP. In this regard, we show that GFP, CFP and YFP themselves form dimers that are readily detectable with cross-linking agents. The present results further demonstrate that a cell-penetrating peptide that targets the MUC1-C CQC cysteines blocks MUC1-C dimerization in cancer cells. These findings provide definitive evidence that: i) the MUC1-C cytoplasmic domain cysteines are necessary and sufficient for MUC1-C dimerization, and ii) these CQC motif cysteines represent an Achilles’ heel for targeting MUC1-C function. PMID:22200620

Relationship between the dimerization of thyroglobulin and its ability to form triiodothyronine.

PubMed

Citterio, Cintia E; Morishita, Yoshiaki; Dakka, Nada; Veluswamy, Balaji; Arvan, Peter

2018-03-30

Thyroglobulin (TG) is the most abundant thyroid gland protein, a dimeric iodoglycoprotein (660 kDa). TG serves as the protein precursor in the synthesis of thyroid hormones tetraiodothyronine (T 4 ) and triiodothyronine (T 3 ). The primary site for T 3 synthesis in TG involves an iodotyrosine acceptor at the antepenultimate Tyr residue (at the extreme carboxyl terminus of the protein). The carboxyl-terminal region of TG comprises a ch olin e sterase- l ike (ChEL) domain followed by a short unique tail sequence. Despite many studies, the monoiodotyrosine donor residue needed for the coupling reaction to create T 3 at this evolutionarily conserved site remains unidentified. In this report, we have utilized a novel, convenient immunoblotting assay to detect T 3 formation after protein iodination in vitro , enabling the study of T 3 formation in recombinant TG secreted from thyrocytes or heterologous cells. With this assay, we confirm the antepenultimate residue of TG as a major T 3 -forming site, but also demonstrate that the side chain of this residue intimately interacts with the same residue in the apposed monomer of the TG dimer. T 3 formation in TG, or the isolated carboxyl-terminal region, is inhibited by mutation of this antepenultimate residue, but we describe the first substitution mutation that actually increases T 3 hormonogenesis by engineering a novel cysteine, 10 residues upstream of the antepenultimate residue, allowing for covalent association of the unique tail sequences, and that helps to bring residues Tyr 2744 from apposed monomers into closer proximity. © 2018 Citterio et al.

Number and brightness image analysis reveals ATF-induced dimerization kinetics of uPAR in the cell membrane

PubMed Central

Hellriegel, Christian; Caiolfa, Valeria R.; Corti, Valeria; Sidenius, Nicolai; Zamai, Moreno

2011-01-01

We studied the molecular forms of the GPI-anchored urokinase plasminogen activator receptor (uPAR-mEGFP) in the human embryo kidney (HEK293) cell membrane and demonstrated that the binding of the amino-terminal fragment (ATF) of urokinase plasminogen activator is sufficient to induce the dimerization of the receptor. We followed the association kinetics and determined precisely the dimeric stoichiometry of uPAR-mEGFP complexes by applying number and brightness (N&B) image analysis. N&B is a novel fluctuation-based approach for measuring the molecular brightness of fluorophores in an image time sequence in live cells. Because N&B is very sensitive to long-term temporal fluctuations and photobleaching, we have introduced a filtering protocol that corrects for these important sources of error. Critical experimental parameters in N&B analysis are illustrated and analyzed by simulation studies. Control experiments are based on mEGFP-GPI, mEGFP-mEGFP-GPI, and mCherry-GPI, expressed in HEK293. This work provides a first direct demonstration of the dimerization of uPAR in live cells. We also provide the first methodological guide on N&B to discern minor changes in molecular composition such as those due to dimerization events, which are involved in fundamental cell signaling mechanisms.—Hellriegel, C., Caiolfa, V. R., Corti, V., Sidenius, N., Zamai, M. Number and brightness image analysis reveals ATF-induced dimerization kinetics of uPAR in the cell membrane. PMID:21602447

Aggregation of γ-crystallins associated with human cataracts via domain swapping at the C-terminal β-strands

PubMed Central

Das, Payel; King, Jonathan A.; Zhou, Ruhong

2011-01-01

The prevalent eye disease age-onset cataract is associated with aggregation of human γD-crystallins, one of the longest-lived proteins. Identification of the γ-crystallin precursors to aggregates is crucial for developing strategies to prevent and reverse cataract. Our microseconds of atomistic molecular dynamics simulations uncover the molecular structure of the experimentally detected aggregation-prone folding intermediate species of monomeric native γD-crystallin with a largely folded C-terminal domain and a mostly unfolded N-terminal domain. About 30 residues including a, b, and c strands from the Greek Key motif 4 of the C-terminal domain experience strong solvent exposure of hydrophobic residues as well as partial unstructuring upon N-terminal domain unfolding. Those strands comprise the domain–domain interface crucial for unusually high stability of γD-crystallin. We further simulate the intermolecular linkage of these monomeric aggregation precursors, which reveals domain-swapped dimeric structures. In the simulated dimeric structures, the N-terminal domain of one monomer is frequently found in contact with residues 135–164 encompassing the a, b, and c strands of the Greek Key motif 4 of the second molecule. The present results suggest that γD-crystallin may polymerize through successive domain swapping of those three C-terminal β-strands leading to age-onset cataract, as an evolutionary cost of its very high stability. Alanine substitutions of the hydrophobic residues in those aggregation-prone β-strands, such as L145 and M147, hinder domain swapping as a pathway toward dimerization. These findings thus provide critical molecular insights onto the initial stages of age-onset cataract, which is important for understanding protein aggregation diseases. PMID:21670251

n-Dopants Based on Dimers of Benzimidazoline Radicals: Structures and Mechanism of Redox Reactions.

PubMed

Zhang, Siyuan; Naab, Benjamin D; Jucov, Evgheni V; Parkin, Sean; Evans, Eric G B; Millhauser, Glenn L; Timofeeva, Tatiana V; Risko, Chad; Brédas, Jean-Luc; Bao, Zhenan; Barlow, Stephen; Marder, Seth R

2015-07-20

Dimers of 2-substituted N,N'-dimethylbenzimidazoline radicals, (2-Y-DMBI)2 (Y=cyclohexyl (Cyc), ferrocenyl (Fc), ruthenocenyl (Rc)), have recently been reported as n-dopants for organic semiconductors. Here their structural and energetic characteristics are reported, along with the mechanisms by which they react with acceptors, A (PCBM, TIPS-pentacene), in solution. X-ray data and DFT calculations both indicate a longer C-C bond for (2-Cyc-DMBI)2 than (2-Fc-DMBI)2 , yet DFT and ESR data show that the latter dissociates more readily due to stabilization of the radical by Fc. Depending on the energetics of dimer (D2 ) dissociation and of D2 -to-A electron transfer, D2 reacts with A to form D(+) and A(-) by either of two mechanisms, differing in whether the first step is endergonic dissociation or endergonic electron transfer. However, the D(+) /0.5 D2 redox potentials-the effective reducing strengths of the dimers-vary little within the series (ca. -1.9 V vs. FeCp2 (+/0) ) (Cp=cyclopentadienyl) due to cancelation of trends in the D(+/0) potential and D2 dissociation energy. The implications of these findings for use of these dimers as n-dopants, and for future dopant design, are discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Altering the N-terminal arms of the polymerase manager protein UmuD modulates protein interactions.

PubMed

Murison, David A; Ollivierre, Jaylene N; Huang, Qiuying; Budil, David E; Beuning, Penny J

2017-01-01

Escherichia coli cells that are exposed to DNA damaging agents invoke the SOS response that involves expression of the umuD gene products, along with more than 50 other genes. Full-length UmuD is expressed as a 139-amino-acid protein, which eventually cleaves its N-terminal 24 amino acids to form UmuD'. The N-terminal arms of UmuD are dynamic and contain recognition sites for multiple partner proteins. Cleavage of UmuD to UmuD' dramatically affects the function of the protein and activates UmuC for translesion synthesis (TLS) by forming DNA Polymerase V. To probe the roles of the N-terminal arms in the cellular functions of the umuD gene products, we constructed additional N-terminal truncated versions of UmuD: UmuD 8 (UmuD Δ1-7) and UmuD 18 (UmuD Δ1-17). We found that the loss of just the N-terminal seven (7) amino acids of UmuD results in changes in conformation of the N-terminal arms, as determined by electron paramagnetic resonance spectroscopy with site-directed spin labeling. UmuD 8 is cleaved as efficiently as full-length UmuD in vitro and in vivo, but expression of a plasmid-borne non-cleavable variant of UmuD 8 causes hypersensitivity to UV irradiation, which we determined is the result of a copy-number effect. UmuD 18 does not cleave to form UmuD', but confers resistance to UV radiation. Moreover, removal of the N-terminal seven residues of UmuD maintained its interactions with the alpha polymerase subunit of DNA polymerase III as well as its ability to disrupt interactions between alpha and the beta processivity clamp, whereas deletion of the N-terminal 17 residues resulted in decreases in binding to alpha and in the ability to disrupt the alpha-beta interaction. We find that UmuD 8 mimics full-length UmuD in many respects, whereas UmuD 18 lacks a number of functions characteristic of UmuD.

Alternative dimerization interfaces in the glucocorticoid receptor-α ligand binding domain.

PubMed

Bianchetti, Laurent; Wassmer, Bianca; Defosset, Audrey; Smertina, Anna; Tiberti, Marion L; Stote, Roland H; Dejaegere, Annick

2018-04-30

Nuclear hormone receptors (NRs) constitute a large family of multi-domain ligand-activated transcription factors. Dimerization is essential for their regulation, and both DNA binding domain (DBD) and ligand binding domain (LBD) are implicated in dimerization. Intriguingly, the glucocorticoid receptor-α (GRα) presents a DBD dimeric architecture similar to that of the homologous estrogen receptor-α (ERα), but an atypical dimeric architecture for the LBD. The physiological relevance of the proposed GRα LBD dimer is a subject of debate. We analyzed all GRα LBD homodimers observed in crystals using an energetic analysis based on the PISA and on the MM/PBSA methods and a sequence conservation analysis, using the ERα LBD dimer as a reference point. Several dimeric assemblies were observed for GRα LBD. The assembly generally taken to be physiologically relevant showed weak binding free energy and no significant residue conservation at the contact interface, while an alternative homodimer mediated by both helix 9 and C-terminal residues showed significant binding free energy and residue conservation. However, none of the GRα LBD assemblies found in crystals are as stable or conserved as the canonical ERα LBD dimer. GRα C-terminal sequence (F-domain) forms a steric obstacle to the canonical dimer assembly in all available structures. Our analysis calls for a re-examination of the currently accepted GRα homodimer structure and experimental investigations of the alternative architectures. This work questions the validity of the currently accepted architecture. This has implications for interpreting physiological data and for therapeutic design pertaining to glucocorticoid research. Copyright © 2018. Published by Elsevier B.V.

Amino-terminal domains of c-myc and N-myc proteins mediate binding to the retinoblastoma gene product

NASA Astrophysics Data System (ADS)

Rustgi, Anil K.; Dyson, Nicholas; Bernards, Rene

1991-08-01

THE proteins encoded by the myc gene family are involved in the control of cell proliferation and differentiation, and aberrant expression of myc proteins has been implicated in the genesis of a variety of neoplasms1. In the carboxyl terminus, myc proteins have two domains that encode a basic domain/helix-loop-helix and a leucine zipper motif, respectively. These motifs are involved both in DNA binding and in protein dimerization2-5. In addition, myc protein family members share several regions of highly conserved amino acids in their amino termini that are essential for transformation6,7. We report here that an N-terminal domain present in both the c-myc and N-myc proteins mediates binding to the retinoblastoma gene product, pRb. We show that the human papilloma virus E7 protein competes with c-myc for binding to pRb, indicating that these proteins share overlapping binding sites on pRb. Furthermore, a mutant Rb protein from a human tumour cell line that carried a 35-amino-acid deletion in its C terminus failed to bind to c-myc. Our results suggest that c-myc and pRb cooperate through direct binding to control cell proliferation.

A designed point mutant in Fis1 disrupts dimerization and mitochondrial fission

PubMed Central

Lees, Jonathan P. B.; Manlandro, Cara Marie; Picton, Lora K.; Ebie Tan, Alexandra Z.; Casares, Salvador; Flanagan, John M.; Fleming, Karen G.; Hill, R. Blake

2012-01-01

Mitochondrial and peroxisomal fission are essential processes with defects resulting in cardiomyopathy and neonatal lethality. Central to organelle fission is Fis1, a monomeric tetratricopeptide-like repeat (TPR) protein whose role in assembly of the fission machinery remains obscure. Two non-functional, Saccharomyces cerevisiae Fis1 mutants (L80P or E78D/I85T/Y88H) were previously identified in genetic screens. Here, we find that these two variants in the cytosolic domain of Fis1 (Fis1ΔTM) are unexpectedly dimeric. A truncation variant of Fis1ΔTM that lacks an N-terminal regulatory domain is also found to be dimeric. The ability to dimerize is a property innate to the native Fis1ΔTM amino acid sequence as we find this domain is dimeric after transient exposure to elevated temperature or chemical denaturants and is kinetically trapped at room temperature. This is the first demonstration of a specific self-association in solution for the Fis1 cytoplasmic domain. We propose a three-dimensional domain-swapped model for dimerization that is validated by a designed mutation, A72P, which potently disrupts dimerization of wild type Fis1. A72P also disrupts dimerization of non-functional variants indicating a common structural basis for dimerization. The obligate monomer variant A72P, like the dimer-promoting variants, is non-functional in fission consistent with a model in which Fis1 activity depends on its ability to interconvert between monomer and dimer species. These studies suggest a new functionally important manner in which TPR containing proteins may reversibly self-associate. PMID:22789569

Accumulation of the Cyclobutane Thymine Dimer in Defined Sequences of Free and Nucleosomal DNA

DTIC Science & Technology

2013-08-01

cyclobutane dimer in a single-stranded vector , Proc. Natl. Acad. Sci. U. S. A., 1988, 85, 8141–8145. 11 C. A. Smith, M. Wang, N. Jiang, L. Che, X. Zhao and...J.-S. Taylor, Mutation spectra of M13 vectors containing site-specific cis–syn, trans–syn-I, (6-4), and Dewar pyrimi- done photoproducts of thymidylyl...Bypass of a site-specific cis–syn thymine dimer in a SV40 vector during in vitro replication by HeLa and XPV cell-free extracts, Biochemistry, 1998

Crystal Structure of Marburg Virus VP40 Reveals a Broad, Basic Patch for Matrix Assembly and a Requirement of the N-Terminal Domain for Immunosuppression.

PubMed

Oda, Shun-Ichiro; Noda, Takeshi; Wijesinghe, Kaveesha J; Halfmann, Peter; Bornholdt, Zachary A; Abelson, Dafna M; Armbrust, Tammy; Stahelin, Robert V; Kawaoka, Yoshihiro; Saphire, Erica Ollmann

2016-02-15

Marburg virus (MARV), a member of the filovirus family, causes severe hemorrhagic fever with up to 90% lethality. MARV matrix protein VP40 is essential for assembly and release of newly copied viruses and also suppresses immune signaling in the infected cell. Here we report the crystal structure of MARV VP40. We found that MARV VP40 forms a dimer in solution, mediated by N-terminal domains, and that formation of this dimer is essential for budding of virus-like particles. We also found the N-terminal domain to be necessary and sufficient for immune antagonism. The C-terminal domains of MARV VP40 are dispensable for immunosuppression but are required for virus assembly. The C-terminal domains are only 16% identical to those of Ebola virus, differ in structure from those of Ebola virus, and form a distinct broad and flat cationic surface that likely interacts with the cell membrane during virus assembly. Marburg virus, a cousin of Ebola virus, causes severe hemorrhagic fever, with up to 90% lethality seen in recent outbreaks. Molecular structures and visual images of the proteins of Marburg virus are essential for the development of antiviral drugs. One key protein in the Marburg virus life cycle is VP40, which both assembles the virus and suppresses the immune system. Here we provide the molecular structure of Marburg virus VP40, illustrate differences from VP40 of Ebola virus, and reveal surfaces by which Marburg VP40 assembles progeny and suppresses immune function. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The Dimeric Architecture of Checkpoint Kinases Mec1ATR and Tel1ATM Reveal a Common Structural Organization.

PubMed

Sawicka, Marta; Wanrooij, Paulina H; Darbari, Vidya C; Tannous, Elias; Hailemariam, Sarem; Bose, Daniel; Makarova, Alena V; Burgers, Peter M; Zhang, Xiaodong

2016-06-24

The phosphatidylinositol 3-kinase-related protein kinases are key regulators controlling a wide range of cellular events. The yeast Tel1 and Mec1·Ddc2 complex (ATM and ATR-ATRIP in humans) play pivotal roles in DNA replication, DNA damage signaling, and repair. Here, we present the first structural insight for dimers of Mec1·Ddc2 and Tel1 using single-particle electron microscopy. Both kinases reveal a head to head dimer with one major dimeric interface through the N-terminal HEAT (named after Huntingtin, elongation factor 3, protein phosphatase 2A, and yeast kinase TOR1) repeat. Their dimeric interface is significantly distinct from the interface of mTOR complex 1 dimer, which oligomerizes through two spatially separate interfaces. We also observe different structural organizations of kinase domains of Mec1 and Tel1. The kinase domains in the Mec1·Ddc2 dimer are located in close proximity to each other. However, in the Tel1 dimer they are fully separated, providing potential access of substrates to this kinase, even in its dimeric form. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Crystal structure and function of an unusual dimeric Hsp20.1 provide insight into the thermal protection mechanism of small heat shock proteins.

PubMed

Liu, Liang; Chen, Jiyun; Yang, Bo; Wang, Yonghua

2015-03-06

Small heat shock proteins (sHSPs) are ubiquitous chaperones that play a vital role in protein homeostasis. sHSPs are characterized by oligomeric architectures and dynamic exchange of subunits. The flexible oligomeric assembling associating with function remains poorly understood. Based on the structural data, it is certainly agreed that two dimerization models depend on the presence or absence of a β6 strand to differentiate nonmetazoan sHSPs from metazoan sHSPs. Here, we report the Sulfolobus solfataricus Hsp20.1 ACD dimer structure, which shows a distinct dimeric interface. We observed that, in the absence of β6, Hsp20.1 dimer does not depend on β7 strand for forming dimer interface as metazoan sHSPs, nor dissociates to monomers. This is in contrast to other published sHSPs. Our structure reveals a variable, highly polar dimer interface that has advantages for rapid subunits exchange and substrate binding. Remarkably, we find that the C-terminal truncation variant has chaperone activity comparable to that of wild-type despite lack of the oligomer structure. Our further study indicates that the N-terminal region is essential for the oligomer and dimer binding to the target protein. Together, the structure and function of Hsp20.1 give more insight into the thermal protection mechanism of sHSPs. Copyright © 2015 Elsevier Inc. All rights reserved.

Pestivirus Npro Directly Interacts with Interferon Regulatory Factor 3 Monomer and Dimer

PubMed Central

Holthauzen, Luis Marcelo F.; Ruggli, Nicolas

2016-01-01

ABSTRACT Interferon regulatory factor 3 (IRF3) is a transcription factor involved in the activation of type I alpha/beta interferon (IFN-α/β) in response to viral infection. Upon viral infection, the IRF3 monomer is activated into a phosphorylated dimer, which induces the transcription of interferon genes in the nucleus. Viruses have evolved several ways to target IRF3 in order to subvert the innate immune response. Pestiviruses, such as classical swine fever virus (CSFV), target IRF3 for ubiquitination and subsequent proteasomal degradation. This is mediated by the viral protein Npro that interacts with IRF3, but the molecular details for this interaction are largely unknown. We used recombinant Npro and IRF3 proteins and show that Npro interacts with IRF3 directly without additional proteins and forms a soluble 1:1 complex. The full-length IRF3 but not merely either of the individual domains is required for this interaction. The interaction between Npro and IRF3 is not dependent on the activation state of IRF3, since Npro binds to a constitutively active form of IRF3 in the presence of its transcriptional coactivator, CREB-binding protein (CBP). The results indicate that the Npro-binding site on IRF3 encompasses a region that is unperturbed by the phosphorylation and subsequent activation of IRF3 and thus excludes the dimer interface and CBP-binding site. IMPORTANCE The pestivirus N-terminal protease, Npro, is essential for evading the host's immune system by facilitating the degradation of interferon regulatory factor 3 (IRF3). However, the nature of the Npro interaction with IRF3, including the IRF3 species (inactive monomer versus activated dimer) that Npro targets for degradation, is largely unknown. We show that classical swine fever virus Npro and porcine IRF3 directly interact in solution and that full-length IRF3 is required for interaction with Npro. Additionally, Npro interacts with a constitutively active form of IRF3 bound to its transcriptional

Structural insights into the human RyR2 N-terminal region involved in cardiac arrhythmias

DOE Office of Scientific and Technical Information (OSTI.GOV)

Borko, Ľubomír; Bauerová-Hlinková, Vladena, E-mail: vladena.bauerova@savba.sk; Hostinová, Eva

2014-11-01

X-ray and solution structures of the human RyR2 N-terminal region were obtained under near-physiological conditions. The structure exhibits a unique network of interactions between its three domains, revealing an important stabilizing role of the central helix. Human ryanodine receptor 2 (hRyR2) mediates calcium release from the sarcoplasmic reticulum, enabling cardiomyocyte contraction. The N-terminal region of hRyR2 (amino acids 1–606) is the target of >30 arrhythmogenic mutations and contains a binding site for phosphoprotein phosphatase 1. Here, the solution and crystal structures determined under near-physiological conditions, as well as a homology model of the hRyR2 N-terminal region, are presented. The N-terminusmore » is held together by a unique network of interactions among its three domains, A, B and C, in which the central helix (amino acids 410–437) plays a prominent stabilizing role. Importantly, the anion-binding site reported for the mouse RyR2 N-terminal region is notably absent from the human RyR2. The structure concurs with the differential stability of arrhythmogenic mutations in the central helix (R420W, I419F and I419F/R420W) which are owing to disparities in the propensity of mutated residues to form energetically favourable or unfavourable contacts. In solution, the N-terminus adopts a globular shape with a prominent tail that is likely to involve residues 545–606, which are unresolved in the crystal structure. Docking the N-terminal domains into cryo-electron microscopy maps of the closed and open RyR1 conformations reveals C{sup α} atom movements of up to 8 Å upon channel gating, and predicts the location of the leucine–isoleucine zipper segment and the interaction site for spinophilin and phosphoprotein phosphatase 1 on the RyR surface.« less

Combinatorial interactions of two amino acids with a single base pair define target site specificity in plant dimeric homeodomain proteins

PubMed Central

Tron, Adriana E.; Bertoncini, Carlos W.; Palena, Claudia M.; Chan, Raquel L.; Gonzalez, Daniel H.

2001-01-01

Four groups of plant homeodomain proteins contain a dimerization motif closely linked to the homeodomain. We here show that two sunflower homeodomain proteins, Hahb-4 and HAHR1, which belong to the Hd-Zip I and GL2/Hd-Zip IV groups, respectively, show different binding preferences at a defined position of a pseudopalindromic DNA-binding site used as a target. HAHR1 shows a preference for the sequence 5′-CATT(A/T)AATG-3′, rather than 5′-CAAT(A/T)ATTG-3′, recognized by Hahb-4. To analyze the molecular basis of this behavior, we have constructed a set of mutants with exchanged residues (Phe→Ile and Ile→Phe) at position 47 of the homeodomain, together with chimeric proteins between HAHR1 and Hahb-4. The results obtained indicate that Phe47, but not Ile47, allows binding to 5′-CATT(A/T)AATG-3′. However, the preference for this sequence is determined, in addition, by amino acids located C-terminal to residue 53 of the HAHR1 homeodomain. A double mutant of Hahb-4 (Ile47→Phe/Ala54→Thr) shows the same binding behavior as HAHR1, suggesting that combinatorial interactions of amino acid residues at positions 47 and 54 of the homeodomain are involved in establishing the affinity and selectivity of plant dimeric homeodomain proteins with different DNA target sequences. PMID:11726696

Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gottipati, Keerthi; Acholi, Sudheer; Ruggli, Nicolas

Pestivirus N{sup pro} is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N{sup pro} blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N{sup pro'}s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N{sup pro}-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N{sup pro} proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 thatmore » forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N{sup pro'}s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N{sup pro} does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N{sup pro'}s autoproteolysis is studied using N{sup pro}-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N{sup pro} prefers small amino acids with non-branched beta carbons at the P1 position.« less

Enhancing action of positive allosteric modulators through the design of dimeric compounds.

PubMed

Drapier, Thomas; Geubelle, Pierre; Bouckaert, Charlotte; Nielsen, Lise; Laulumaa, Saara; Goffin, Eric; Dilly, Sébastien; Francotte, Pierre; Hanson, Julien; Pochet, Lionel; Kastrup, Jette Sandholm; Pirotte, Bernard

2018-05-18

The present study describes the identification of highly potent dimeric 1,2,4-benzothiadiazine 1,1-dioxide (BTD)-type positive allosteric modulators of the AMPA receptors (AMPApams) obtained by linking two monomeric BTD scaffolds through their respective 6-positions. Using previous X-ray data from monomeric BTDs co-crystallized with the GluA2o ligand-binding domain (LBD), a molecular modeling approach was performed to predict the preferred dimeric combinations. Two 6,6-ethylene-linked dimeric BTD compounds (16 and 22) were prepared and evaluated as AMPApams on HEK293 cells expressing GluA2o(Q) (calcium flux experiment). These compounds were found to be about 10,000 times more potent than their respective monomers, the most active dimeric compound being the bis-4-cyclopropyl-substituted compound 22 [6,6'-(ethane-1,2-diyl)bis(4-cyclopropyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide], with an EC50 value of 1.4 nM. As a proof of concept, the bis-4-methyl-substituted dimeric compound 16 (EC50 = 13 nM) was successfully co-crystallized with the GluA2o-LBD and was found to occupy the two BTD binding sites at the LBD dimer interface.

Mutations of the phage lambda nutL region that prevent the action of Nun, a site-specific transcription termination factor.

PubMed Central

Baron, J; Weisberg, R A

1992-01-01

Phage HK022 encodes a protein, Nun, that promotes transcription termination within the pL and pR operons of its relative, phage lambda. The lambda sequences required for termination had previously been shown to overlap the nut sites, which are essential for transcription antitermination during normal lambda growth. To further specify the Nun target and to determine its relation to the nut sites, we constructed deletion and base substitution mutations of the lambda nutL region and measured Nun-dependent reduction of the expression of a downstream reporter gene. The shortest construct that retained full Nun responsiveness was a 42-bp segment that included both boxA and boxB, sequences that have been implicated in lambda antitermination. Deletion of boxA reduced Nun termination, and deletion of both sequences eliminated Nun termination. Base substitutions in boxA and the proximal portion of boxB impaired Nun termination, while base substitutions between boxA and boxB, in the distal portion of boxB, and immediately downstream from boxB had no appreciable effect. The termination defect of all of the base substitution mutations was relieved by increasing the level of Nun protein; in contrast, the deletions and a multiple-base substitution did not regain full Nun responsiveness at elevated Nun concentrations. We also asked if these mutant nut regions retained their ability to interact with N, the lambda-encoded antitermination protein. A qualitative assay showed that mutations within boxA or boxB reduced interaction, while mutations outside boxA and boxB did not. These data show that (i) the recognition sites for N and Nun overlap to a very considerable extent but are probably not identical and (ii) a high concentration of Nun promotes its interaction with mutant nut sites, a behavior also reported to be characteristic of N. PMID:1532174

Resin-assisted Enrichment of N-terminal Peptides for Characterizing Proteolytic Processing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Jong Seo; Dai, Ziyu; Aryal, Uma K.

2013-06-17

Proteolytic processing is a ubiquitous, irreversible posttranslational modification that plays an important role in cellular regulation in all living organisms. Herein we report a resin-assisted positive selection method for specifically enriching protein N-terminal peptides to facilitate the characterization of proteolytic processing events by liquid chromatography-tandem mass spectrometry. In this approach, proteins are initially reduced and alkylated and their lysine residues are converted to homoarginines. Then, protein N-termini are selectively converted to reactive thiol groups. We demonstrate that these sequential reactions were achieved with nearly quantitative efficiencies. Thiol-containing N-terminal peptides are then captured (>98% efficiency) by a thiol-affinity resin, a significantmore » improvement over the traditional avidin/biotin enrichment. Application to cell lysates of Aspergillus niger, a filamentous fungus of interest for biomass degradation, enabled the identification of 1672 unique protein N-termini and proteolytic cleavage sites from 690 unique proteins.« less

Membrane interaction of the N-terminal domain of chemokine receptor CXCR1.

PubMed

Haldar, Sourav; Raghuraman, H; Namani, Trishool; Rajarathnam, Krishna; Chattopadhyay, Amitabha

2010-06-01

The N-terminal domain of chemokine receptors constitutes one of the two critical ligand binding sites, and plays important roles by mediating binding affinity, receptor selectivity, and regulating function. In this work, we monitored the organization and dynamics of a 34-mer peptide of the CXC chemokine receptor 1 (CXCR1) N-terminal domain and its interaction with membranes by utilizing a combination of fluorescence-based approaches and surface pressure measurements. Our results show that the CXCR1 N-domain 34-mer peptide binds vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and upon binding, the tryptophan residues of the peptide experience motional restriction and exhibit red edge excitation shift (REES) of 19nm. These results are further supported by increase in fluorescence anisotropy and mean fluorescence lifetime upon membrane binding. These results constitute one of the first reports demonstrating membrane interaction of the N-terminal domain of CXCR1 and gain relevance in the context of the emerging role of cellular membranes in chemokine signaling.

C-terminal interactions mediate the quaternary dynamics of αB-crystallin

PubMed Central

Hilton, Gillian R.; Hochberg, Georg K. A.; Laganowsky, Arthur; McGinnigle, Scott I.; Baldwin, Andrew J.; Benesch, Justin L. P.

2013-01-01

αB-crystallin is a highly dynamic, polydisperse small heat-shock protein that can form oligomers ranging in mass from 200 to 800 kDa. Here we use a multifaceted mass spectrometry approach to assess the role of the C-terminal tail in the self-assembly of αB-crystallin. Titration experiments allow us to monitor the binding of peptides representing the C-terminus to the αB-crystallin core domain, and observe individual affinities to both monomeric and dimeric forms. Notably, we find that binding the second peptide equivalent to the core domain dimer is considerably more difficult than the first, suggesting a role of the C-terminus in regulating assembly. This finding motivates us to examine the effect of point mutations in the C-terminus in the full-length protein, by quantifying the changes in oligomeric distribution and corresponding subunit exchange rates. Our results combine to demonstrate that alterations in the C-terminal tail have a significant impact on the thermodynamics and kinetics of αB-crystallin. Remarkably, we find that there is energy compensation between the inter- and intra-dimer interfaces: when one interaction is weakened, the other is strengthened. This allosteric communication between binding sites on αB-crystallin is likely important for its role in binding target proteins. PMID:23530258

Structure and Function of the Sterol Carrier Protein-2 N-Terminal Presequence†

PubMed Central

Martin, Gregory G.; Hostetler, Heather A.; McIntosh, Avery L.; Tichy, Shane E.; Williams, Brad J.; Russell, David H.; Berg, Jeremy M.; Spencer, Thomas A.; Ball, Judith; Kier, Ann B.; Schroeder, Friedhelm

2008-01-01

Although sterol carrier protein-2 (SCP-2) is encoded as a precursor protein (proSCP-2), little is known regarding the structure and function of the 20-amino acid N-terminal presequence. As shown herein, the presequence contains significant secondary structure and alters SCP-2: (i) secondary structure (CD), (ii) tertiary structure (aqueous exposure of Trp shown by UV absorbance, fluorescence, fluorescence quenching), (iii) ligand binding site [Trp response to ligands, peptide cross-linked by photoactivatable free cholesterol (FCBP)], (iv) selectivity for interaction with anionic phospholipid-rich membranes, (v) interaction with a peroxisomal import protein [FRET studies of Pex5p(C) binding], the N-terminal presequence increased SCP-2’s affinity for Pex5p(C) by 10-fold, and (vi) intracellular targeting in living and fixed cells (confocal microscopy). Nearly 5-fold more SCP-2 than proSCP-2 colocalized with plasma membrane lipid rafts/caveolae (AF488-CTB), 2.8-fold more SCP-2 than proSCP-2 colocalized with a mitochondrial marker (Mitotracker), but nearly 2-fold less SCP-2 than proSCP-2 colocalized with peroxisomes (AF488-antibody to PMP70). These data indicate the importance of the N-terminal presequence in regulating SCP-2 structure, cholesterol localization within the ligand binding site, membrane association, and, potentially, intracellular targeting. PMID:18465878

The N-terminal region of the dopamine D2 receptor, a rhodopsin-like GPCR, regulates correct integration into the plasma membrane and endocytic routes

PubMed Central

Cho, DI; Min, C; Jung, KS; Cheong, SY; Zheng, M; Cheong, SJ; Oak, MH; Cheong, JH; Lee, BK; Kim, KM

2012-01-01

BACKGROUND AND PURPOSE Functional roles of the N-terminal region of rhodopsin-like GPCR family remain unclear. Using dopamine D2 and D3 receptors as a model system, we probed the roles of the N-terminal region in the signalling, intracellular trafficking of receptor proteins, and explored the critical factors that determine the functionality of the N-terminal region. EXPERIMENTAL APPROACH The N-terminal region of the D2 receptor was gradually shortened or switched with that of the D3 receptor or a non-specific sequence (FLAG), or potential N-terminal glycosylation sites were mutated. Effects of these manipulations on surface expression, internalization, post-endocytic behaviours and signalling were determined. KEY RESULTS Shortening the N-terminal region of the D2 receptor enhanced receptor internalization and impaired surface expression and signalling; ligand binding, desensitization and down-regulation were not affected but their association with a particular microdomain, caveolae, was disrupted. Replacement of critical residues within the N-terminal region with the FLAG epitope failed to restore surface expression but partially restored the altered internalization and signalling. When the N-terminal regions were switched between D2 and D3 receptors, cell surface expression pattern of each receptor was switched. Mutations of potential N-terminal glycosylation sites inhibited surface expression but enhanced internalization of D2 receptors. CONCLUSIONS AND IMPLICATIONS Shortening of N-terminus or mutation of glycosylation sites located within the N-terminus enhanced receptor internalization but impaired the surface expression of D2 receptors. The N-terminal region of the D2 receptor, in a sequence-specific manner, controls the receptor's conformation and integration into the plasma membrane, which determine its subcellular localization, intracellular trafficking and signalling properties. PMID:22117524

N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis

PubMed Central

Taggart, David J.; Dayeh, Daniel M.; Fredrickson, Saul W.; Suo, Zucai

2014-01-01

The X-family DNA polymerases λ (Polλ) and β (Polβ) possess similar 5′-2-deoxyribose-5-phosphatelyase (dRPase) and polymerase domains. Besides these domains, Polλ also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Polλ. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Polβ, Polλ and two N-terminal deletion constructs of Polλ during the bypass of either an abasic site or a 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Polλ enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Polλ did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Polλ and Polβ preferentially generated −1 or −2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Polλ cooperatively promoted the generation of −2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Polλ increased the generation of −1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Polλ opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Polλ act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Polλ as a gap-filling polymerase

Structure, Dynamics, and Allosteric Potential of Ionotropic Glutamate Receptor N-Terminal Domains

PubMed Central

Krieger, James; Bahar, Ivet; Greger, Ingo H.

2015-01-01

Ionotropic glutamate receptors (iGluRs) are tetrameric cation channels that mediate synaptic transmission and plasticity. They have a unique modular architecture with four domains: the intracellular C-terminal domain (CTD) that is involved in synaptic targeting, the transmembrane domain (TMD) that forms the ion channel, the membrane-proximal ligand-binding domain (LBD) that binds agonists such as L-glutamate, and the distal N-terminal domain (NTD), whose function is the least clear. The extracellular portion, comprised of the LBD and NTD, is loosely arranged, mediating complex allosteric regulation and providing a rich target for drug development. Here, we briefly review recent work on iGluR NTD structure and dynamics, and further explore the allosteric potential for the NTD in AMPA-type iGluRs using coarse-grained simulations. We also investigate mechanisms underlying the established NTD allostery in NMDA-type iGluRs, as well as the fold-related metabotropic glutamate and GABAB receptors. We show that the clamshell motions intrinsically favored by the NTD bilobate fold are coupled to dimeric and higher-order rearrangements that impact the iGluR LBD and ultimately the TMD. Finally, we explore the dynamics of intact iGluRs and describe how it might affect receptor operation in a synaptic environment. PMID:26255587

Role of the ancillary ligand N,N-dimethylaminoethanol in the sensitization of Eu(III) and Tb(III) luminescence in dimeric beta-diketonates.

PubMed

Eliseeva, Svetlana V; Kotova, Oxana V; Gumy, Frédéric; Semenov, Sergey N; Kessler, Vadim G; Lepnev, Leonid S; Bünzli, Jean-Claude G; Kuzmina, Natalia P

2008-04-24

Two types of dimeric complexes [Ln2(hfa)6(mu2-O(CH2)2NHMe2)2] and [Ln(thd)2(mu2,eta2-O(CH2)2NMe2)]2 (Ln = YIII, EuIII, GdIII, TbIII, TmIII, LuIII; hfa- = hexafluoroacetylacetonato, thd- = dipivaloylmethanato) are obtained by reacting [Ln(hfa)3(H2O)2] and [Ln(thd)3], respectively, with N,N-dimethylaminoethanol in toluene and are fully characterized. X-ray single crystal analysis performed for the TbIII compounds confirms their dimeric structure. The coordination mode of N,N-dimethylaminoethanol depends on the nature of the beta-diketonate. In [Tb2(hfa)6(mu2-O(CH2)2NHMe2)2], eight-coordinate TbIII ions adopt distorted square antiprismatic coordination environments and are O-bridged by two zwitterionic N,N-dimethylaminoethanol ligands with a Tb1...Tb2 separation of 3.684(1) A. In [Tb(thd)2(mu2,eta2-O(CH2)2NMe2)]2, the N,N-dimethylaminoethanol acts as chelating-bridging O,N-donor anion and the TbIII ions are seven-coordinate; the Tb1...Tb1A separation amounts to 3.735(2) A within centrosymmetric dimers. The dimeric complexes are thermally stable up to 180 degrees C, as shown by thermogravimetric analysis, and their volatility is sufficient for quantitative sublimation under reduced pressure. The EuIII and TbIII dimers display metal-centered luminescence, particularly [Eu2(hfa)6(O(CH2)2NHMe2)2] (quantum yield Q(L)Ln = 58%) and [Tb(thd)2(O(CH2)2NMe2)]2 (32%). Consideration of energy migration paths within the dimers, based on the study of both pure and EuIII- or TbIII-doped (0.01-0.1 mol %) LuIII analogues, leads to the conclusion that both the beta-diketone and N,N-dimethylaminoethanol ligands contribute significantly to the sensitization process of the EuIII luminescence. The ancillary ligand increases considerably the luminescence of [Eu2(hfa)6(O(CH2)2NHMe2)2], compared to [Ln(hfa)3(H2O)2], through the formation of intra-ligand states while it is detrimental to TbIII luminescence in both beta-diketonates. Thin films of the most luminescent compound [Eu2(hfa)6(O(CH2

Regulatory interactions in the dimeric cytochrome bc(1) complex: the advantages of being a twin.

PubMed

Covian, Raul; Trumpower, Bernard L

2008-09-01

The dimeric cytochrome bc(1) complex catalyzes the oxidation-reduction of quinol and quinone at sites located in opposite sides of the membrane in which it resides. We review the kinetics of electron transfer and inhibitor binding that reveal functional interactions between the quinol oxidation site at center P and quinone reduction site at center N in opposite monomers in conjunction with electron equilibration between the cytochrome b subunits of the dimer. A model for the mechanism of the bc(1) complex has emerged from these studies in which binding of ligands that mimic semiquinone at center N regulates half-of-the-sites reactivity at center P and binding of ligands that mimic catalytically competent binding of ubiquinol at center P regulates half-of-the-sites reactivity at center N. An additional feature of this model is that inhibition of quinol oxidation at the quinone reduction site is avoided by allowing catalysis in only one monomer at a time, which maximizes the number of redox acceptor centers available in cytochrome b for electrons coming from quinol oxidation reactions at center P and minimizes the leakage of electrons that would result in the generation of damaging oxygen radicals.

PSD-95 uncoupling from NMDA receptors by Tat- N-dimer ameliorates neuronal depolarization in cortical spreading depression.

PubMed

Kucharz, Krzysztof; Søndergaard Rasmussen, Ida; Bach, Anders; Strømgaard, Kristian; Lauritzen, Martin

2017-05-01

Cortical spreading depression is associated with activation of NMDA receptors, which interact with the postsynaptic density protein 95 (PSD-95) that binds to nitric oxide synthase (nNOS). Here, we tested whether inhibition of the nNOS/PSD-95/NMDA receptor complex formation by anti-ischemic compound, UCCB01-144 (Tat- N-dimer) ameliorates the persistent effects of cortical spreading depression on cortical function. Using in vivo two-photon microscopy in somatosensory cortex in mice, we show that fluorescently labelled Tat- N-dimer readily crosses blood-brain barrier and accumulates in nerve cells during the first hour after i.v. injection. The Tat- N-dimer suppressed stimulation-evoked synaptic activity by 2-20%, while cortical blood flow and cerebral oxygen metabolic (CMRO 2 ) responses were preserved. During cortical spreading depression, the Tat- N-dimer reduced the average amplitude of the negative shift in direct current potential by 33% (4.1 mV). Furthermore, the compound diminished the average depression of spontaneous electrocorticographic activity by 11% during first 40 min of post-cortical spreading depression recovery, but did not mitigate the suppressing effect of cortical spreading depression on cortical blood flow and CMRO 2 . We suggest that uncoupling of PSD-95 from NMDA receptors reduces overall neuronal excitability and the amplitude of the spreading depolarization wave. These findings may be of interest for understanding the neuroprotective effects of the nNOS/PSD-95 uncoupling in stroke.

Quantifying Dimer and Trimer Formation by Tri- n -butyl Phosphates in n -Dodecane: Molecular Dynamics Simulations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vo, Quynh N.; Dang, Liem X.; Nilsson, Mikael

2016-07-21

Tri-n-butyl phosphate (TBP), a representative of neutral organophosphorous ligands, is an important extractant used in solvent extraction process for the recovery of uranium and plutonium from spent nuclear fuel. Microscopic pictures of TBP isomerism and its behavior in n-dodecane diluent were investigated utilizing MD simulations with previously optimized force field parameters for TBP and n-dodecane. Potential Mean Force (PMF) calculations on a single TBP molecule show seven probable TBP isomers. Radial Distribution Functions (RDF) of TBP suggests the existence of TBP trimers at high TBP concentrations in addition to dimers. 2D PMF calculations were performed to determine the angle andmore » distance criteria for TBP trimers. The dimerization and trimerization constants of TBP in n-dodecane were obtained and match our own experimental values using FTIR technique. The new insights into the conformational behaviors of TBP molecule as a monomer and as part of an aggregate could greatly aid the understanding of the complexation between TBP and metal ions in solvent extraction system. The U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences and Biosciences funded the work performed by LXD.« less

Dimerization of tetracationic porphyrins: ionic strength dependence.

PubMed

Dixon, D W; Steullet, V

1998-02-01

Cationic porphyrins are under study in a number of contexts including their interaction with biological targets, as possible therapeutic agents and as building blocks for molecular devices such as molecular photodiodes and solar cells. Many cationic porphyrins dimerize readily in aqueous solution. Dimerization in turn can control the properties of the porphyrin as well as its binding to its target. The propensity of a porphyrin to dimerize in aqueous solution can be estimated by recording the optical spectrum of the solution as a function of the concentration of added salt. Analysis of the data in terms of the Debye-Hückel formalism gives an estimate of the extent of dimerization as a function of ionic strength. Data for TMPyP4 [meso-tetrakis(4-N-methylpyridinium)porphyrin] and its butyl and octyl homologs; TMAP [meso-tetrakis(4-N,N,N-trimethylanilinium)porphyrin]; T theta PP [meso-tetrakis[4-N-[(3-(trimethyl-ammonio)propyl)oxy]phenyl]porphyrin] and the ferrocenyl porphyrin P3Fc are discussed. Dimerization may affect binding of the cationic porphyrins to their targets, e.g., DNA.

Dimerization Controls Marburg Virus VP24-dependent Modulation of Host Antioxidative Stress Responses.

PubMed

Johnson, Britney; Li, Jing; Adhikari, Jagat; Edwards, Megan R; Zhang, Hao; Schwarz, Toni; Leung, Daisy W; Basler, Christopher F; Gross, Michael L; Amarasinghe, Gaya K

2016-08-28

Marburg virus (MARV), a member of the Filoviridae family that also includes Ebola virus (EBOV), causes lethal hemorrhagic fever with case fatality rates that have exceeded 50% in some outbreaks. Within an infected cell, there are numerous host-viral interactions that contribute to the outcome of infection. Recent studies identified MARV protein 24 (mVP24) as a modulator of the host antioxidative responses, but the molecular mechanism remains unclear. Using a combination of biochemical and mass spectrometry studies, we show that mVP24 is a dimer in solution that directly binds to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) to regulate nuclear factor (erythroid-derived 2)-like 2 (Nrf2). This interaction between Keap1 and mVP24 occurs through the Kelch interaction loop (K-Loop) of mVP24 leading to upregulation of antioxidant response element transcription, which is distinct from other Kelch binders that regulate Nrf2 activity. N-terminal truncations disrupt mVP24 dimerization, allowing monomeric mVP24 to bind Kelch with higher affinity and stimulate higher antioxidative stress response element (ARE) reporter activity. Mass spectrometry-based mapping of the interface revealed overlapping binding sites on Kelch for mVP24 and the Nrf2 proteins. Substitution of conserved cysteines, C209 and C210, to alanine in the mVP24 K-Loop abrogates Kelch binding and ARE activation. Our studies identify a shift in the monomer-dimer equilibrium of MARV VP24, driven by its interaction with Keap1 Kelch domain, as a critical determinant that modulates host responses to pathogenic Marburg viral infections. Copyright © 2016 Elsevier Ltd. All rights reserved.

Dimerization Controls Marburg Virus VP24-dependent Modulation of Host Antioxidative Stress Responses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, Britney; Li, Jing; Adhikari, Jagat

Marburg virus (MARV), a member of the Filoviridae family that also includes Ebola virus (EBOV), causes lethal hemorrhagic fever with case fatality rates that have exceeded 50% in some outbreaks. Within an infected cell, there are numerous host-viral interactions that contribute to the outcome of infection. Recent studies identified MARV protein 24 (mVP24) as a modulator of the host antioxidative responses, but the molecular mechanism remains unclear. Using a combination of biochemical and mass spectrometry studies, we show that mVP24 is a dimer in solution that directly binds to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) to regulatemore » nuclear factor (erythroid-derived 2)-like 2 (Nrf2). This interaction between Keap1 and mVP24 occurs through the Kelch interaction loop (K-Loop) of mVP24 leading to upregulation of antioxidant response element transcription, which is distinct from other Kelch binders that regulate Nrf2 activity. N-terminal truncations disrupt mVP24 dimerization, allowing monomeric mVP24 to bind Kelch with higher affinity and stimulate higher antioxidative stress response element (ARE) reporter activity. Mass spectrometry-based mapping of the interface revealed overlapping binding sites on Kelch for mVP24 and the Nrf2 proteins. Substitution of conserved cysteines, C209 and C210, to alanine in the mVP24 K-Loop abrogates Kelch binding and ARE activation. Our studies identify a shift in the monomer-dimer equilibrium of MARV VP24, driven by its interaction with Keap1 Kelch domain, as a critical determinant that modulates host responses to pathogenic Marburg viral infections.« less

Radiation-induced tetramer-to-dimer transition of Escherichia coli lactose repressor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goffinont, S.; Davidkova, M.; Spotheim-Maurizot, M., E-mail: spotheim@cnrs-orleans.fr

2009-08-21

The wild type lactose repressor of Escherichia coli is a tetrameric protein formed by two identical dimers. They are associated via a C-terminal 4-helix bundle (called tetramerization domain) whose stability is ensured by the interaction of leucine zipper motifs. Upon in vitro {gamma}-irradiation the repressor losses its ability to bind the operator DNA sequence due to damage of its DNA-binding domains. Using an engineered dimeric repressor for comparison, we show here that irradiation induces also the change of repressor oligomerisation state from tetramer to dimer. The splitting of the tetramer into dimers can result from the oxidation of the leucinemore » residues of the tetramerization domain.« less

Dynamics and asymmetry in the dimer of the norovirus major capsid protein.

PubMed

Tubiana, Thibault; Boulard, Yves; Bressanelli, Stéphane

2017-01-01

Noroviruses are the major cause of non-bacterial acute gastroenteritis in humans and livestock worldwide, despite being physically among the simplest animal viruses. The icosahedral capsid encasing the norovirus RNA genome is made of 90 dimers of a single ca 60-kDa polypeptide chain, VP1, arranged with T = 3 icosahedral symmetry. Here we study the conformational dynamics of this main building block of the norovirus capsid. We use molecular modeling and all-atom molecular dynamics simulations of the VP1 dimer for two genogroups with 50% sequence identity. We focus on the two points of flexibility in VP1 known from the crystal structure of the genogroup I (GI, human) capsid and from subsequent cryo-electron microscopy work on the GII capsid (also human). First, with a homology model of the GIII (bovine) VP1 dimer subjected to simulated annealing then classical molecular dynamics simulations, we show that the N-terminal arm conformation seen in the GI crystal structure is also favored in GIII VP1 but depends on the protonation state of critical residues. Second, simulations of the GI dimer show that the VP1 spike domain will not keep the position found in the GII electron microscopy work. Our main finding is a consistent propensity of the VP1 dimer to assume prominently asymmetric conformations. In order to probe this result, we obtain new SAXS data on GI VP1 dimers. These data are not interpretable as a population of symmetric dimers, but readily modeled by a highly asymmetric dimer. We go on to discuss possible implications of spontaneously asymmetric conformations in the successive steps of norovirus capsid assembly. Our work brings new lights on the surprising conformational range encoded in the norovirus major capsid protein.

Structure of the Fibrillin-1 N-Terminal Domains Suggests that Heparan Sulfate Regulates the Early Stages of Microfibril Assembly

PubMed Central

Yadin, David A.; Robertson, Ian B.; McNaught-Davis, Joanne; Evans, Paul; Stoddart, David; Handford, Penny A.; Jensen, Sacha A.; Redfield, Christina

2013-01-01

Summary The human extracellular matrix glycoprotein fibrillin-1 is the primary component of the 10- to 12-nm-diameter microfibrils, which perform key structural and regulatory roles in connective tissues. Relatively little is known about the molecular mechanisms of fibrillin assembly into microfibrils. Studies using recombinant fibrillin fragments indicate that an interaction between the N- and C-terminal regions drives head-to-tail assembly. Here, we present the structure of a fibrillin N-terminal fragment comprising the fibrillin unique N-terminal (FUN) and the first three epidermal growth factor (EGF)-like domains (FUN-EGF3). Two rod-like domain pairs are separated by a short, flexible linker between the EGF1 and EGF2 domains. We also show that the binding site for the C-terminal region spans multiple domains and overlaps with a heparin interaction site. These data suggest that heparan sulfate may sequester fibrillin at the cell surface via FUN-EGF3 prior to aggregation of the C terminus, thereby regulating microfibril assembly. PMID:24035709

N-terminal nesprin-2 variants regulate β-catenin signalling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Qiuping; Minaisah, Rose-Marie; Ferraro, Elisa

2016-07-15

The spatial compartmentalisation of biochemical signalling pathways is essential for cell function. Nesprins are a multi-isomeric family of proteins that have emerged as signalling scaffolds, herein, we investigate the localisation and function of novel nesprin-2 N-terminal variants. We show that these nesprin-2 variants display cell specific distribution and reside in both the cytoplasm and nucleus. Immunofluorescence microscopy revealed that nesprin-2 N-terminal variants colocalised with β-catenin at cell-cell junctions in U2OS cells. Calcium switch assays demonstrated that nesprin-2 and β-catenin are lost from cell-cell junctions in low calcium conditions whereas emerin localisation at the NE remained unaltered, furthermore, an N-terminal fragmentmore » of nesprin-2 was sufficient for cell-cell junction localisation and interacted with β-catenin. Disruption of these N-terminal nesprin-2 variants, using siRNA depletion resulted in loss of β-catenin from cell-cell junctions, nuclear accumulation of active β-catenin and augmented β-catenin transcriptional activity. Importantly, we show that U2OS cells lack nesprin-2 giant, suggesting that the N-terminal nesprin-2 variants regulate β-catenin signalling independently of the NE. Together, these data identify N-terminal nesprin-2 variants as novel regulators of β-catenin signalling that tether β-catenin to cell-cell contacts to inhibit β-catenin transcriptional activity. - Highlights: • N-terminal nesprin-2 variants display cell specific expression patterns. • N-terminal spectrin repeats of nesprin-2 interact with β-catenin. • N-terminal nesprin-2 variants scaffold β-catenin at cell-cell junctions.. • Nesprin-2 variants play multiple roles in β-catenin signalling.« less

Specificity and Versatility of Substrate Binding Sites in Four Catalytic Domains of Human N-Terminal Acetyltransferases

PubMed Central

Grauffel, Cédric; Abboud, Angèle; Liszczak, Glen; Marmorstein, Ronen; Arnesen, Thomas; Reuter, Nathalie

2012-01-01

Nt-acetylation is among the most common protein modifications in eukaryotes. Although thought for a long time to protect proteins from degradation, the role of Nt-acetylation is still debated. It is catalyzed by enzymes called N-terminal acetyltransferases (NATs). In eukaryotes, several NATs, composed of at least one catalytic domain, target different substrates based on their N-terminal sequences. In order to better understand the substrate specificity of human NATs, we investigated in silico the enzyme-substrate interactions in four catalytic subunits of human NATs (Naa10p, Naa20p, Naa30p and Naa50p). To date hNaa50p is the only human subunit for which X-ray structures are available. We used the structure of the ternary hNaa50p/AcCoA/MLG complex and a structural model of hNaa10p as a starting point for multiple molecular dynamics simulations of hNaa50p/AcCoA/substrate (substrate = MLG, EEE, MKG), hNaa10p/AcCoA/substrate (substrate = MLG, EEE). Nine alanine point-mutants of the hNaa50p/AcCoA/MLG complex were also simulated. Homology models of hNaa20p and hNaa30p were built and compared to hNaa50p and hNaa10p. The simulations of hNaa50p/AcCoA/MLG reproduce the interactions revealed by the X-ray data. We observed strong hydrogen bonds between MLG and tyrosines 31, 138 and 139. Yet the tyrosines interacting with the substrate’s backbone suggest that their role in specificity is limited. This is confirmed by the simulations of hNaa50p/AcCoA/EEE and hNaa10p/AcCoA/MLG, where these hydrogen bonds are still observed. Moreover these tyrosines are all conserved in hNaa20p and hNaa30p. Other amino acids tune the specificity of the S1’ sites that is different for hNaa10p (acidic), hNaa20p (hydrophobic/basic), hNaa30p (basic) and hNaa50p (hydrophobic). We also observe dynamic correlation between the ligand binding site and helix that tightens under substrate binding. Finally, by comparing the four structures we propose maps of the peptide-enzyme interactions that

Concerted regulation of ISWI by an autoinhibitory domain and the H4 N-terminal tail

PubMed Central

Ludwigsen, Johanna; Pfennig, Sabrina; Singh, Ashish K; Schindler, Christina; Harrer, Nadine; Forné, Ignasi; Zacharias, Martin; Mueller-Planitz, Felix

2017-01-01

ISWI-family nucleosome remodeling enzymes need the histone H4 N-terminal tail to mobilize nucleosomes. Here we mapped the H4-tail binding pocket of ISWI. Surprisingly the binding site was adjacent to but not overlapping with the docking site of an auto-regulatory motif, AutoN, in the N-terminal region (NTR) of ISWI, indicating that AutoN does not act as a simple pseudosubstrate as suggested previously. Rather, AutoN cooperated with a hitherto uncharacterized motif, termed AcidicN, to confer H4-tail sensitivity and discriminate between DNA and nucleosomes. A third motif in the NTR, ppHSA, was functionally required in vivo and provided structural stability by clamping the NTR to Lobe 2 of the ATPase domain. This configuration is reminiscent of Chd1 even though Chd1 contains an unrelated NTR. Our results shed light on the intricate structural and functional regulation of ISWI by the NTR and uncover surprising parallels with Chd1. DOI: http://dx.doi.org/10.7554/eLife.21477.001 PMID:28109157

Dimerization and actin-bundling properties of villin and its role in the assembly of epithelial cell brush borders.

PubMed

George, Sudeep P; Wang, Yaohong; Mathew, Sijo; Srinivasan, Kamalakkannan; Khurana, Seema

2007-09-07

Villin is a major actin-bundling protein in the brush border of epithelial cells. In this study we demonstrate for the first time that villin can bundle actin filaments using a single F-actin binding site, because it has the ability to self-associate. Using fluorescence resonance energy transfer, we demonstrate villin self-association in living cells in microvilli and in growth factor-stimulated cells in membrane ruffles and lamellipodia. Using sucrose density gradient, size-exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight, the majority of villin was identified as a monomer or dimer. Villin dimers were also identified in Caco-2 cells, which endogenously express villin and Madin-Darby canine kidney cells that ectopically express villin. Using truncation mutants of villin, site-directed mutagenesis, and fluorescence resonance energy transfer, an amino-terminal dimerization site was identified that regulated villin self-association in parallel conformation as well as actin bundling by villin. This detailed analysis describes for the first time microvillus assembly by villin, redefines the actin-bundling function of villin, and provides a molecular mechanism for actin bundling by villin, which could have wider implications for other actin cross-linking proteins that share a villin-like headpiece domain. Our study also provides a molecular basis to separate the morphologically distinct actin-severing and actin-bundling properties of villin.

The bZIP dimer localizes at DNA full-sites where each basic region can alternately translocate and bind to subsites at the half-site

PubMed Central

Chan, I-San; Al-Sarraj, Taufik; Shahravan, S. Hesam; Fedorova, Anna V.; Shin, Jumi A.

2012-01-01

Crystal structures of the GCN4 bZIP (basic region/leucine zipper) with the AP-1 or CRE site show how each GCN4 basic region binds to a 4-bp cognate half-site as a single DNA target; however, this may not always fully describe how bZIP proteins interact with their target sites. Previously, we showed that the GCN4 basic region interacts with all 5 bp in half-site TTGCG (termed 5H-LR), and that 5H-LR comprises two 4-bp subsites, TTGC and TGCG, which individually are also target sites of the basic region. In this work, we explored how the basic region interacts with 5H-LR when the bZIP dimer localizes to full-sites. Using AMBER molecular modeling, we simulated GCN4 bZIP complexes with full-sites containing 5H-LR to investigate in silico the interface between the basic region and 5H-LR. We also performed in vitro investigation of bZIP–DNA interactions at a number of full-sites that contain 5H-LR vs. either subsite: we analyzed results from DNase I footprinting and electrophoretic mobility shift assay (EMSA) and from EMSA titrations to quantify binding affinities. Our computational and experimental results together support a highly dynamic DNA-binding model: when a bZIP dimer localizes to its target full-site, the basic region can alternately recognize either subsite as a distinct target at 5H-LR and translocate between the subsites, potentially by sliding and hopping. This model provides added insights into how α-helical DNA-binding domains of transcription factors can localize to their gene regulatory sequences in vivo. PMID:22856882

The bZIP dimer localizes at DNA full-sites where each basic region can alternately translocate and bind to subsites at the half-site.

PubMed

Chan, I-San; Al-Sarraj, Taufik; Shahravan, S Hesam; Fedorova, Anna V; Shin, Jumi A

2012-08-21

Crystal structures of the GCN4 bZIP (basic region/leucine zipper) with the AP-1 or CRE site show how each GCN4 basic region binds to a 4 bp cognate half-site as a single DNA target; however, this may not always fully describe how bZIP proteins interact with their target sites. Previously, we showed that the GCN4 basic region interacts with all 5 bp in half-site TTGCG (termed 5H-LR) and that 5H-LR comprises two 4 bp subsites, TTGC and TGCG, which individually are also target sites of the basic region. In this work, we explore how the basic region interacts with 5H-LR when the bZIP dimer localizes to full-sites. Using AMBER molecular modeling, we simulated GCN4 bZIP complexes with full-sites containing 5H-LR to investigate in silico the interface between the basic region and 5H-LR. We also performed in vitro investigation of bZIP-DNA interactions at a number of full-sites that contain 5H-LR versus either subsite: we analyzed results from DNase I footprinting and electrophoretic mobility shift assay (EMSA) and from EMSA titrations to quantify binding affinities. Our computational and experimental results together support a highly dynamic DNA-binding model: when a bZIP dimer localizes to its target full-site, the basic region can alternately recognize either subsite as a distinct target at 5H-LR and translocate between the subsites, potentially by sliding and hopping. This model provides added insights into how α-helical DNA-binding domains of transcription factors can localize to their gene regulatory sequences in vivo.

The N-Terminal Residues 43 to 60 Form the Interface for Dopamine Mediated α-Synuclein Dimerisation

PubMed Central

Leong, Su Ling; Hinds, Mark G.; Connor, Andrea R.; Smith, David P.; Illes-Toth, Eva; Pham, Chi L. L.; Barnham, Kevin J.; Cappai, Roberto

2015-01-01

α-synuclein (α-syn) is a major component of the intracellular inclusions called Lewy bodies, which are a key pathological feature in the brains of Parkinson’s disease patients. The neurotransmitter dopamine (DA) inhibits the fibrillisation of α-syn into amyloid, and promotes α-syn aggregation into SDS-stable soluble oligomers. While this inhibition of amyloid formation requires the oxidation of both DA and the methionines in α-syn, the molecular basis for these processes is still unclear. This study sought to define the protein sequences required for the generation of oligomers. We tested N- (α-syn residues 43–140) and C-terminally (1–95) truncated α-syn, and found that similar to full-length protein both truncated species formed soluble DA:α-syn oligomers, albeit 1–95 had a different profile. Using nuclear magnetic resonance (NMR), and the N-terminally truncated α-syn 43–140 protein, we analysed the structural characteristics of the DA:α-syn 43–140 dimer and α-syn 43–140 monomer and found the dimerisation interface encompassed residues 43 to 60. Narrowing the interface to this small region will help define the mechanism by which DA mediates the formation of SDS-stable soluble DA:α-syn oligomers. PMID:25679387

Nickel Ligation of the N-Terminal Amine of HypA Is Required for Urease Maturation in Helicobacter pylori.

PubMed

Hu, Heidi Q; Johnson, Ryan C; Merrell, D Scott; Maroney, Michael J

2017-02-28

The human pathogen Helicobacter pylori requires nickel for colonization of the acidic environment of the stomach. HypA, a Ni metallochaperone that is typically associated with hydrogenase maturation, is also required for urease maturation and acid survival of H. pylori. There are two proposed Ni site structures for HypA; one is a paramagnetic six-coordinate site characterized by X-ray absorption spectroscopy (XAS) in unmodified HypA, while another is a diamagnetic four-coordinate planar site characterized by solution nuclear magnetic resonance in an N-terminally modified HypA construct. To determine the role of the N-terminal amine in Ni binding of HypA, an N-terminal extension variant, L2*-HypA, in which a leucine residue was inserted into the second position of the amino acid sequence in the proposed Ni-binding motif, was characterized in vitro and in vivo. Structural characterization of the Ni site using XAS showed a coordination change from six-coordinate in wild-type HypA (WT-HypA) to five-coordinate pyramidal in L2*-HypA, which was accompanied by the loss of two N/O donor protein ligands and the addition of an exogenous bromide ligand from the buffer. The magnetic properties of the Ni sites in WT-HypA compared to those of the Ni sites in L2*-HypA confirmed that a spin-state change from high to low spin accompanied this change in structure. The L2*-HypA H. pylori strain was shown to be acid sensitive and deficient in urease activity in vivo. In vitro characterization showed that L2*-HypA did not disrupt the HypA-UreE interaction that is essential for urease maturation but was at least 20-fold weaker in Ni binding than WT-HypA. Characterization of the L2*-HypA variant clearly demonstrates that the N-terminal amine of HypA is involved in proper Ni coordination and is necessary for urease activity and acid survival.

Nickel Ligation of the N-Terminal Amine of HypA Is Required for Urease Maturation in Helicobacter pylori

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Heidi Q.; Johnson, Ryan C.; Merrell, D. Scott

The human pathogen Helicobacter pylori requires nickel for colonization of the acidic environment of the stomach. HypA, a Ni metallochaperone that is typically associated with hydrogenase maturation, is also required for urease maturation and acid survival of H. pylori. There are two proposed Ni site structures for HypA; one is a paramagnetic six-coordinate site characterized by X-ray absorption spectroscopy (XAS) in unmodified HypA, while another is a diamagnetic four-coordinate planar site characterized by solution nuclear magnetic resonance in an N-terminally modified HypA construct. To determine the role of the N-terminal amine in Ni binding of HypA, an N-terminal extension variant,more » L2*-HypA, in which a leucine residue was inserted into the second position of the amino acid sequence in the proposed Ni-binding motif, was characterized in vitro and in vivo. Structural characterization of the Ni site using XAS showed a coordination change from six-coordinate in wild-type HypA (WT-HypA) to five-coordinate pyramidal in L2*-HypA, which was accompanied by the loss of two N/O donor protein ligands and the addition of an exogenous bromide ligand from the buffer. The magnetic properties of the Ni sites in WT-HypA compared to those of the Ni sites in L2*-HypA confirmed that a spin-state change from high to low spin accompanied this change in structure. The L2*-HypA H. pylori strain was shown to be acid sensitive and deficient in urease activity in vivo. In vitro characterization showed that L2*-HypA did not disrupt the HypA–UreE interaction that is essential for urease maturation but was at least 20-fold weaker in Ni binding than WT-HypA. Characterization of the L2*-HypA variant clearly demonstrates that the N-terminal amine of HypA is involved in proper Ni coordination and is necessary for urease activity and acid survival.« less

Kosterlitz-Thouless transitions and phase diagrams of the interacting monomer-dimer model on a checkerboard lattice

NASA Astrophysics Data System (ADS)

Li, Sazi; Li, Wei; Chen, Ziyu

2014-11-01

Using the tensor network approach, we investigate the monomer-dimer models on a checkerboard lattice, in which there are interactions (with strength v ) between the parallel dimers on half of the plaquettes. For the fully packed interacting dimer model, we observe a Kosterlitz-Thouless (KT) transition between the low-temperature symmetry breaking and the high-temperature critical phases; for the doped monomer-dimer case with finite chemical potential μ , we also find an order-disorder phase transition which is of second order instead. We use the boundary matrix product state approach to detect the KT and second-order phase transitions and obtain the phase diagrams v -T and μ -T . Moreover, for the noninteracting monomer-dimer model (setting μ =ν =0 ), we get an extraordinarily accurate determination of the free energy per site (negative of the monomer-dimer constant h2) as f =-0.662 798 972 833 746 with the dimer density n =0.638 123 109 228 547 , both of 15 correct digits.

Effect of site disorder on the ground state of a frustrated spin dimer quantum magnet

NASA Astrophysics Data System (ADS)

Hristov, Alexander; Shapiro, Maxwell; Lee, Minseong; Rodenbach, Linsey; Choi, Eun Sang; Park, Ju-Hyun; Munsie, Tim; Luke, Graeme; Fisher, Ian

Ba3Mn2O8 is a geometrically frustrated spin dimer quantum magnet. Pairs of Mn 5+ (S = 1) ions are strongly coupled via antiferromagnetic exchange to yield a singlet ground state, with excited triplet and quintuplet states. Isovalent substitution of V5+ (S = 0) for Mn breaks dimers, resulting in unpaired S = 1 spins, the ground state of which is investigated here for compositions spanning the range 0 <= x <= 1 of Ba3(Mn1-xVx)2O8. From a theoretical perspective, for dimers occupying an unfrustrated bipartite lattice, such site disorder is anticipated to yield long range magnetism for unpaired Mn spins both in the dilute limit where x is small, a phenomena known as order-by-disorder, and in the proximity of x = 1 / 2 where the system is maximally disordered and close to a percolation threshold. In this frustrated system, however, our experiments find evidence of spin freezing for six compositions 0 . 05 <= x <= 0 . 85 . In this regime, we find entropy removed at an energy scale independent of the freezing temperature. We discuss the possibility of a spin-glass to random singlet transition for critical compositions in the two dilute limits x -> 0 and x -> 1 . NSF DMR-Award 1205165.

PRINT: A Protein Bioconjugation Method with Exquisite N-terminal Specificity

PubMed Central

Sur, Surojit; Qiao, Yuan; Fries, Anja; O’Meally, Robert N.; Cole, Robert N.; Kinzler, Kenneth W.; Vogelstein, Bert; Zhou, Shibin

2015-01-01

Chemical conjugation is commonly used to enhance the pharmacokinetics, biodistribution, and potency of protein therapeutics, but often leads to non-specific modification or loss of bioactivity. Here, we present a simple, versatile and widely applicable method that allows exquisite N-terminal specific modification of proteins. Combining reversible side-chain blocking and protease mediated cleavage of a commonly used HIS tag appended to a protein, we generate with high yield and purity exquisitely site specific and selective bio-conjugates of TNF-α by using amine reactive NHS ester chemistry. We confirm the N terminal selectivity and specificity using mass spectral analyses and show near complete retention of the biological activity of our model protein both in vitro and in vivo murine models. We believe that this methodology would be applicable to a variety of potentially therapeutic proteins and the specificity afforded by this technique would allow for rapid generation of novel biologics. PMID:26678960

Structural Determinants Underlying Constitutive Dimerization of Unoccupied Human Follitropin Receptors

PubMed Central

Guan, Rongbin; Wu, Xueqing; Feng, Xiuyan; Zhang, Meilin; Hébert, Terence E.; Segaloff, Deborah L.

2009-01-01

The human follitropin receptor (hFSHR) is a G protein-coupled receptor (GPCR) central to reproductive physiology that is composed of an extracellular domain (ECD) fused to a serpentine region. Using bioluminescence resonance energy transfer (BRET) in living cells, we show that hFSHR dimers form constitutively during their biosynthesis. Mutations in TM1 and TM4 had no effect on hFSHR dimerization, alone or when combined with mutation of Tyr110 in the ECD, a residue predicted to mediate dimerization of the soluble hormone-binding portion of the ECD complexed with FSH (Q. Fan and W. Hendrickson, Nature 433:269–277, 2005). Expressed individually, the serpentine region and a membrane-anchored form of the hFSHR ECD each exhibited homodimerization, suggesting that both domains contribute to dimerization of the full-length receptor. However, even in the context of only the membrane-anchored ECD, mutation of Tyr110 to alanine did not inhibit dimerization. The full-length hFSHR and the membrane-anchored ECD were then each engineered to introduce a consensus site for N-linked glycosylation at residue 110. Despite experimental validation of the presence of carbohydrate on residue 110, we failed to observe disruption of dimerization of either the full-length hFSHR or membrane-anchored ECD containing the inserted glycan wedge. Taken altogether, our data suggest that both the serpentine region and the ECD contribute to hFSHR dimerization and that the dimerization interface of the unoccupied hFSHR does not involve Tyr110 of the ECD. PMID:19800402

Effects of Dimerization of Serratia marcescens Endonuclease on Water Dynamics.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Chuanying; Beck, Brian W.; Krause, Kurt

2007-02-15

The research described in this product was performed in part in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. The dynamics and structure of Serratia marcescens endonuclease and its neighboring solvent are investigated by molecular dynamics (MD). Comparisons are made with structural and biochemical experiments. The dimer form is physiologic and functions more processively than the monomer. We previously found a channel formed by connected clusters of waters from the active site to the dimer interface. Here, we showmore » that dimerization clearly changes correlations in the water structure and dynamics in the active site not seen in the monomer. Our results indicate that water at the active sites of the dimer is less affected compared with bulk solvent than in the monomer where it has much slower characteristic relaxation times. Given that water is a required participant in the reaction, this gives a clear advantage to dimerization in the absence of an apparent ability to use both active sites simultaneously.« less

Structure, Dynamics, and Allosteric Potential of Ionotropic Glutamate Receptor N-Terminal Domains.

PubMed

Krieger, James; Bahar, Ivet; Greger, Ingo H

2015-09-15

Ionotropic glutamate receptors (iGluRs) are tetrameric cation channels that mediate synaptic transmission and plasticity. They have a unique modular architecture with four domains: the intracellular C-terminal domain (CTD) that is involved in synaptic targeting, the transmembrane domain (TMD) that forms the ion channel, the membrane-proximal ligand-binding domain (LBD) that binds agonists such as L-glutamate, and the distal N-terminal domain (NTD), whose function is the least clear. The extracellular portion, comprised of the LBD and NTD, is loosely arranged, mediating complex allosteric regulation and providing a rich target for drug development. Here, we briefly review recent work on iGluR NTD structure and dynamics, and further explore the allosteric potential for the NTD in AMPA-type iGluRs using coarse-grained simulations. We also investigate mechanisms underlying the established NTD allostery in NMDA-type iGluRs, as well as the fold-related metabotropic glutamate and GABAB receptors. We show that the clamshell motions intrinsically favored by the NTD bilobate fold are coupled to dimeric and higher-order rearrangements that impact the iGluR LBD and ultimately the TMD. Finally, we explore the dynamics of intact iGluRs and describe how it might affect receptor operation in a synaptic environment. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Light-regulated synthesis of cyclic-di-GMP by a bidomain construct of the cyanobacteriochrome Tlr0924 (SesA) without stable dimerization

DOE PAGES

Blain-Hartung, Matthew D.; Rockwell, Nathan Clarke; Lagarias, J. Clark

2017-10-26

Here, phytochromes and cyanobacteriochromes (CBCRs) use double bond photoisomerization of their linear tetrapyrrole (bilin) chromophores within cGMP-specific phosphodiesterases/Adenylyl cyclases/FhlA (GAF) domain-containing photosensory modules to regulate activity of C-terminal output domains. CBCRs exhibit much more diverse photocycles than phytochromes, and are often found in large modular proteins such as Tlr0924 (SesA), one of three blue light regulators of cell aggregation in the cyanobacterium Thermosynechococcus elongatus. Tlr0924 contains a single bilin-binding GAF domain adjacent to a C-terminal diguanylate cyclase (GGDEF) domain whose catalytic activity requires formation of a dimeric transition state presumably supported by a multi-domain extension at its N-terminus. To probemore » the structural basis of light-mediated signal propagation from the photosensory input domain to a signaling output domain for a representative CBCR, these studies explore the properties of a bidomain GAF-GGDEF construct of Tlr0924 (Tlr0924Δ) that retains light-regulated diguanylate cyclase activity. Surprisingly, CD spectroscopy and size exclusion chromatography data do not support formation of stable dimers in the either the blue-absorbing 15ZP b dark state or the green-absorbing 15EP g photoproduct state of Tlr0924Δ. Analysis of variants containing site-specific mutations reveals that proper signal transmission requires both chromophorylation of the GAF domain and individual residues within the amphipathic linker region between GAF and GGDEF domains. Based on these data, we propose a model in which bilin binding and light signals are propagated from the GAF domain via the linker region to alter the equilibrium and interconversion dynamics between active and inactive conformations of the GGDEF domain to favor or disfavor formation of catalytic competent dimers.« less

Light-regulated synthesis of cyclic-di-GMP by a bidomain construct of the cyanobacteriochrome Tlr0924 (SesA) without stable dimerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blain-Hartung, Matthew D.; Rockwell, Nathan Clarke; Lagarias, J. Clark

Here, phytochromes and cyanobacteriochromes (CBCRs) use double bond photoisomerization of their linear tetrapyrrole (bilin) chromophores within cGMP-specific phosphodiesterases/Adenylyl cyclases/FhlA (GAF) domain-containing photosensory modules to regulate activity of C-terminal output domains. CBCRs exhibit much more diverse photocycles than phytochromes, and are often found in large modular proteins such as Tlr0924 (SesA), one of three blue light regulators of cell aggregation in the cyanobacterium Thermosynechococcus elongatus. Tlr0924 contains a single bilin-binding GAF domain adjacent to a C-terminal diguanylate cyclase (GGDEF) domain whose catalytic activity requires formation of a dimeric transition state presumably supported by a multi-domain extension at its N-terminus. To probemore » the structural basis of light-mediated signal propagation from the photosensory input domain to a signaling output domain for a representative CBCR, these studies explore the properties of a bidomain GAF-GGDEF construct of Tlr0924 (Tlr0924Δ) that retains light-regulated diguanylate cyclase activity. Surprisingly, CD spectroscopy and size exclusion chromatography data do not support formation of stable dimers in the either the blue-absorbing 15ZP b dark state or the green-absorbing 15EP g photoproduct state of Tlr0924Δ. Analysis of variants containing site-specific mutations reveals that proper signal transmission requires both chromophorylation of the GAF domain and individual residues within the amphipathic linker region between GAF and GGDEF domains. Based on these data, we propose a model in which bilin binding and light signals are propagated from the GAF domain via the linker region to alter the equilibrium and interconversion dynamics between active and inactive conformations of the GGDEF domain to favor or disfavor formation of catalytic competent dimers.« less

Effect of D23N mutation on the dimer conformation of amyloid β-proteins: ab initio molecular simulations in water.

PubMed

Okamoto, Akisumi; Yano, Atsushi; Nomura, Kazuya; Higai, Shin'ichi; Kurita, Noriyuki

2014-05-01

The molecular pathogenesis of Alzheimer's disease (AD) is deeply involved in aggregations of amyloid β-proteins (Aβ) in a diseased brain. The recent experimental studies indicated that the mutation of Asp23 by Asn (D23N) within the coding sequence of Aβ increases the risk for the pathogeny of cerebral amyloid angiopathy and early-onset familial ADs. Fibrils of the D23N mutated Aβs can form both parallel and antiparallel structures, and the parallel one is considered to be associated with the pathogeny. However, the structure and the aggregation mechanism of the mutated Aβ fibrils are not elucidated at atomic and electronic levels. We here investigated solvated structures of the two types of Aβ dimers, each of which is composed of the wild-type or the D23N mutated Aβ, using classical molecular mechanics and ab initio fragment molecular orbital (FMO) methods, in order to reveal the effect of the D23N mutation on the structure of Aβ dimer as well as the specific interactions between the Aβ monomers. The results elucidate that the effect of the D23N mutation is significant for the parallel structure of Aβ dimer and that the solvating water molecules around the Aβ dimer have significant contribution to the stability of Aβ dimer. Copyright © 2014 Elsevier Inc. All rights reserved.

A pause site for RNA polymerase II is associated with termination of transcription.

PubMed Central

Enriquez-Harris, P; Levitt, N; Briggs, D; Proudfoot, N J

1991-01-01

Termination of transcription by RNA polymerase II has been postulated to involve a pausing process. We have identified such a pause signal, 350 bp into the 3' flanking region of the human alpha 2 globin gene at a position where termination is thought to occur. We show that this pause signal enhances the utilization of an upstream poly(A) site which is otherwise out-competed by a stronger downstream poly(A) site. We also demonstrate that the pause site rescues a poly(A) site that is inactive due to its location within an intron. Using nuclear run-on analysis we show that elongating RNA polymerase II molecules accumulate over this pause signal. Furthermore we show that when the pause site is positioned immediately downstream of a strong poly(A) signal, significant levels of transcription termination take place. Images PMID:2050120

The N-Terminal Domain of the Flo1 Flocculation Protein from Saccharomyces cerevisiae Binds Specifically to Mannose Carbohydrates ▿

PubMed Central

Goossens, Katty V. Y.; Stassen, Catherine; Stals, Ingeborg; Donohue, Dagmara S.; Devreese, Bart; De Greve, Henri; Willaert, Ronnie G.

2011-01-01

Saccharomyces cerevisiae cells possess a remarkable capacity to adhere to other yeast cells, which is called flocculation. Flocculation is defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended. These cell-cell interactions are mediated by a class of specific cell wall proteins, called flocculins, that stick out of the cell walls of flocculent cells. The N-terminal part of the three-domain protein is responsible for carbohydrate binding. We studied the N-terminal domain of the Flo1 protein (N-Flo1p), which is the most important flocculin responsible for flocculation of yeast cells. It was shown that this domain is both O and N glycosylated and is structurally composed mainly of β-sheets. The binding of N-Flo1p to d-mannose, α-methyl-d-mannoside, various dimannoses, and mannan confirmed that the N-terminal domain of Flo1p is indeed responsible for the sugar-binding activity of the protein. Moreover, fluorescence spectroscopy data suggest that N-Flo1p contains two mannose carbohydrate binding sites with different affinities. The carbohydrate dissociation constants show that the affinity of N-Flo1p for mono- and dimannoses is in the millimolar range for the binding site with low affinity and in the micromolar range for the binding site with high affinity. The high-affinity binding site has a higher affinity for low-molecular-weight (low-MW) mannose carbohydrates and no affinity for mannan. However, mannan as well as low-MW mannose carbohydrates can bind to the low-affinity binding site. These results extend the cellular flocculation model on the molecular level. PMID:21076009

The dimerization domain in DapE enzymes is required for catalysis.

PubMed

Nocek, Boguslaw; Starus, Anna; Makowska-Grzyska, Magdalena; Gutierrez, Blanca; Sanchez, Stephen; Jedrzejczak, Robert; Mack, Jamey C; Olsen, Kenneth W; Joachimiak, Andrzej; Holz, Richard C

2014-01-01

The emergence of antibiotic-resistant bacterial strains underscores the importance of identifying new drug targets and developing new antimicrobial compounds. Lysine and meso-diaminopimelic acid are essential for protein production and bacterial peptidoglycan cell wall remodeling and are synthesized in bacteria by enzymes encoded within dap operon. Therefore dap enzymes may serve as excellent targets for developing a new class of antimicrobial agents. The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) converts N-succinyl-L,L-diaminopimelic acid to L,L-diaminopimelic acid and succinate. The enzyme is composed of catalytic and dimerization domains, and belongs to the M20 peptidase family. To understand the specific role of each domain of the enzyme we engineered dimerization domain deletion mutants of DapEs from Haemophilus influenzae and Vibrio cholerae, and characterized these proteins structurally and biochemically. No activity was observed for all deletion mutants. Structural comparisons of wild-type, inactive monomeric DapE enzymes with other M20 peptidases suggest that the dimerization domain is essential for DapE enzymatic activity. Structural analysis and molecular dynamics simulations indicate that removal of the dimerization domain increased the flexibility of a conserved active site loop that may provide critical interactions with the substrate.

Cloning and determination of the transcription termination site of ribosomal RNA gene of the mouse.

PubMed Central

Kominami, R; Mishima, Y; Urano, Y; Sakai, M; Muramatsu, M

1982-01-01

A Eco RI 6.6 kb DNA fragment containing the 3'-end of 28S ribosomal RNA gene of the mouse was detected by Southern blot hybridization, and cloned in a lambda-phage vector. The site of transcription termination and the processed 3'-end of 28S RNA were determined on the cloned fragment and the surrounding nucleotide sequence determined. The 3'-terminal nucleotides of mouse 28S RNA are similar to those of yeast, Drosophila and Xenopus although the homology was lost drastically beyond the 3'-end of 28S RNA. 45S precursor RNA terminated at 30 nucleotides downstream from the 3'-end of 28S RNA gene. A structure of a dyad symmetry with a loop was found immediately prior to the termination site of 45S RNA. The rDNA termination site thus shares some common features with termination sites recognized by other RNA polymerases. Images PMID:6281727

The C-Terminal Domain of the Virulence Factor MgtC Is a Divergent ACT Domain

PubMed Central

Yang, Yinshan; Labesse, Gilles; Carrère-Kremer, Séverine; Esteves, Kevin; Kremer, Laurent

2012-01-01

MgtC is a virulence factor of unknown function important for survival inside macrophages in several intracellular bacterial pathogens, including Mycobacterium tuberculosis. It is also involved in adaptation to Mg2+ deprivation, but previous work suggested that MgtC is not a Mg2+ transporter. In this study, we demonstrated that the amount of the M. tuberculosis MgtC protein is not significantly increased by Mg2+ deprivation. Members of the MgtC protein family share a conserved membrane N-terminal domain and a more divergent cytoplasmic C-terminal domain. To get insights into MgtC functional and structural organization, we have determined the nuclear magnetic resonance (NMR) structure of the C-terminal domain of M. tuberculosis MgtC. This structure is not affected by the Mg2+ concentration, indicating that it does not bind Mg2+. The structure of the C-terminal domain forms a βαββαβ fold found in small molecule binding domains called ACT domains. However, the M. tuberculosis MgtC ACT domain differs from canonical ACT domains because it appears to lack the ability to dimerize and to bind small molecules. We have shown, using a bacterial two-hybrid system, that the M. tuberculosis MgtC protein can dimerize and that the C-terminal domain somehow facilitates this dimerization. Taken together, these results indicate that M. tuberculosis MgtC does not have an intrinsic function related to Mg2+ uptake or binding but could act as a regulatory factor based on protein-protein interaction that could be facilitated by its ACT domain. PMID:22984256

Regulation of presynaptic Ca2+, synaptic plasticity and contextual fear conditioning by a N-terminal β-amyloid fragment.

PubMed

Lawrence, James L M; Tong, Mei; Alfulaij, Naghum; Sherrin, Tessi; Contarino, Mark; White, Michael M; Bellinger, Frederick P; Todorovic, Cedomir; Nichols, Robert A

2014-10-22

Soluble β-amyloid has been shown to regulate presynaptic Ca(2+) and synaptic plasticity. In particular, picomolar β-amyloid was found to have an agonist-like action on presynaptic nicotinic receptors and to augment long-term potentiation (LTP) in a manner dependent upon nicotinic receptors. Here, we report that a functional N-terminal domain exists within β-amyloid for its agonist-like activity. This sequence corresponds to a N-terminal fragment generated by the combined action of α- and β-secretases, and resident carboxypeptidase. The N-terminal β-amyloid fragment is present in the brains and CSF of healthy adults as well as in Alzheimer's patients. Unlike full-length β-amyloid, the N-terminal β-amyloid fragment is monomeric and nontoxic. In Ca(2+) imaging studies using a model reconstituted rodent neuroblastoma cell line and isolated mouse nerve terminals, the N-terminal β-amyloid fragment proved to be highly potent and more effective than full-length β-amyloid in its agonist-like action on nicotinic receptors. In addition, the N-terminal β-amyloid fragment augmented theta burst-induced post-tetanic potentiation and LTP in mouse hippocampal slices. The N-terminal fragment also rescued LTP inhibited by elevated levels of full-length β-amyloid. Contextual fear conditioning was also strongly augmented following bilateral injection of N-terminal β-amyloid fragment into the dorsal hippocampi of intact mice. The fragment-induced augmentation of fear conditioning was attenuated by coadministration of nicotinic antagonist. The activity of the N-terminal β-amyloid fragment appears to reside largely in a sequence surrounding a putative metal binding site, YEVHHQ. These findings suggest that the N-terminal β-amyloid fragment may serve as a potent and effective endogenous neuromodulator. Copyright © 2014 the authors 0270-6474/14/3414210-09$15.00/0.

Monitoring Environmental Recovery at Terminated Produced Water Discharge Sites in Coastal Louisiana Waters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Continental Shelf Associates, Inc.

1999-08-16

This report presents the results of a study of terminated produced water discharge sites in the coastal waters of Louisiana. Environmental recovery at the sites is documented by comparing pre-termination and post-termination (six months and one year) data. Produced water, sediments, and sediment interstitial water samples were analyzed for radionuclides, metals, and hydrocarbons. Benthic infauna were identified from samples collected in the vicinity of the discharge and reference sites. Radium isotope activities were determined in fish and crustacean samples. In addition, an environmental risk assessment is made on the basis of the concentrations of metals and hydrocarbons determined in themore » samples.« less

Annealing to sequences within the primer binding site loop promotes an HIV-1 RNA conformation favoring RNA dimerization and packaging

PubMed Central

Seif, Elias; Niu, Meijuan; Kleiman, Lawrence

2013-01-01

The 5′ untranslated region (5′ UTR) of HIV-1 genomic RNA (gRNA) includes structural elements that regulate reverse transcription, transcription, translation, tRNALys3 annealing to the gRNA, and gRNA dimerization and packaging into viruses. It has been reported that gRNA dimerization and packaging are regulated by changes in the conformation of the 5′-UTR RNA. In this study, we show that annealing of tRNALys3 or a DNA oligomer complementary to sequences within the primer binding site (PBS) loop of the 5′ UTR enhances its dimerization in vitro. Structural analysis of the 5′-UTR RNA using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) shows that the annealing promotes a conformational change of the 5′ UTR that has been previously reported to favor gRNA dimerization and packaging into virus. The model predicted by SHAPE analysis is supported by antisense experiments designed to test which annealed sequences will promote or inhibit gRNA dimerization. Based on reports showing that the gRNA dimerization favors its incorporation into viruses, we tested the ability of a mutant gRNA unable to anneal to tRNALys3 to be incorporated into virions. We found a ∼60% decrease in mutant gRNA packaging compared with wild-type gRNA. Together, these data further support a model for viral assembly in which the initial annealing of tRNALys3 to gRNA is cytoplasmic, which in turn aids in the promotion of gRNA dimerization and its incorporation into virions. PMID:23960173

Changes at the KinA PAS-A Dimerization Interface Influence Histidine Kinase Function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, James; Tomchick, Diana R.; Brautigam, Chad A.

2008-11-12

The Bacillus subtilis KinA protein is a histidine protein kinase that controls the commitment of this organism to sporulate in response to nutrient deprivation and several other conditions. Prior studies indicated that the N-terminal Per-ARNT-Sim domain (PAS-A) plays a critical role in the catalytic activity of this enzyme, as demonstrated by the significant decrease of the autophosphorylation rate of a KinA protein lacking this domain. On the basis of the environmental sensing role played by PAS domains in a wide range of proteins, including other bacterial sensor kinases, it has been suggested that the PAS-A domain plays an important regulatorymore » role in KinA function. We have investigated this potential by using a combination of biophysical and biochemical methods to examine PAS-A structure and function, both in isolation and within the intact protein. Here, we present the X-ray crystal structure of the KinA PAS-A domain, showing that it crystallizes as a homodimer using {beta}-sheet/{beta}-sheet packing interactions as observed for several other PAS domain complexes. Notably, we observed two dimers with tertiary and quaternary structure differences in the crystalline lattice, indicating significant structural flexibility in these domains. To confirm that KinA PAS-A also forms dimers in solution, we used a combination of NMR spectroscopy, gel filtration chromatography, and analytical ultracentrifugation, the results of which are all consistent with the crystallographic results. We experimentally tested the importance of several residues at the dimer interface using site-directed mutagenesis, finding changes in the PAS-A domain that significantly alter KinA enzymatic activity in vitro and in vivo. These results support the importance of PAS domains within KinA and other histidine kinases and suggest possible routes for natural or artificial regulation of kinase activity.« less

Crystal structure of ryanodine receptor N-terminal domain from Plutella xylostella reveals two potential species-specific insecticide-targeting sites.

PubMed

Lin, Lianyun; Liu, Chen; Qin, Juan; Wang, Jie; Dong, Shengjie; Chen, Wei; He, Weiyi; Gao, Qingzhi; You, Minsheng; Yuchi, Zhiguang

2018-01-01

Ryanodine receptors (RyRs) are large calcium-release channels located in sarcoplasmic reticulum membrane. They play a central role in excitation-contraction coupling of muscle cells. Three commercialized insecticides targeting pest RyRs generate worldwide sales over 2 billion U.S. dollars annually, but the structure of insect RyRs remains elusive, hindering our understanding of the mode of action of RyR-targeting insecticides and the development of insecticide resistance in pests. Here we present the crystal structure of RyR N-terminal domain (NTD) (residue 1-205) at 2.84 Å resolution from the diamondback moth (DBM), Plutella xylostella, a destructive pest devouring cruciferous crops all over the world. Similar to its mammalian homolog, DBM RyR NTD consists of a beta-trefoil folding motif and a flanking alpha helix. Interestingly, two regions in NTD interacting with neighboring domains showed distinguished conformations in DBM relative to mammalian RyRs. Using homology modeling and molecular dynamics simulation, we created a structural model of the N-terminal three domains, showing two unique binding pockets that could be targeted by potential species-specific insecticides. Thermal melt experiment showed that the stability of DBM RyR NTD was higher than mammalian RyRs, probably due to a stable intra-domain disulfide bond observed in the crystal structure. Previously DBM NTD was shown to be one of the two critical regions to interact with insecticide flubendiamide, but isothermal titration calorimetry experiments negated DBM NTD alone as a major binding site for flubendiamide. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification and Characterization of Sites Where Persistent Atrial Fibrillation Is Terminated by Localized Ablation.

PubMed

Zaman, Junaid A B; Sauer, William H; Alhusseini, Mahmood I; Baykaner, Tina; Borne, Ryan T; Kowalewski, Christopher A B; Busch, Sonia; Zei, Paul C; Park, Shirley; Viswanathan, Mohan N; Wang, Paul J; Brachmann, Johannes; Krummen, David E; Miller, John M; Rappel, Wouter Jan; Narayan, Sanjiv M; Peters, Nicholas S

2018-01-01

The mechanisms by which persistent atrial fibrillation (AF) terminates via localized ablation are not well understood. To address the hypothesis that sites where localized ablation terminates persistent AF have characteristics identifiable with activation mapping during AF, we systematically examined activation patterns acquired only in cases of unequivocal termination by ablation. We recruited 57 patients with persistent AF undergoing ablation, in whom localized ablation terminated AF to sinus rhythm or organized tachycardia. For each site, we performed an offline analysis of unprocessed unipolar electrograms collected during AF from multipolar basket catheters using the maximum -dV/dt assignment to construct isochronal activation maps for multiple cycles. Additional computational modeling and phase analysis were used to study mechanisms of map variability. At all sites of AF termination, localized repetitive activation patterns were observed. Partial rotational circuits were observed in 26 of 57 (46%) cases, focal patterns in 19 of 57 (33%), and complete rotational activity in 12 of 57 (21%) cases. In computer simulations, incomplete segments of partial rotations coincided with areas of slow conduction characterized by complex, multicomponent electrograms, and variations in assigning activation times at such sites substantially altered mapped mechanisms. Local activation mapping at sites of termination of persistent AF showed repetitive patterns of rotational or focal activity. In computer simulations, complete rotational activation sequence was observed but was sensitive to assignment of activation timing particularly in segments of slow conduction. The observed phenomena of repetitive localized activation and the mechanism by which local ablation terminates putative AF drivers require further investigation. © 2018 American Heart Association, Inc.

C-Terminal Tyrosine Residue Modifications Modulate the Protective Phosphorylation of Serine 129 of α-Synuclein in a Yeast Model of Parkinson's Disease

PubMed Central

Lázaro, Diana F.; Pinho, Raquel; Valerius, Oliver; Outeiro, Tiago F.; Braus, Gerhard H.

2016-01-01

Parkinson´s disease (PD) is characterized by the presence of proteinaceous inclusions called Lewy bodies that are mainly composed of α-synuclein (αSyn). Elevated levels of oxidative or nitrative stresses have been implicated in αSyn related toxicity. Phosphorylation of αSyn on serine 129 (S129) modulates autophagic clearance of inclusions and is prominently found in Lewy bodies. The neighboring tyrosine residues Y125, Y133 and Y136 are phosphorylation and nitration sites. Using a yeast model of PD, we found that Y133 is required for protective S129 phosphorylation and for S129-independent proteasome clearance. αSyn can be nitrated and form stable covalent dimers originating from covalent crosslinking of two tyrosine residues. Nitrated tyrosine residues, but not di-tyrosine-crosslinked dimers, contributed to αSyn cytotoxicity and aggregation. Analysis of tyrosine residues involved in nitration and crosslinking revealed that the C-terminus, rather than the N-terminus of αSyn, is modified by nitration and di-tyrosine formation. The nitration level of wild-type αSyn was higher compared to that of A30P mutant that is non-toxic in yeast. A30P formed more dimers than wild-type αSyn, suggesting that dimer formation represents a cellular detoxification pathway in yeast. Deletion of the yeast flavohemoglobin gene YHB1 resulted in an increase of cellular nitrative stress and cytotoxicity leading to enhanced aggregation of A30P αSyn. Yhb1 protected yeast from A30P-induced mitochondrial fragmentation and peroxynitrite-induced nitrative stress. Strikingly, overexpression of neuroglobin, the human homolog of YHB1, protected against αSyn inclusion formation in mammalian cells. In total, our data suggest that C-terminal Y133 plays a major role in αSyn aggregate clearance by supporting the protective S129 phosphorylation for autophagy and by promoting proteasome clearance. C-terminal tyrosine nitration increases pathogenicity and can only be partially detoxified by �

C-Terminal Tyrosine Residue Modifications Modulate the Protective Phosphorylation of Serine 129 of α-Synuclein in a Yeast Model of Parkinson's Disease.

PubMed

Kleinknecht, Alexandra; Popova, Blagovesta; Lázaro, Diana F; Pinho, Raquel; Valerius, Oliver; Outeiro, Tiago F; Braus, Gerhard H

2016-06-01

Parkinson´s disease (PD) is characterized by the presence of proteinaceous inclusions called Lewy bodies that are mainly composed of α-synuclein (αSyn). Elevated levels of oxidative or nitrative stresses have been implicated in αSyn related toxicity. Phosphorylation of αSyn on serine 129 (S129) modulates autophagic clearance of inclusions and is prominently found in Lewy bodies. The neighboring tyrosine residues Y125, Y133 and Y136 are phosphorylation and nitration sites. Using a yeast model of PD, we found that Y133 is required for protective S129 phosphorylation and for S129-independent proteasome clearance. αSyn can be nitrated and form stable covalent dimers originating from covalent crosslinking of two tyrosine residues. Nitrated tyrosine residues, but not di-tyrosine-crosslinked dimers, contributed to αSyn cytotoxicity and aggregation. Analysis of tyrosine residues involved in nitration and crosslinking revealed that the C-terminus, rather than the N-terminus of αSyn, is modified by nitration and di-tyrosine formation. The nitration level of wild-type αSyn was higher compared to that of A30P mutant that is non-toxic in yeast. A30P formed more dimers than wild-type αSyn, suggesting that dimer formation represents a cellular detoxification pathway in yeast. Deletion of the yeast flavohemoglobin gene YHB1 resulted in an increase of cellular nitrative stress and cytotoxicity leading to enhanced aggregation of A30P αSyn. Yhb1 protected yeast from A30P-induced mitochondrial fragmentation and peroxynitrite-induced nitrative stress. Strikingly, overexpression of neuroglobin, the human homolog of YHB1, protected against αSyn inclusion formation in mammalian cells. In total, our data suggest that C-terminal Y133 plays a major role in αSyn aggregate clearance by supporting the protective S129 phosphorylation for autophagy and by promoting proteasome clearance. C-terminal tyrosine nitration increases pathogenicity and can only be partially detoxified by �

Theoretical realization of cluster-assembled hydrogen storage materials based on terminated carbon atomic chains.

PubMed

Liu, Chun-Sheng; An, Hui; Guo, Ling-Ju; Zeng, Zhi; Ju, Xin

2011-01-14

The capacity of carbon atomic chains with different terminations for hydrogen storage is studied using first-principles density functional theory calculations. Unlike the physisorption of H(2) on the H-terminated chain, we show that two Li (Na) atoms each capping one end of the odd- or even-numbered carbon chain can hold ten H(2) molecules with optimal binding energies for room temperature storage. The hybridization of the Li 2p states with the H(2)σ orbitals contributes to the H(2) adsorption. However, the binding mechanism of the H(2) molecules on Na arises only from the polarization interaction between the charged Na atom and the H(2). Interestingly, additional H(2) molecules can be bound to the carbon atoms at the chain ends due to the charge transfer between Li 2s2p (Na 3s) and C 2p states. More importantly, dimerization of these isolated metal-capped chains does not affect the hydrogen binding energy significantly. In addition, a single chain can be stabilized effectively by the C(60) fullerenes termination. With a hydrogen uptake of ∼10 wt.% on Li-coated C(60)-C(n)-C(60) (n = 5, 8), the Li(12)C(60)-C(n)-Li(12)C(60) complex, keeping the number of adsorbed H(2) molecules per Li and stabilizing the dispersion of individual Li atoms, can serve as better building blocks of polymers than the (Li(12)C(60))(2) dimer. These findings suggest a new route to design cluster-assembled hydrogen storage materials based on terminated sp carbon chains.

N-terminal RASSF family

PubMed Central

Underhill-Day, Nicholas; Hill, Victoria

2011-01-01

Epigenetic inactivation of tumor suppressor genes is a hallmark of cancer development. RASSF1A (Ras Association Domain Family 1 isoform A) tumor suppressor gene is one of the most frequently epigenetically inactivated genes in a wide range of adult and children's cancers and could be a useful molecular marker for cancer diagnosis and prognosis. RASSF1A has been shown to play a role in several biological pathways, including cell cycle control, apoptosis and microtubule dynamics. RASSF2, RASSF4, RASSF5 and RASSF6 are also epigenetically inactivated in cancer but have not been analyzed in as wide a range of malignancies as RASSF1A. Recently four new members of the RASSF family were identified these are termed N-Terminal RASSF genes (RASSF7–RASSF10). Molecular and biological analysis of these newer members has just begun. This review highlights what we currently know in respects to structural, functional and molecular properties of the N-Terminal RASSFs. PMID:21116130

Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates.

PubMed

Kleifeld, Oded; Doucet, Alain; Prudova, Anna; auf dem Keller, Ulrich; Gioia, Magda; Kizhakkedathu, Jayachandran N; Overall, Christopher M

2011-09-22

Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo-N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and

Amide-N-oxide heterosynthon and amide dimer homosynthon in cocrystals of carboxamide drugs and pyridine N-oxides.

PubMed

Babu, N Jagadeesh; Reddy, L Sreenivas; Nangia, Ashwini

2007-01-01

The carboxamide-pyridine N-oxide heterosynthon is sustained by syn(amide)N-H...O-(oxide) hydrogen bond and auxiliary (N-oxide)C-H...O(amide) interaction (Reddy, L. S.; Babu, N. J.; Nangia, A. Chem. Commun. 2006, 1369). We evaluate the scope and utility of this heterosynthon in amide-containing molecules and drugs (active pharmaceutical ingredients, APIs) with pyridine N-oxide cocrystal former molecules (CCFs). Out of 10 cocrystals in this study and 7 complexes from previous work, amide-N-oxide heterosynthon is present in 12 structures and amide dimer homosynthon occurs in 5 structures. The amide dimer is favored over amide-N-oxide synthon in cocrystals when there is competition from another H-bonding functional group, e.g., 4-hydroxybenzamide, or because of steric factors, as in carbamazepine API. The molecular organization in carbamazepine.quinoxaline N,N'-dioxide 1:1 cocrystal structure is directed by amide homodimer and anti(amide)N-H...O-(oxide) hydrogen bond. Its X-ray crystal structure matches with the third lowest energy frame calculated in Polymorph Predictor (Cerius(2), COMPASS force field). Apart from generating new and diverse supramolecular structures, hydration is controlled in one substance. 4-Picoline N-oxide deliquesces within a day, but its cocrystal with barbital does not absorb moisture at 50% RH and 30 degrees C up to four weeks. Amide-N-oxide heterosynthon has potential utility in both amide and N-oxide type drug molecules with complementary CCFs. Its occurrence probability in the Cambridge Structural Database is 87% among 27 structures without competing acceptors and 78% in 41 structures containing OH, NH, H(2)O functional groups.

Energetic Coupling between Ligand Binding and Dimerization in E. coli Phosphoglycerate Mutase

PubMed Central

Gardner, Nathan W.; Monroe, Lyman K.; Kihara, Daisuke; Park, Chiwook

2016-01-01

Energetic coupling of two molecular events in a protein molecule is ubiquitous in biochemical reactions mediated by proteins, such as catalysis and signal transduction. Here, we investigate energetic coupling between ligand binding and folding of a dimer using a model system that shows three-state equilibrium unfolding in an exceptional quality. The homodimeric E. coli cofactor-dependent phosphoglycerate mutase (dPGM) was found to be stabilized by ATP in a proteome-wide screen, although dPGM does not require or utilize ATP for enzymatic function. We investigated the effect of ATP on the thermodynamic stability of dPGM using equilibrium unfolding. In the absence of ATP, dPGM populates a partially unfolded, monomeric intermediate during equilibrium unfolding. However, addition of 1.0 mM ATP drastically reduces the population of the intermediate by selectively stabilizing the native dimer. Using a computational ligand docking method, we predicted ATP binds to the active site of the enzyme using the triphosphate group. By performing equilibrium unfolding and isothermal titration calorimetry with active-site variants of dPGM, we confirmed that active-site residues are involved in ATP binding. Our findings show that ATP promotes dimerization of the protein by binding to the active site, which is distal from the dimer interface. This cooperativity suggests an energetic coupling between the active-site and the dimer interface. We also propose a structural link to explain how ligand binding to the active site is energetically coupled with dimerization. PMID:26919584

Electronic and mechanical characteristics of stacked dimer molecular junctions.

PubMed

Magyarkuti, András; Adak, Olgun; Halbritter, Andras; Venkataraman, Latha

2018-02-15

Break-junction measurements are typically aimed at characterizing electronic properties of single molecules bound between two metal electrodes. Although these measurements have provided structure-function relationships for such devices, there is little work that studies the impact of molecule-molecule interactions on junction characteristics. Here, we use a scanning tunneling microscope based break-junction technique to study pi-stacked dimer junctions formed with two amine-terminated conjugated molecules. We show that the conductance, force and flicker noise of such dimers differ dramatically when compared with the corresponding monomer junctions and discuss the implications of these results on intra- and inter-molecular charge transport.

Effect of the coordination of the superficial site in the monomer dimer reaction on a disordered substrate

NASA Astrophysics Data System (ADS)

Valencia, Eliana; Cortés, Joaquín.; Puschmann, Heinrich

2000-12-01

Using Monte Carlo simulation experiments, a study is made of the effect of the superficial coordination number in a square lattice of sites for the monomer-dimer surface reaction (Ziff, Gulari and Barshad model) in the case of disordered substrates showing geometric heterogeneity of the sites, such as the percolation clusters. An analysis is made of the change in character of the phase transitions and in the size of the reactive window in the phase diagram, and the results were also compared with mean field theoretical calculations for disordered systems.

Identification of the Dimer Exchange Interface of the Bacterial DNA Damage Response Protein UmuD.

PubMed

Murison, David A; Timson, Rebecca C; Koleva, Bilyana N; Ordazzo, Michael; Beuning, Penny J

2017-09-12

The Escherichia coli SOS response, an induced DNA damage response pathway, confers survival on bacterial cells by providing accurate repair mechanisms as well as the potentially mutagenic pathway translesion synthesis (TLS). The umuD gene products are upregulated after DNA damage and play roles in both nonmutagenic and mutagenic aspects of the SOS response. Full-length UmuD is expressed as a homodimer of 139-amino-acid subunits, which eventually cleaves its N-terminal 24 amino acids to form UmuD'. The cleavage product UmuD' and UmuC form the Y-family polymerase DNA Pol V (UmuD' 2 C) capable of performing TLS. UmuD and UmuD' exist as homodimers, but their subunits can readily exchange to form UmuDD' heterodimers preferentially. Heterodimer formation is an essential step in the degradation pathway of UmuD'. The recognition sequence for ClpXP protease is located within the first 24 amino acids of full-length UmuD, and the partner of full-length UmuD, whether UmuD or UmuD', is degraded by ClpXP. To better understand the mechanism by which UmuD subunits exchange, we measured the kinetics of exchange of a number of fluorescently labeled single-cysteine UmuD variants as detected by Förster resonance energy transfer. Labeling sites near the dimer interface correlate with increased rates of exchange, indicating that weakening the dimer interface facilitates exchange, whereas labeling sites on the exterior decrease the rate of exchange. In most but not all cases, homodimer and heterodimer exchange exhibit similar rates, indicating that somewhat different molecular surfaces mediate homodimer exchange and heterodimer formation.

Site-specific Isopeptide Bridge Tethering of Chimeric gp41 N-terminal Heptad Repeat Helical Trimers for the Treatment of HIV-1 Infection.

PubMed

Wang, Chao; Li, Xue; Yu, Fei; Lu, Lu; Jiang, Xifeng; Xu, Xiaoyu; Wang, Huixin; Lai, Wenqing; Zhang, Tianhong; Zhang, Zhenqing; Ye, Ling; Jiang, Shibo; Liu, Keliang

2016-08-26

Peptides derived from the N-terminal heptad repeat (NHR) of HIV-1 gp41 can be potent inhibitors against viral entry when presented in a nonaggregating trimeric coiled-coil conformation via the introduction of exogenous trimerization motifs and intermolecular disulfide bonds. We recently discovered that crosslinking isopeptide bridges within the de novo helical trimers added exceptional resistance to unfolding. Herein, we attempted to optimize (CCIZN17)3, a representative disulfide bond-stabilized chimeric NHR-trimer, by incorporating site-specific interhelical isopeptide bonds as the redox-sensitive disulfide surrogate. In this process, we systematically examined the effect of isopeptide bond position and molecular sizes of auxiliary trimeric coiled-coil motif and NHR fragments on the antiviral potency of these NHR-trimers. Pleasingly, (IZ14N24N)3 possessed promising inhibitory activity against HIV-1 infection and markedly increased proteolytic stability relative to its disulfide-tethered counterpart, suggesting good potential for further development as an effective antiviral agent for treatment of HIV-1 infection.

Site-specific Isopeptide Bridge Tethering of Chimeric gp41 N-terminal Heptad Repeat Helical Trimers for the Treatment of HIV-1 Infection

PubMed Central

Wang, Chao; Li, Xue; Yu, Fei; Lu, Lu; Jiang, Xifeng; Xu, Xiaoyu; Wang, Huixin; Lai, Wenqing; Zhang, Tianhong; Zhang, Zhenqing; Ye, Ling; Jiang, Shibo; Liu, Keliang

2016-01-01

Peptides derived from the N-terminal heptad repeat (NHR) of HIV-1 gp41 can be potent inhibitors against viral entry when presented in a nonaggregating trimeric coiled-coil conformation via the introduction of exogenous trimerization motifs and intermolecular disulfide bonds. We recently discovered that crosslinking isopeptide bridges within the de novo helical trimers added exceptional resistance to unfolding. Herein, we attempted to optimize (CCIZN17)3, a representative disulfide bond-stabilized chimeric NHR-trimer, by incorporating site-specific interhelical isopeptide bonds as the redox-sensitive disulfide surrogate. In this process, we systematically examined the effect of isopeptide bond position and molecular sizes of auxiliary trimeric coiled-coil motif and NHR fragments on the antiviral potency of these NHR-trimers. Pleasingly, (IZ14N24N)3 possessed promising inhibitory activity against HIV-1 infection and markedly increased proteolytic stability relative to its disulfide-tethered counterpart, suggesting good potential for further development as an effective antiviral agent for treatment of HIV-1 infection. PMID:27562370

Biochemistry of terminal deoxynucleotidyltransferase. Identification and unity of ribo- and deoxyribonucleoside triphosphate binding site in terminal deoxynucleotidyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pandey, V.N.; Modak, M.J.

Terminal deoxynucleotidyltransferase is the only DNA polymerase that is strongly inhibited in the presence of ATP. We have labeled calf terminal deoxynucleotidyltransferase with (/sup 32/P)ATP in order to identify its binding site in terminal deoxynucleotidyltransferase. The specificity of ATP cross-linking to terminal deoxynucleotidyltransferase is shown by the competitive inhibition of the overall cross-linking reaction by deoxynucleoside triphosphates, as well as the ATP analogs Ap4A and Ap5A. Tryptic peptide mapping of (/sup 32/P)ATP-labeled enzyme revealed a peptide fraction that contained the majority of cross-linked ATP. The properties, chromatographic characteristics, amino acid composition, and sequence analysis of this peptide fraction were identicalmore » with those found associated with dTTP cross-linked terminal deoxynucleotidyl-transferase peptide. The involvement of the same 2 cysteine residues in the crosslinking of both nucleotides further confirmed the unity of the ATP and dTTP binding domain that contains residues 224-237 in the primary amino acid sequence of calf terminal deoxynucleotidyltransferase.« less

A cyclobutane thymine–N4-methylcytosine dimer is resistant to hydrolysis but strongly blocks DNA synthesis

PubMed Central

Yamamoto, Junpei; Oyama, Tomoko; Kunishi, Tomohiro; Masutani, Chikahide; Hanaoka, Fumio; Iwai, Shigenori

2014-01-01

Exposure of DNA to ultraviolet light produces harmful crosslinks between adjacent pyrimidine bases, to form cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6–4)pyrimidone photoproducts. The CPD is frequently formed, and its repair mechanisms have been exclusively studied by using a CPD formed at a TT site. On the other hand, biochemical analyses using CPDs formed within cytosine-containing sequence contexts are practically difficult, because saturated cytosine easily undergoes hydrolytic deamination. Here, we found that N-alkylation of the exocyclic amino group of 2′-deoxycytidine prevents hydrolysis in CPD formation, and an N-methylated cytosine-containing CPD was stable enough to be derivatized into its phosphoramidite building block and incorporated into oligonucleotides. Kinetic studies of the CPD-containing oligonucleotide indicated that its lifetime under physiological conditions is relatively long (∼7 days). In biochemical analyses using human DNA polymerase η, incorporation of TMP opposite the N-methylcytosine moiety of the CPD was clearly detected, in addition to dGMP incorporation, and the incorrect TMP incorporation blocked DNA synthesis. The thermodynamic parameters confirmed the formation of this unusual base pair. PMID:24185703

Crystal structure of inhibitor of growth 4 (ING4) dimerization domain reveals functional organization of ING family of chromatin-binding proteins.

PubMed

Culurgioni, Simone; Muñoz, Inés G; Moreno, Alberto; Palacios, Alicia; Villate, Maider; Palmero, Ignacio; Montoya, Guillermo; Blanco, Francisco J

2012-03-30

The protein ING4 binds to histone H3 trimethylated at Lys-4 (H3K4me3) through its C-terminal plant homeodomain, thus recruiting the HBO1 histone acetyltransferase complex to target promoters. The structure of the plant homeodomain finger bound to an H3K4me3 peptide has been described, as well as the disorder and flexibility in the ING4 central region. We report the crystal structure of the ING4 N-terminal domain, which shows an antiparallel coiled-coil homodimer with each protomer folded into a helix-loop-helix structure. This arrangement suggests that ING4 can bind simultaneously two histone tails on the same or different nucleosomes. Dimerization has a direct impact on ING4 tumor suppressor activity because monomeric mutants lose the ability to induce apoptosis after genotoxic stress. Homology modeling based on the ING4 structure suggests that other ING dimers may also exist.

The intrinsically disordered C-terminal domain of the measles virus nucleoprotein interacts with the C-terminal domain of the phosphoprotein via two distinct sites and remains predominantly unfolded

PubMed Central

Bourhis, Jean-Marie; Receveur-Bréchot, Véronique; Oglesbee, Michael; Zhang, Xinsheng; Buccellato, Matthew; Darbon, Hervé; Canard, Bruno; Finet, Stéphanie; Longhi, Sonia

2005-01-01

Measles virus is a negative-sense, single-stranded RNA virus within theMononegavirales order,which includes several human pathogens, including rabies, Ebola, Nipah, and Hendra viruses. Themeasles virus nucleoprotein consists of a structured N-terminal domain, and of an intrinsically disordered C-terminal domain, NTAIL (aa 401–525), which undergoes induced folding in the presence of the C-terminal domain (XD, aa 459–507) of the viral phosphoprotein. With in NTAIL, an α-helical molecular recognition element (α-MoRE, aa 488–499) involved in binding to P and in induced folding was identified and then observed in the crystal structure of XD. Using small-angle X-ray scattering, we have derived a low-resolution structural model of the complex between XD and NTAIL, which shows that most of NTAIL remains disordered in the complex despite P-induced folding within the α-MoRE. The model consists of an extended shape accommodating the multiple conformations adopted by the disordered N-terminal region of NTAIL, and of a bulky globular region, corresponding to XD and to the C terminus of NTAIL (aa 486–525). Using surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and heteronuclear magnetic resonance, we show that NTAIL has an additional site (aa 517–525) involved in binding to XD but not in the unstructured-to-structured transition. This work provides evidence that intrinsically disordered domains can establish complex interactions with their partners, and can contact them through multiple sites that do not all necessarily gain regular secondary structure. PMID:16046624

(19)F NMR reveals multiple conformations at the dimer interface of the nonstructural protein 1 effector domain from influenza A virus.

PubMed

Aramini, James M; Hamilton, Keith; Ma, Li-Chung; Swapna, G V T; Leonard, Paul G; Ladbury, John E; Krug, Robert M; Montelione, Gaetano T

2014-04-08

Nonstructural protein 1 of influenza A virus (NS1A) is a conserved virulence factor comprised of an N-terminal double-stranded RNA (dsRNA)-binding domain and a multifunctional C-terminal effector domain (ED), each of which can independently form symmetric homodimers. Here we apply (19)F NMR to NS1A from influenza A/Udorn/307/1972 virus (H3N2) labeled with 5-fluorotryptophan, and we demonstrate that the (19)F signal of Trp187 is a sensitive, direct monitor of the ED helix:helix dimer interface. (19)F relaxation dispersion data reveal the presence of conformational dynamics within this functionally important protein:protein interface, whose rate is more than three orders of magnitude faster than the kinetics of ED dimerization. (19)F NMR also affords direct spectroscopic evidence that Trp187, which mediates intermolecular ED:ED interactions required for cooperative dsRNA binding, is solvent exposed in full-length NS1A at concentrations below aggregation. These results have important implications for the diverse roles of this NS1A epitope during influenza virus infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

Dimerization in Highly Concentrated Solutions of Phosphoimidazolide Activated Mononucleotides

NASA Technical Reports Server (NTRS)

Kanavarioti, Anastassia

1997-01-01

Phosphoimidazolide activated ribomononucleotides (*pN) are useful substrates for the non-enzymatic synthesis of polynucleotides. However, dilute neutral aqueous solutions of *pN typically yield small amounts of dimers and traces of polymers; most of *pN hydrolyzes to yield nucleoside 5'-monophosphate. Here we report the self-condensation of nucleoside 5'-phosphate 2- methylimidazolide (2-MeImpN with N = cytidine, uridine or guanosine) in the presence of Mg2(+) in concentrated solutions, such as might have been found in an evaporating lagoon on prebiotic Earth. The product distribution indicates that oligomerization is favored at the expense of hydrolysis. At 1.0 M, 2-MelmpU and 2-MelmpC produce about 65% of oligomers including 4% of the 3',5'-Iinked dimer. Examination of the product distribution of the three isomeric dimers in a self-condensation allows identification of reaction pathways that lead to dimer formation. Condensations in a concentrated mixture of all three nucleotides (U,C,G mixtures) is made possible by the enhanced solubility of 2-MeImpG in such mixtures. Although percent yield of intemucleotide linked dimers is enhanced as a function of initial monomer concentration, pyrophosphate dimer yields remain practically unchanged at about 20% for 2-MelmpU, 16% for 2-MeImpC and 25% of the total pyrophosphate in the U,C,G mixtures. The efficiency by which oligomers are produced in these concentrated solutions makes the evaporating lagoon scenario a potentially interesting medium for the prebiotic synthesis of dimers and short RNAs.

Dimerization in Highly Concentrated Solutions of Phosphoimidazolide Activated Monomucleotides

NASA Astrophysics Data System (ADS)

Kanavarioti, Anastassia

1997-08-01

Phosphoimidazolide activated ribomononucleotides (*pN) are useful substrates for the non-enzymatic synthesis of polynucleotides. However, dilute neutral aqueous solutions of *pN typically yield small amounts of dimers and traces of polymers; most of *pN hydrolyzes to yield nucleoside 5'-monophosphate. Here we report the self-condensation of nucleoside 5'-phosphate 2-methylimidazolide (2-MeImpN with N = cytidine, uridine or guanosine) in the presence of Mg2+ in concentrated solutions, such as might have been found in an evaporating lagoon on prebiotic Earth. The product distribution indicates that oligomerization is favored at the expense of hydrolysis. At 1.0 M, 2-MeImpU and 2-MeImpC produce about 65% of oligomers including 4% of the 3',5'-linked dimer. Examination of the product distribution of the three isomeric dimers in a self-condensation allows identification of reaction pathways that lead to dimer formation. Condensations in a concentrated mixture of all three nucleotides (U,C,G mixtures) is made possible by the enhanced solubility of 2-MeImpG in such mixtures. Although percent yield of internucleotide linked dimers is enhanced as a function of initial monomer concentration, pyrophosphate dimer yields remain practically unchanged at about 20% for 2-MeImpU, 16% for 2-MeImpC and 25% of the total pyrophosphate in the U,C,G mixtures. The efficiency by which oligomers are produced in these concentrated solutions makes the evaporating lagoon scenario a potentially interesting medium for the prebiotic synthesis of dimers and short RNAs.

Chloroperoxidase-catalyzed oxidation of 4,6-dimethyldibenzothiophene as dimer complexes: evidence for kinetic cooperativity.

PubMed

Torres, Eduardo; Aburto, Jorge

2005-05-15

A sigmoidal kinetic behavior of chloroperoxidase for the oxidation of 4,6-dimethyldibenzothiophene (4,6-DMDBT) in water-miscible organic solvent is for the first time reported. Kinetics of 4,6-DMDBT oxidation showed a cooperative profile probably due to the capacity of chloroperoxidase to recognize a substrate dimer (pi-pi dimer) in its active site. Experimental evidence is given for dimer formation and its presence in the active site of chloroperoxidase. The kinetic data were adjusted for a binding site able to interact with either monomer or dimer substrates, producing a cooperative model describing a one-site binding of two related species. Determination of kinetics constants by iterative calculations of possible oxidation paths of 4,6-DMDBT suggests that kinetics oxidation of dimer substrate is preferred when compared to monomer oxidation. Steady-state fluorometry of substrate in the absence and presence of chloroperoxidase, described by the spectral center of mass, supports this last conclusion.

Degradation pattern of photosystem II reaction center protein D1 in intact leaves. The major photoinhibition-induced cleavage site in D1 polypeptide is located amino terminally of the DE loop.

PubMed

Kettunen, R; Tyystjärvi, E; Aro, E M

1996-08-01

Photoinhibition-induced degradation of the D1 protein of the photosystem II reaction center was studied in intact pumpkin (Cucurbita pepo L.) leaves. Photoinhibition was observed to cause the cleavage of the D1 protein at two distinct sites. The main cleavage generated an 18-kD N-terminal and a 20-kD C-terminal degradation fragment of the D1 protein. this cleavage site was mapped to be located clearly N terminally of the DE loop. The other, less-frequent cleavage occurred at the DE loop and produced the well-documented 23-kD, N-terminal D1 degradation product. Furthermore, the 23-kD, N-terminal D1 fragment appears to be phosphorylated and can be detected only under severe photoinhibition in vivo. Comparison of the D1 degradation pattern after in vivo photoinhibition to that after in vitro acceptor-side and donor-side photoinhibition, performed with isolated photosystem II core particles, gives indirect evidence in support of donor-side photoinhibition in intact leaves.

Select human cancer mutants of NRMT1 alter its catalytic activity and decrease N-terminal trimethylation.

PubMed

Shields, Kaitlyn M; Tooley, John G; Petkowski, Janusz J; Wilkey, Daniel W; Garbett, Nichola C; Merchant, Michael L; Cheng, Alan; Schaner Tooley, Christine E

2017-08-01

A subset of B-cell lymphoma patients have dominant mutations in the histone H3 lysine 27 (H3K27) methyltransferase EZH2, which change it from a monomethylase to a trimethylase. These mutations occur in aromatic resides surrounding the active site and increase growth and alter transcription. We study the N-terminal trimethylase NRMT1 and the N-terminal monomethylase NRMT2. They are 50% identical, but differ in key aromatic residues in their active site. Given how these residues affect EZH2 activity, we tested whether they are responsible for the distinct catalytic activities of NRMT1/2. Additionally, NRMT1 acts as a tumor suppressor in breast cancer cells. Its loss promotes oncogenic phenotypes but sensitizes cells to DNA damage. Mutations of NRMT1 naturally occur in human cancers, and we tested a select group for altered activity. While directed mutation of the aromatic residues had minimal catalytic effect, NRMT1 mutants N209I (endometrial cancer) and P211S (lung cancer) displayed decreased trimethylase and increased monomethylase/dimethylase activity. Both mutations are located in the peptide-binding channel and indicate a second structural region impacting enzyme specificity. The NRMT1 mutants demonstrated a slower rate of trimethylation and a requirement for higher substrate concentration. Expression of the mutants in wild type NRMT backgrounds showed no change in N-terminal methylation levels or growth rates, demonstrating they are not acting as dominant negatives. Expression of the mutants in cells lacking endogenous NRMT1 resulted in minimal accumulation of N-terminal trimethylation, indicating homozygosity could help drive oncogenesis or serve as a marker for sensitivity to DNA damaging chemotherapeutics or γ-irradiation. © 2017 The Protein Society.

Isomeric N-Annulated Perylene Diimide Dimers for Organic Solar Cells.

PubMed

Ma, Zetong; Fu, Huiting; Meng, Dong; Jiang, Wei; Sun, Yanming; Wang, Zhaohui

2018-04-16

Two isomeric N-annulated perylene diimide dimers, namely, p-BDNP and m-BDNP were designed and synthesized via geometric tuning. The distinct molecular geometry and packing arrangements of isomers with almost identical optical and electrochemical properties rendered us an in-depth understanding of the molecular structure-aggregation state-photovoltaic performance relationship. Blended with the commercially available donor PCE-10, p-BDNP and m-BDNP showed distinct differences in photovoltaic performance with power conversion efficiencies (PCEs) of 5.01 % and 4.15 %, respectively. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure and energetics of InN and GaN dimers

NASA Astrophysics Data System (ADS)

Šimová, Lucia; Tzeli, Demeter; Urban, Miroslav; Černušák, Ivan; Theodorakopoulos, Giannoula; Petsalakis, Ioannis D.

2008-06-01

Large-scale mapping of various dimers of indium nitride and gallium nitride in singlet and triplet electronic states is reported. Second-order perturbation theory with Møller-Plesset partitioning of the Hamiltonian (MP2) and coupled-cluster with single and double excitations corrected for the triple excitations (CCSD(T)) are used for the geometry determinations and evaluation of excitation and dissociation energies. For gallium and nitrogen we have used the singly augmented correlation-consistent triple-zeta basis set (aug-cc-pVTZ), for indium we have used the aug-cc-pVTZ-pseudopotential basis set. The dissociation energies are corrected for basis set superposition error (BBSE) including geometrical relaxation of the monomers. We compare and discuss the similarities and dissimilarities in the structural patterns and energetics of both groups of isomers, including the effect of the BSSE. Our computations show that there are not only different ground states for In 2N 2 and Ga 2N 2 but also different numbers of stable stationary points on their potential energy surface. We compare our results with the molecular data published so far for these systems.

X-ray-structure of a cytidylyl-3',5'-adenosine-proflavine complex: a self-paired parallel-chain double helical dimer with an intercalated acridine dye.

PubMed Central

Westhof, E; Sundaralingam, M

1980-01-01

The non-self-complementary dinucleoside monophosphate cytidylyl-3',5'-adenosine (CpA) forms a base-paired parallel-chain dimer with an intercalated proflavine. The dimer complex possesses a right-handed helical twist. The dimer helix has an irregular girth with a neutral adenine-adenine (A-A) pair, hydrogen-bonded through the N6 and N7 sites (C1'...C1' separation of 10.97 A), and a triply hydrogen-bonded protonated cytosine-cytosine (C-C) pair with a proton shared between the base N3 sites (Cl'...Cl' separation of 9.59 A). The torsion angles of the sugar-phosphate backbone are within their most preferred ranges and the sugar puckering sequence (5' leads to 3') is C3'-endo, C2'-endo. There is also a second proflavine molecule sandwiched between CpA dimers on the 21-axis. Both proflavines are necessarily disordered, being on dyad axis, and this suggests possible insights into the dynamics of intercalation of planar drugs. This structure shows that intercalation of planar drugs in nucleic acids may not be restricted to antiparallel complementary Watson-Crick pairing regions and provides additional mechanisms for acridine mutagenesis. PMID:6929524

Dimerization model of the C-terminal RNA Recognition Motif of HuR.

PubMed

Díaz-Quintana, Antonio; García-Mauriño, Sofía M; Díaz-Moreno, Irene

2015-04-28

Human antigen R (HuR) is a ubiquitous 32 kDa protein comprising three RNA Recognition Motifs (RRMs), whose main function is to bind Adenylate and uridylate Rich Elements (AREs) in 3' UnTranslated Regions (UTRs) of mRNAs. In addition to binding RNA molecules, the third domain (RRM3) is involved in HuR oligomerization and apoptotic signaling. The RRM3 monomer is able to dimerize, with its self-binding affinity being dependent on ionic strength. Here we provide a deeper structural insight into the nature of the encounter complexes leading to the formation of RRM3 dimers by using Brownian Dynamics and Molecular Dynamics. Our computational data show that the initial unspecific encounter follows a downhill pathway until reaching an optimum conformation stabilized by hydrophobic interactions. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Salt bridge residues between I-Ak dimer of dimers alpha-chains modulate antigen presentation.

PubMed

Yadati, S; Nydam, T; Demian, D; Wade, T K; Gabriel, J L; Barisas, B G; Wade, W F

1999-03-15

Class II dimers of dimers are predicted to have functional significance in antigen presentation. The putative contact amino acids of the I-Ak class II dimer of dimers have been identified by molecular modeling based on the DR1 crystal structure (Nydam et al., Int. Immunol. 10, 1237,1998). We have previously reported the role in antigen presentation of dimer of dimers contact amino acids located in the C-terminal domains of the alpha- and beta-chains of class II. Our calculations show that residues Ealpha89 and Ralpha145 in the alpha2-domain form an inter alpha-chain salt bridge between pairs of alphabeta-heterodimers. Other residues, Qalpha92 and Nalpha115, may be involved in close association in that part of the alpha-chain. We investigated the role of these amino acids on class II expression and antigen presentation. Class II composed of an Ealpha89K substituted alpha-chain paired with a wt beta-chain exhibited inhibited antigen presentation and expression of alpha-chain serologic epitopes. In contrast, mutation of Ralpha145E had less affect on antigen presentation and did not affect I-Ak serologic epitopes. Interchanging charges of the salt bridge residues by expressing both Ralpha145E and Ealpha89K on the same chain obviated the large negative effect of the Ealpha89K mutation on antigen presentation but not on the serologic epitopes. Our results are similar for those reported for mutation of DR3's inter-chain salt bridge with the exception that double mutants did not moderate the DR3 defect. Interestingly, the amino acids differences between I-A and DR change the location of the inter-chain salt bridges. In DR1 these residues are located at positions Ealpha88 and Kalpha111; in I-Ak these residues are located at position Ealpha89 and Ralpha145. Inter alpha-chain salt bridges are thus maintained in various class II molecules by amino acids located in different parts of the alpha2-domain. This conservation of structure suggests that considerable functional

The epitope of monoclonal antibodies blocking erythrocyte invasion by Plasmodium falciparum map to the dimerization and receptor glycan binding sites of EBA-175.

PubMed

Ambroggio, Xavier; Jiang, Lubin; Aebig, Joan; Obiakor, Harold; Lukszo, Jan; Narum, David L

2013-01-01

The malaria parasite, Plasmodium falciparum, and related parasites use a variety of proteins with Duffy-Binding Like (DBL) domains to bind glycoproteins on the surface of host cells. Among these proteins, the 175 kDa erythrocyte binding antigen, EBA-175, specifically binds to glycophorin A on the surface of human erythrocytes during the process of merozoite invasion. The domain responsible for glycophorin A binding was identified as region II (RII) which contains two DBL domains, F1 and F2. The crystal structure of this region revealed a dimer that is presumed to represent the glycophorin A binding conformation as sialic acid binding sites and large cavities are observed at the dimer interface. The dimer interface is largely composed of two loops from within each monomer, identified as the F1 and F2 β-fingers that contact depressions in the opposing monomers in a similar manner. Previous studies have identified a panel of five monoclonal antibodies (mAbs) termed R215 to R218 and R256 that bind to RII and inhibit invasion of erythrocytes to varying extents. In this study, we predict the F2 β-finger region as the conformational epitope for mAbs, R215, R217, and R256, and confirm binding for the most effective blocking mAb R217 and R215 to a synthetic peptide mimic of the F2 β-finger. Localization of the epitope to the dimerization and glycan binding sites of EBA-175 RII and site-directed mutagenesis within the predicted epitope are consistent with R215 and R217 blocking erythrocyte invasion by Plasmodium falciparum by preventing formation of the EBA-175- glycophorin A complex.

Properties of an intergenic terminator and start site switch that regulate IMD2 transcription in yeast.

PubMed

Jenks, M Harley; O'Rourke, Thomas W; Reines, Daniel

2008-06-01

The IMD2 gene in Saccharomyces cerevisiae is regulated by intracellular guanine nucleotides. Regulation is exerted through the choice of alternative transcription start sites that results in synthesis of either an unstable short transcript terminating upstream of the start codon or a full-length productive IMD2 mRNA. Start site selection is dictated by the intracellular guanine nucleotide levels. Here we have mapped the polyadenylation sites of the upstream, unstable short transcripts that form a heterogeneous family of RNAs of approximately 200 nucleotides. The switch from the upstream to downstream start sites required the Rpb9 subunit of RNA polymerase II. The enzyme's ability to locate the downstream initiation site decreased exponentially as the start was moved downstream from the TATA box. This suggests that RNA polymerase II's pincer grip is important as it slides on DNA in search of a start site. Exosome degradation of the upstream transcripts was highly dependent upon the distance between the terminator and promoter. Similarly, termination was dependent upon the Sen1 helicase when close to the promoter. These findings extend the emerging concept that distinct modes of termination by RNA polymerase II exist and that the distance of the terminator from the promoter, as well as its sequence, is important for the pathway chosen.

Protein Folding Mechanism of the Dimeric AmphiphysinII/Bin1 N-BAR Domain

PubMed Central

Gruber, Tobias; Balbach, Jochen

2015-01-01

The human AmphyphisinII/Bin1 N-BAR domain belongs to the BAR domain superfamily, whose members sense and generate membrane curvatures. The N-BAR domain is a 57 kDa homodimeric protein comprising a six helix bundle. Here we report the protein folding mechanism of this protein as a representative of this protein superfamily. The concentration dependent thermodynamic stability was studied by urea equilibrium transition curves followed by fluorescence and far-UV CD spectroscopy. Kinetic unfolding and refolding experiments, including rapid double and triple mixing techniques, allowed to unravel the complex folding behavior of N-BAR. The equilibrium unfolding transition curve can be described by a two-state process, while the folding kinetics show four refolding phases, an additional burst reaction and two unfolding phases. All fast refolding phases show a rollover in the chevron plot but only one of these phases depends on the protein concentration reporting the dimerization step. Secondary structure formation occurs during the three fast refolding phases. The slowest phase can be assigned to a proline isomerization. All kinetic experiments were also followed by fluorescence anisotropy detection to verify the assignment of the dimerization step to the respective folding phase. Based on these experiments we propose for N-BAR two parallel folding pathways towards the homodimeric native state depending on the proline conformation in the unfolded state. PMID:26368922

The Tip of the Four N-Terminal α-Helices of Clostridium sordellii Lethal Toxin Contains the Interaction Site with Membrane Phosphatidylserine Facilitating Small GTPases Glucosylation

PubMed Central

Varela Chavez, Carolina; Haustant, Georges Michel; Baron, Bruno; England, Patrick; Chenal, Alexandre; Pauillac, Serge; Blondel, Arnaud; Popoff, Michel-Robert

2016-01-01

Clostridium sordellii lethal toxin (TcsL) is a powerful virulence factor responsible for severe toxic shock in man and animals. TcsL belongs to the large clostridial glucosylating toxin (LCGT) family which inactivates small GTPases by glucosylation with uridine-diphosphate (UDP)-glucose as a cofactor. Notably, TcsL modifies Rac and Ras GTPases, leading to drastic alteration of the actin cytoskeleton and cell viability. TcsL enters cells via receptor-mediated endocytosis and delivers the N-terminal glucosylating domain (TcsL-cat) into the cytosol. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to the lipid membrane. We have previously reported that the N-terminal four helical bundle structure (1–93 domain) recognizes a broad range of lipids, but that TcsL-cat specifically binds to PS and phosphatidic acid. Here, we show using mutagenesis that the PS binding site is localized on the tip of the four-helix bundle which is rich in positively-charged amino acids. Residues Y14, V15, F17, and R18 on loop 1, between helices 1 and 2, in coordination with R68 from loop 3, between helices 3 and 4, form a pocket which accommodates L-serine. The functional PS-binding site is required for TcsL-cat binding to the plasma membrane and subsequent cytotoxicity. TcsL-cat binding to PS facilitates a high enzymatic activity towards membrane-anchored Ras by about three orders of magnitude as compared to Ras in solution. The PS-binding site is conserved in LCGTs, which likely retain a common mechanism of binding to the membrane for their full activity towards membrane-bound GTPases. PMID:27023605

Structural basis for substrate recognition by the human N-terminal methyltransferase 1

DOE PAGES

Dong, Cheng; Mao, Yunfei; Tempel, Wolfram; ...

2015-11-05

α-N-terminal methylation represents a highly conserved and prevalent post-translational modification, yet its biological function has remained largely speculative. The recent discovery of α-N-terminal methyltransferase 1 (NTMT1) and its physiological substrates propels the elucidation of a general role of α-N-terminal methylation in mediating DNA-binding ability of the modified proteins. The phenotypes, observed from both NTMT1 knockdown in breast cancer cell lines and knockout mouse models, suggest the potential involvement of α-N-terminal methylation in DNA damage response and cancer development. In this study, we report the first crystal structures of human NTMT1 in complex with cofactor S-adenosyl-L-homocysteine (SAH) and six substrate peptides,more » respectively, and reveal that NTMT1 contains two characteristic structural elements (a β hairpin and an N-terminal extension) that contribute to its substrate specificity. Our complex structures, coupled with mutagenesis, binding, and enzymatic studies, also present the key elements involved in locking the consensus substrate motif XPK (X indicates any residue type other than D/E) into the catalytic pocket for α-N-terminal methylation and explain why NTMT1 prefers an XPK sequence motif. We propose a catalytic mechanism for α-N-terminal methylation. Overall, this study gives us the first glimpse of the molecular mechanism of α-N-terminal methylation and potentially contributes to the advent of therapeutic agents for human diseases associated with deregulated α-N-terminal methylation.« less

pH-Dependent Binding of Chloride to a Marine Alkaline Phosphatase Affects the Catalysis, Active Site Stability, and Dimer Equilibrium.

PubMed

Hjörleifsson, Jens G; Ásgeirsson, Bjarni

2017-09-26

The effect of ionic strength on enzyme activity and stability varies considerably between enzymes. Ionic strength is known to affect the catalytic activity of some alkaline phosphatases (APs), such as Escherichia coli AP, but how ions affect APs is debated. Here, we studied the effect of various ions on a cold-adapted AP from Vibrio splendidus (VAP). Previously, we have found that the active form of VAP is extremely unstable at low ionic strengths. Here we show that NaCl increased the activity and stability of VAP and that the effect was pH-dependent in the range of pH 7-10. The activity profile as a function of pH formed two maxima, indicating a possible conformational change. Bringing the pH from the neutral to the alkaline range was accompanied by a large increase in both the K i for inorganic phosphate (product inhibition) and the K M for p-nitrophenyl phosphate. The activity transitions observed as the pH was varied correlated with structural changes as monitored by tryptophan fluorescence. Thermal and urea-induced inactivation was shown to be accompanied by neither dissociation of the active site metal ions nor dimer dissociation. This would suggest that the inactivation involved subtle changes in active site conformation. Furthermore, the VAP dimer equilibrium was studied for the first time and shown to highly favor dimerization, which was dependent on pH and NaCl concentration. Taken together, the data support a model in which anions bind to some specific acceptor in the active site of VAP, resulting in great stabilization and catalytic rate enhancement, presumably through a different mechanism.

Structural insights into the intertwined dimer of fyn SH2.

PubMed

Huculeci, Radu; Garcia-Pino, Abel; Buts, Lieven; Lenaerts, Tom; van Nuland, Nico

2015-12-01

Src homology 2 domains are interaction modules dedicated to the recognition of phosphotyrosine sites incorporated in numerous proteins found in intracellular signaling pathways. Here we provide for the first time structural insight into the dimerization of Fyn SH2 both in solution and in crystalline conditions, providing novel crystal structures of both the dimer and peptide-bound structures of Fyn SH2. Using nuclear magnetic resonance chemical shift analysis, we show how the peptide is able to eradicate the dimerization, leading to monomeric SH2 in its bound state. Furthermore, we show that Fyn SH2's dimer form differs from other SH2 dimers reported earlier. Interestingly, the Fyn dimer can be used to construct a completed dimer model of Fyn without any steric clashes. Together these results extend our understanding of SH2 dimerization, giving structural details, on one hand, and suggesting a possible physiological relevance of such behavior, on the other hand. © 2015 The Protein Society.

Structural model of dodecameric heat-shock protein Hsp21: Flexible N-terminal arms interact with client proteins while C-terminal tails maintain the dodecamer and chaperone activity.

PubMed

Rutsdottir, Gudrun; Härmark, Johan; Weide, Yoran; Hebert, Hans; Rasmussen, Morten I; Wernersson, Sven; Respondek, Michal; Akke, Mikael; Højrup, Peter; Koeck, Philip J B; Söderberg, Christopher A G; Emanuelsson, Cecilia

2017-05-12

Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended I X V X I motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the I X V X I motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Oxidative Folding and N-terminal Cyclization of Onconase+

PubMed Central

Welker, Ervin; Hathaway, Laura; Xu, Guoqiang; Narayan, Mahesh; Pradeep, Lovy; Shin, Hang-Cheol; Scheraga, Harold A.

2008-01-01

Cyclization of the N-terminal glutamine residue to pyroglutamic acid in onconase, an anti-cancer chemotherapeutic agent, increases the activity and stability of the protein. Here, we examine the correlated effects of the folding/unfolding process and the formation of this N-terminal pyroglutamic acid. The results in this study indicate that cyclization of the N-terminal glutamine has no significant effect on the rate of either reductive unfolding or oxidative folding of the protein. Both the cyclized and uncyclized proteins seem to follow the same oxidative folding pathways; however, cyclization altered the relative flux of the protein in these two pathways by increasing the rate of formation of a kinetically trapped intermediate. Glutaminyl cyclase (QC) catalyzed the cyclization of the unfolded, reduced protein, but had no effect on the disulfide-intact, uncyclized, folded protein. The structured intermediates of uncyclized onconase were also resistant to QC-catalysis, consistent with their having a native-like fold. These observations suggest that, in vivo, cyclization takes place during the initial stages of oxidative folding, specifically, before the formation of structured intermediates. The competition between oxidative folding and QC-mediated cyclization suggests that QC-catalyzed cyclization of the N-terminal glutamine in onconase occurs in the endoplasmic reticulum, probably co-translationally. PMID:17439243

Binding Energies of Proton-Bound Dimers of Imidazole and n-Acetylalanine Methyl Ester Obtained by Blackbody Infrared Radiative Dissociation

PubMed Central

Jockusch, Rebecca A.; Williams*, Evan R.

2005-01-01

The dissociation kinetics of protonated n-acetyl-L-alanine methyl ester dimer (AcAlaMEd), imidazole dimer, and their cross dimer were measured using blackbody infrared radiative dissociation (BIRD). Master equation modeling of these data was used to extract threshold dissociation energies (Eo) for the dimers. Values of 1.18 ± 0.06, 1.11 ± 0.04, and 1.12 ± 0.08 eV were obtained for AcAlaMEd, imidazole dimer, and the cross dimer, respectively. Assuming that the reverse activation barrier for dissociation of the ion–molecule complex is negligible, the value of Eo can be compared to the dissociation enthalpy (ΔHd°) from HPMS data. The Eo values obtained for the imidazole dimer and the cross dimer are in agreement with HPMS values; the value for AcAlaMEd is somewhat lower. Radiative rate constants used in the master equation modeling were determined using transition dipole moments calculated at the semiempirical (AM1) level for all dimers and compared to ab initio (RHF/3-21G*) calculations where possible. To reproduce the experimentally measured dissociation rates using master equation modeling, it was necessary to multiply semiempirical transition dipole moments by a factor between 2 and 3. Values for transition dipole moments from the ab initio calculations could be used for two of the dimers but appear to be too low for AcAlaMEd. These results demonstrate that BIRD, in combination with master equation modeling, can be used to determine threshold dissociation energies for intermediate size ions that are in neither the truncated Boltzmann nor the rapid energy exchange limit. PMID:16604163

Efficient sortase-mediated N-terminal labeling of TEV protease cleaved recombinant proteins.

PubMed

Sarpong, Kwabena; Bose, Ron

2017-03-15

A major challenge in attaching fluorophores or other handles to proteins is the availability of a site-specific labeling strategy that provides stoichiometric modification without compromising protein integrity. We developed a simple approach that combines TEV protease cleavage, sortase modification and affinity purification to N-terminally label proteins. To achieve stoichiometrically-labeled protein, we included a short affinity tag in the fluorophore-containing peptide for post-labeling purification of the modified protein. This strategy can be easily applied to any recombinant protein with a TEV site and we demonstrate this on Epidermal Growth Factor Receptor (EGFR) and Membrane Scaffold Protein (MSP) constructs. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase*

PubMed Central

Zhang, Shengnan; Fitzpatrick, Paul F.

2016-01-01

Liver phenylalanine hydroxylase (PheH) is an allosteric enzyme that requires activation by phenylalanine for full activity. The location of the allosteric site for phenylalanine has not been established. NMR spectroscopy of the isolated regulatory domain (RDPheH(25–117) is the regulatory domain of PheH lacking residues 1–24) of the rat enzyme in the presence of phenylalanine is consistent with formation of a side-by-side ACT dimer. Six residues in RDPheH(25–117) were identified as being in the phenylalanine-binding site on the basis of intermolecular NOEs between unlabeled phenylalanine and isotopically labeled protein. The location of these residues is consistent with two allosteric sites per dimer, with each site containing residues from both monomers. Site-specific variants of five of the residues (E44Q, A47G, L48V, L62V, and H64N) decreased the affinity of RDPheH(25–117) for phenylalanine based on the ability to stabilize the dimer. Incorporation of the A47G, L48V, and H64N mutations into the intact protein increased the concentration of phenylalanine required for activation. The results identify the location of the allosteric site as the interface of the regulatory domain dimer formed in activated PheH. PMID:26823465

The N-terminal strand modulates immunoglobulin light chain fibrillogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pozo-Yauner, Luis del, E-mail: ldelpozo@inmegen.gob.mx; Wall, Jonathan S.; González Andrade, Martín

2014-01-10

Highlights: •We evaluated the impact of mutations in the N-terminal strand of 6aJL2 protein. •Mutations destabilized the protein in a position-dependent manner. •Destabilizing mutations accelerated the fibrillogenesis by shortening the lag time. •The effect on the kinetic of fibril elongation by seeding was of different nature. •The N-terminal strand is buried in the fibrillar state of 6aJL2 protein. -- Abstract: It has been suggested that the N-terminal strand of the light chain variable domain (V{sub L}) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stabilitymore » and kinetic of fibrillogenesis of the V{sub L} protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein.« less

Roles of N-terminal fatty acid acylations in membrane compartment partitioning: Arabidopsis h-type thioredoxins as a case study.

PubMed

Traverso, José A; Micalella, Chiara; Martinez, Aude; Brown, Spencer C; Satiat-Jeunemaître, Béatrice; Meinnel, Thierry; Giglione, Carmela

2013-03-01

N-terminal fatty acylations (N-myristoylation [MYR] and S-palmitoylation [PAL]) are crucial modifications affecting 2 to 4% of eukaryotic proteins. The role of these modifications is to target proteins to membranes. Predictive tools have revealed unexpected targets of these acylations in Arabidopsis thaliana and other plants. However, little is known about how N-terminal lipidation governs membrane compartmentalization of proteins in plants. We show here that h-type thioredoxins (h-TRXs) cluster in four evolutionary subgroups displaying strictly conserved N-terminal modifications. It was predicted that one subgroup undergoes only MYR and another undergoes both MYR and PAL. We used plant TRXs as a model protein family to explore the effect of MYR alone or MYR and PAL in the same family of proteins. We used a high-throughput biochemical strategy to assess MYR of specific TRXs. Moreover, various TRX-green fluorescent protein fusions revealed that MYR localized protein to the endomembrane system and that partitioning between this membrane compartment and the cytosol correlated with the catalytic efficiency of the N-myristoyltransferase acting at the N terminus of the TRXs. Generalization of these results was obtained using several randomly selected Arabidopsis proteins displaying a MYR site only. Finally, we demonstrated that a palmitoylatable Cys residue flanking the MYR site is crucial to localize proteins to micropatching zones of the plasma membrane.

Crystal Structures of Apo and Metal-Bound Forms of the UreE Protein from Helicobacter pylori: Role of Multiple Metal Binding Sites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Rong; Munger, Christine; Asinas, Abdalin

2010-10-22

The crystal structure of the urease maturation protein UreE from Helicobacter pylori has been determined in its apo form at 2.1 {angstrom} resolution, bound to Cu{sup 2+} at 2.7 {angstrom} resolution, and bound to Ni{sup 2+} at 3.1 {angstrom} resolution. Apo UreE forms dimers, while the metal-bound enzymes are arranged as tetramers that consist of a dimer of dimers associated around the metal ion through coordination by His102 residues from each subunit of the tetramer. Comparison of independent subunits from different crystal forms indicates changes in the relative arrangement of the N- and C-terminal domains in response to metal binding.more » The improved ability of engineered versions of UreE containing hexahistidine sequences at either the N-terminal or C-terminal end to provide Ni{sup 2+} for the final metal sink (urease) is eliminated in the H102A version. Therefore, the ability of the improved Ni{sup 2+}-binding versions to deliver more nickel is likely an effect of an increased local concentration of metal ions that can rapidly replenish transferred ions bound to His102.« less

Half-of-the-sites reactivity of outer-membrane phospholipase A against an active-site-directed inhibitor.

PubMed

Ubarretxena-Belandia, I; Cox, R C; Dijkman, R; Egmond, M R; Verheij, H M; Dekker, N

1999-03-01

The reaction of a novel active-site-directed phospholipase A1 inhibitor with the outer-membrane phospholipase A (OMPLA) was investigated. The inhibitor 1-p-nitrophenyl-octylphosphonate-2-tridecylcarbamoyl-3-et hanesulfonyl -amino-3-deoxy-sn-glycerol irreversibly inactivated OMPLA. The inhibition reaction did not require the cofactor calcium or an unprotonated active-site His142. The inhibition of the enzyme solubilized in hexadecylphosphocholine micelles was characterized by a rapid (t1/2 = 20 min) and complete loss of enzymatic activity, concurrent with the covalent modification of 50% of the active-site serines, as judged from the amount of p-nitrophenolate (PNP) released. Modification of the remaining 50% occurred at a much lower rate, indicative of half-of-the-sites reactivity against the inhibitor of this dimeric enzyme. Inhibition of monomeric OMPLA solubilized in hexadecyl-N,N-dimethyl-1-ammonio-3-propanesulfonate resulted in an equimolar monophasic release of PNP, concurrent with the loss of enzymatic activity (t1/2 = 14 min). The half-of-the-sites reactivity is discussed in view of the dimeric nature of this enzyme.

Two Drosophila chorion genes terminate transcription in discrete regions near their poly(A) sites.

PubMed Central

Osheim, Y N; Miller, O L; Beyer, A L

1986-01-01

We have examined transcription termination of two closely linked Drosophila melanogaster chorion genes, s36-1 and s38-1, using the electron microscope. Our method is unusual and is independent of in vitro nuclear run-on transcription. By measuring transcription unit lengths in chromatin spreads, we can localize efficient termination sites to a region of approximately 210 bp for s36-1 and approximately 365 bp for s38-1. The center of this region is approximately 105 nucleotides downstream of the poly(A) site for the s36-1 gene, and approximately 400 nucleotides downstream for the s38-1 gene. Thus, these two Drosophila chorion genes terminate more closely to their poly(A) addition sites and in a shorter region than many other polyadenylated genes examined to date. Images Fig. 1. Fig. 2. PMID:3104029

In vitro resolution of the dimer bridge of the minute virus of mice (MVM) genome supports the modified rolling hairpin model for MVM replication.

PubMed

Liu, Q; Yong, C B; Astell, C R

1994-06-01

Previous characterization of the terminal sequences of the minute virus of mice (MVM) genome demonstrated that the right hand palindrome contains two sequences, each the inverted complement of the other. However, the left hand palindrome was shown to exist as a unique sequence [Astell et al., J. Virol. 54: 179-185 (1985)]. The modified rolling hairpin (MRH) model for MVM replication provided an explanation of how the right hand palindrome could undergo hairpin transfer to generate two sequences, while the left end palindrome within the dimer bridge could undergo asymmetric resolution and retain the unique left end sequence. This report describes in vitro resolution of the wild-type dimer bridge sequence of MVM using recombinant (baculovirus) expressed NS-1 and a replication extract from LA9 cells. The resolution products are consistent with those predicted by the MRH model, providing support for this replication mechanism. In addition, mutant dimer bridge clones were constructed and used in the resolution assay. The mutant structures included removal of the asymmetry in the hairpin stem, inversion of the sequence at the initiating nick site, and a 2-bp deletion within one stem of the dimer bridge. In all cases, the mutant dimer bridge structures are resolved; however, the resolution pattern observed with the mutant dimer bridge compared with the wild-type dimer bridge is shifted toward symmetrical resolution. These results suggest that sequences within the left hand hairpin (and hence dimer bridge sequence) are responsible for asymmetric resolution and conservation of the unique sequence within the left hand palindrome of the MVM genome.

Identification and Functional Characterization of N-Terminally Acetylated Proteins in Drosophila melanogaster

PubMed Central

Gerrits, Bertran; Roschitzki, Bernd; Mohanty, Sonali; Niederer, Eva M.; Laczko, Endre; Timmerman, Evy; Lange, Vinzenz; Hafen, Ernst; Aebersold, Ruedi; Vandekerckhove, Joël; Basler, Konrad; Ahrens, Christian H.; Gevaert, Kris; Brunner, Erich

2009-01-01

Protein modifications play a major role for most biological processes in living organisms. Amino-terminal acetylation of proteins is a common modification found throughout the tree of life: the N-terminus of a nascent polypeptide chain becomes co-translationally acetylated, often after the removal of the initiating methionine residue. While the enzymes and protein complexes involved in these processes have been extensively studied, only little is known about the biological function of such N-terminal modification events. To identify common principles of N-terminal acetylation, we analyzed the amino-terminal peptides from proteins extracted from Drosophila Kc167 cells. We detected more than 1,200 mature protein N-termini and could show that N-terminal acetylation occurs in insects with a similar frequency as in humans. As the sole true determinant for N-terminal acetylation we could extract the (X)PX rule that indicates the prevention of acetylation under all circumstances. We could show that this rule can be used to genetically engineer a protein to study the biological relevance of the presence or absence of an acetyl group, thereby generating a generic assay to probe the functional importance of N-terminal acetylation. We applied the assay by expressing mutated proteins as transgenes in cell lines and in flies. Here, we present a straightforward strategy to systematically study the functional relevance of N-terminal acetylations in cells and whole organisms. Since the (X)PX rule seems to be of general validity in lower as well as higher eukaryotes, we propose that it can be used to study the function of N-terminal acetylation in all species. PMID:19885390

Magnetic and superconducting competition within the Hubbard dimer. Exact solution

NASA Astrophysics Data System (ADS)

Matlak, M.; Somska, T.; Grabiec, B.

2005-02-01

We express the Hubbard dimer Hamiltonian in the second quantization with theuse of the Hubbard and spin operators. We consider the case of positive and negative U. We decompose the resulting Hamiltonian into several parts collecting all the terms belonging to the same energy level. Such a decomposition visualizes explicitely all intrinsic interactions competing together and deeply hidden in the original form of the dimer Hamiltonian. Among them are competitive ferromagnetic and antiferromagnetic interactions. There are also hopping terms present which describe Cooper pairs hopping between sites 1 and 2 with positive and negative coupling constants (similar as in Kulik-Pedan, Penson-Kolb models). We show that the competition between intrinsic interactions strongly depends on the model parametrs and the averaged occupation number of electrons n [0, 4] resulting in different regimes of the model (as e.g. t-J model regime, etc.).

Identification of the WW domain-interaction sites in the unstructured N-terminal domain of EBV LMP 2A.

PubMed

Seo, Min-Duk; Park, Sung Jean; Kim, Hyun-Jung; Lee, Bong Jin

2007-01-09

Epstein-Barr virus latency is maintained by the latent membrane protein (LMP) 2A, which mimics the B-cell receptor (BCR) and perturbs BCR signaling. The cytoplasmic N-terminal domain of LMP2A is composed of 119 amino acids. The N-terminal domain of LMP2A (LMP2A NTD) contains two PY motifs (PPPPY) that interact with the WW domains of Nedd4 family ubiquitin-protein ligases. Based on our analysis of NMR data, we found that the LMP2A NTD adopts an overall random-coil structure in its native state. However, the region between residues 60 and 90 was relatively ordered, and seemed to form the hydrophobic core of the LMP2A NTD. This region resides between two PY motifs and is important for WW domain binding. Mapping of the residues involved in the interaction between the LMP2A NTD and WW domains was achieved by chemical shift perturbation, by the addition of WW2 and WW3 peptides. Interestingly, the binding of the WW domains mainly occurred in the hydrophobic core of the LMP2A NTD. In addition, we detected a difference in the binding modes of the two PY motifs against the two WW peptides. The binding of the WW3 peptide caused the resonances of five residues (Tyr(60), Glu(61), Asp(62), Trp(65), and Gly(66)) just behind the N-terminal PY motif of the LMP2A NTD to disappear. A similar result was obtained with WW2 binding. However, near the C-terminal PY motif, the chemical shift perturbation caused by WW2 binding was different from that due to WW3 binding, indicating that the residues near the PY motifs are involved in selective binding of WW domains. The present work represents the first structural study of the LMP2A NTD and provides fundamental structural information about its interaction with ubiquitin-protein ligase.

Structure of the N-terminal fragment of Escherichia coli Lon protease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Mi; Basic Research Program, SAIC-Frederick, Frederick, MD 21702; Gustchina, Alla

2010-08-01

The medium-resolution structure of the N-terminal fragment of E. coli Lon protease shows that this part of the enzyme consists of two compact domains and a very long α-helix. The structure of a recombinant construct consisting of residues 1–245 of Escherichia coli Lon protease, the prototypical member of the A-type Lon family, is reported. This construct encompasses all or most of the N-terminal domain of the enzyme. The structure was solved by SeMet SAD to 2.6 Å resolution utilizing trigonal crystals that contained one molecule in the asymmetric unit. The molecule consists of two compact subdomains and a very longmore » C-terminal α-helix. The structure of the first subdomain (residues 1–117), which consists mostly of β-strands, is similar to that of the shorter fragment previously expressed and crystallized, whereas the second subdomain is almost entirely helical. The fold and spatial relationship of the two subdomains, with the exception of the C-terminal helix, closely resemble the structure of BPP1347, a 203-amino-acid protein of unknown function from Bordetella parapertussis, and more distantly several other proteins. It was not possible to refine the structure to satisfactory convergence; however, since almost all of the Se atoms could be located on the basis of their anomalous scattering the correctness of the overall structure is not in question. The structure reported here was also compared with the structures of the putative substrate-binding domains of several proteins, showing topological similarities that should help in defining the binding sites used by Lon substrates.« less

Crystal Structure of the Pseudomonas aeruginosa Virulence Factor Regulator

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cordes, Timothy J.; Worzalla, Gregory A.; Ginster, Aaron M.

2012-09-07

Virulence factor regulator (Vfr) enhances Pseudomonas aeruginosa pathogenicity through its role as a global transcriptional regulator. The crystal structure of Vfr shows that it is a winged-helix DNA-binding protein like its homologue cyclic AMP receptor protein (CRP). In addition to an expected primary cyclic AMP-binding site, a second ligand-binding site is nestled between the N-terminal domain and the C-terminal helix-turn-helix domain. Unlike CRP, Vfr is a symmetric dimer in the absence of DNA. Removal of seven disordered N-terminal residues of Vfr prvents the growth of P. aeruginosa.

[Correlation between thermostability of the xylanase EvXyn11(TS) and its N-terminal disulfide bridge].

PubMed

Min, Rou; Li, Jianfang; Gao, Shujuan; Zhang, Huimin; Wu, Jing; Wu, Minchen

2013-04-04

To reveal the correlation between thermostability of xylanase EvXyn11(TS) and its N-terminal disulfide bridge, an EvXyn11(TS)-encoding gene (Syxyn11) was synthesized and subjected to site-directed mutagenesis. Multiple homology alignment of protein primary structures between the EvXyn11(TS) and several GH family 11 xylanases displayed that, in their N-termini, only EvXyn11(TS) contained a disulfide bridge (Cys5-Cys32), whose effect on the xylanase thermostability was predicted by molecular dynamics simulation. We constructed a gene Syxyn11(M), encoding the mutated xylanase (EvXyn11(M)) without N-terminal disulfide bridge. Then, Syxyn11 and Syxyn11(M) were expressed in Pichia pastoris GS115, and temperature and pH properties of the expressed enzymes were analyzed. The analytical results displayed that the temperature optimum of EvXyn11(M) was 70 degrees C, which was 15 degrees C lower than that of EvXyn11(TS). The half-life (t1/2(90)) of EvXyn11(TS) at 90 degrees C was 32 min, while the t1/2(70) of EvXyn11(M) at 70 degrees C was only 8.0 min. The important role of the N-terminal disulfide bridge on the thermostability of EvXyn11(TS) was first predicted by molecular dynamics simulation, and confirmed by site-directed mutagenesis. This work provided a novel strategy to improve thermostabilities of the mesophilic family 11 xylanases with high specific activities.

Analytical study of avian reticuloendotheliosis virus dimeric RNA generated in vivo and in vitro.

PubMed

Darlix, J L; Gabus, C; Allain, B

1992-12-01

The retroviral genome consists of two identical RNA molecules associated at their 5' ends by a stable structure called the dimer linkage structure. The dimer linkage structure, while maintaining the dimer state of the retroviral genome, might also be involved in packaging and reverse transcription, as well as recombination during proviral DNA synthesis. To study the dimer structure of the retroviral genome and the mechanism of dimerization, we analyzed features of the dimeric genome of reticuloendotheliosis virus (REV) type A and identified elements required for its dimerization. Here we report that the REV dimeric genome extracted from virions and infected cells, as well as that synthesized in vitro, is more resistant to heat denaturation than avian sarcoma and leukemia virus, murine leukemia virus, or human immunodeficiency virus type 1 dimeric RNA. The minimal domain required to form a stable REV RNA dimer in vitro was found to map between positions 268 and 452 (KpnI and SalI sites), thus corresponding to the E encapsidation sequence (J. E. Embretson and H. M. Temin, J. Virol. 61:2675-2683, 1987). In addition, both the 5' and 3' halves of E are necessary in cis for RNA dimerization and the extent of RNA dimerization is influenced by viral sequences flanking E. Rapid and efficient dimerization of REV RNA containing gag sequences in addition to the E sequences and annealing of replication primer tRNA(Pro) to the primer-binding site necessitate the nucleocapsid protein.

Analytical study of avian reticuloendotheliosis virus dimeric RNA generated in vivo and in vitro.

PubMed Central

Darlix, J L; Gabus, C; Allain, B

1992-01-01

The retroviral genome consists of two identical RNA molecules associated at their 5' ends by a stable structure called the dimer linkage structure. The dimer linkage structure, while maintaining the dimer state of the retroviral genome, might also be involved in packaging and reverse transcription, as well as recombination during proviral DNA synthesis. To study the dimer structure of the retroviral genome and the mechanism of dimerization, we analyzed features of the dimeric genome of reticuloendotheliosis virus (REV) type A and identified elements required for its dimerization. Here we report that the REV dimeric genome extracted from virions and infected cells, as well as that synthesized in vitro, is more resistant to heat denaturation than avian sarcoma and leukemia virus, murine leukemia virus, or human immunodeficiency virus type 1 dimeric RNA. The minimal domain required to form a stable REV RNA dimer in vitro was found to map between positions 268 and 452 (KpnI and SalI sites), thus corresponding to the E encapsidation sequence (J. E. Embretson and H. M. Temin, J. Virol. 61:2675-2683, 1987). In addition, both the 5' and 3' halves of E are necessary in cis for RNA dimerization and the extent of RNA dimerization is influenced by viral sequences flanking E. Rapid and efficient dimerization of REV RNA containing gag sequences in addition to the E sequences and annealing of replication primer tRNA(Pro) to the primer-binding site necessitate the nucleocapsid protein. Images PMID:1331519

pH-Responsive Dimeric Zinc(II) Phthalocyanine in Mesoporous Silica Nanoparticles as an Activatable Nanophotosensitizing System for Photodynamic Therapy.

PubMed

Wong, Roy C H; Chow, Sun Y S; Zhao, Shirui; Fong, Wing-Ping; Ng, Dennis K P; Lo, Pui-Chi

2017-07-19

An acid-cleavable acetal-linked zinc(II) phthalocyanine dimer with an azido terminal group (cPc) was prepared and conjugated to alkyne-modified mesoporous silica nanoparticles via copper(I)-catalyzed alkyne-azide cycloaddition reaction. For comparison, an amine-linked analogue (nPc) was also prepared as a non-acid-cleavable counterpart. These dimeric phthalocyanines were significantly self-quenched due to the close proximity of the phthalocyanine units inside the mesopores, resulting in much weaker fluorescence emission and singlet oxygen generation, both in N,N-dimethylformamide and in phosphate-buffered saline (PBS), compared with the free molecular counterparts. Under acidic conditions in PBS, the cPc-encapsulated nanosystem was activated in terms of fluorescence emission and singlet oxygen production. After internalization into human colon adenocarcinoma HT29 cells, it exhibited much higher intracellular fluorescence and photocytotoxicity compared to the nanosystem entrapped with nPc. The activation of this nanosystem was also demonstrated in tumor-bearing nude mice. The intratumoral fluorescence intensity increased gradually over 24 h, while for the nPc counterpart the fluorescence remained very weak. The results suggest that this nanosystem serves as a promising activatable nanophotosensitizing agent for photodynamic therapy.

Structural Properties of Human IAPP Dimer in Membrane Environment Studied by All-Atom Molecular Dynamics Simulations.

PubMed

Liu, Na; Duan, Mojie; Yang, Minghui

2017-08-11

The aggregation of human islet amyloid polypeptide (hIAPP) can damage the membrane of the β-cells in the pancreatic islets and induce type 2 diabetes (T2D). Growing evidences indicated that the major toxic species are small oligomers of IAPP. Due to the fast aggregation nature, it is hard to characterize the structures of IAPP oligomers by experiments, especially in the complex membrane environment. On the other side, molecular dynamics simulation can provide atomic details of the structure and dynamics of the aggregation of IAPP. In this study, all-atom bias-exchange metadynamics (BE-Meta) and unbiased molecular dynamics simulations were employed to study the structural properties of IAPP dimer in the membranes environments. A number of intermediates, including α-helical states, β-sheet states, and fully disordered states, are identified. The formation of N-terminal β-sheet structure is prior to the C-terminal β-sheet structure towards the final fibril-like structures. The α-helical intermediates have lower propensity in the dimeric hIAPP and are off-pathway intermediates. The simulations also demonstrate that the β-sheet intermediates induce more perturbation on the membrane than the α-helical and disordered states and thus pose higher disruption ability.

N-Terminal Acetylation Inhibits Protein Targeting to the Endoplasmic Reticulum

PubMed Central

Forte, Gabriella M. A.; Pool, Martin R.; Stirling, Colin J.

2011-01-01

Amino-terminal acetylation is probably the most common protein modification in eukaryotes with as many as 50%–80% of proteins reportedly altered in this way. Here we report a systematic analysis of the predicted N-terminal processing of cytosolic proteins versus those destined to be sorted to the secretory pathway. While cytosolic proteins were profoundly biased in favour of processing, we found an equal and opposite bias against such modification for secretory proteins. Mutations in secretory signal sequences that led to their acetylation resulted in mis-sorting to the cytosol in a manner that was dependent upon the N-terminal processing machinery. Hence N-terminal acetylation represents an early determining step in the cellular sorting of nascent polypeptides that appears to be conserved across a wide range of species. PMID:21655302

A first-principles study of electronic properties of H and F-terminated zigzag BNC nanoribbons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alaal, Naresh; Department of Physics, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India.; Department of Materials Engineering, Monash University, Clayton, Victoria -3800, Australia.

2016-05-06

Nanoribbons are quasi one-dimensional structures which have interesting electronic properties on the basis of their edge geometries, and width. We studied the electronic properties of hydrogen and fluorine-terminated zigzag BNC nanoribbons (BNCNRs) using a first-principles based density functional theory approach. We considered BNCNRs that were composed of an equal number of C-C and B-N dimers; one of the edges ends with an N atom and opposite edge ends with a C atom. These two edge atoms are passivated by H or F atoms. Our results suggest that hydrogen-terminated BNCNRs (H-BNCNRs) and flourine-terminated BNCNRs (F-BNCNRs) have different electronic properties. H-BNCNRs exhibitmore » intrinsic half-metallic behavior while F-BNCNRs are indirect band gap semiconductors. Chemical functionalization of BNCNRs with H and F atoms show that BNCNRs have a diverse range of electronic properties.« less

UNC-45/CRO1/She4p (UCS) Protein Forms Elongated Dimer and Joins Two Myosin Heads Near Their Actin Binding Region

DOE Office of Scientific and Technical Information (OSTI.GOV)

H Shi; G Blobel

2011-12-31

UNC-45/CRO1/She4p (UCS) proteins have variously been proposed to affect the folding, stability, and ATPase activity of myosins. They are the only proteins known to interact directly with the motor domain. To gain more insight into UCS function, we determined the atomic structure of the yeast UCS protein, She4p, at 2.9 {angstrom} resolution. We found that 16 helical repeats are organized into an L-shaped superhelix with an amphipathic N-terminal helix dangling off the short arm of the L-shaped molecule. In the crystal, She4p forms a 193-{angstrom}-long, zigzag-shaped dimer through three distinct and evolutionary conserved interfaces. We have identified She4p's C-terminal regionmore » as a ligand for a 27-residue-long epitope on the myosin motor domain. Remarkably, this region consists of two adjacent, but distinct, binding epitopes localized at the nucleotide-responsive cleft between the nucleotide- and actin-filament-binding sites. One epitope is situated inside the cleft, the other outside the cleft. After ATP hydrolysis and Pi ejection, the cleft narrows at its base from 20 to 12 {angstrom} thereby occluding the inside the cleft epitope, while leaving the adjacent, outside the cleft binding epitope accessible to UCS binding. Hence, one cycle of higher and lower binding affinity would accompany one ATP hydrolysis cycle and a single step in the walk on an actin filament rope. We propose that a UCS dimer links two myosins at their motor domains and thereby functions as one of the determinants for step size of myosin on actin filaments.« less

D-dimer test

MedlinePlus

... vein thrombosis - D-dimer; Pulmonary embolism - D-dimer; Blood clot to the lungs - D-dimer ... dimer test if you are showing symptoms of blood clots, such as: Swelling, pain, warmth, and changes in ...

Positive selection in the N-terminal extramembrane domain of lung surfactant protein C (SP-C) in marine mammals.

PubMed

Foot, Natalie J; Orgeig, Sandra; Donnellan, Stephen; Bertozzi, Terry; Daniels, Christopher B

2007-07-01

Maximum-likelihood models of codon and amino acid substitution were used to analyze the lung-specific surfactant protein C (SP-C) from terrestrial, semi-aquatic, and diving mammals to identify lineages and amino acid sites under positive selection. Site models used the nonsynonymous/synonymous rate ratio (omega) as an indicator of selection pressure. Mechanistic models used physicochemical distances between amino acid substitutions to specify nonsynonymous substitution rates. Site models strongly identified positive selection at different sites in the polar N-terminal extramembrane domain of SP-C in the three diving lineages: site 2 in the cetaceans (whales and dolphins), sites 7, 9, and 10 in the pinnipeds (seals and sea lions), and sites 2, 9, and 10 in the sirenians (dugongs and manatees). The only semi-aquatic contrast to indicate positive selection at site 10 was that including the polar bear, which had the largest body mass of the semi-aquatic species. Analysis of the biophysical properties that were influential in determining the amino acid substitutions showed that isoelectric point, chemical composition of the side chain, polarity, and hydrophobicity were the crucial determinants. Amino acid substitutions at these sites may lead to stronger binding of the N-terminal domain to the surfactant phospholipid film and to increased adsorption of the protein to the air-liquid interface. Both properties are advantageous for the repeated collapse and reinflation of the lung upon diving and resurfacing and may reflect adaptations to the high hydrostatic pressures experienced during diving.

A strategy for complex dimer formation when biomimicry fails: total synthesis of ten coccinellid alkaloids.

PubMed

Sherwood, Trevor C; Trotta, Adam H; Snyder, Scott A

2014-07-09

Although dimeric natural products can often be synthesized in the laboratory by directly merging advanced monomers, these approaches sometimes fail, leading instead to non-natural architectures via incorrect unions. Such a situation arose during our studies of the coccinellid alkaloids, when attempts to directly dimerize Nature's presumed monomeric precursors in a putative biomimetic sequence afforded only a non-natural analogue through improper regiocontrol. Herein, we outline a unique strategy for dimer formation that obviates these difficulties, one which rapidly constructs the coccinellid dimers psylloborine A and isopsylloborine A through a terminating sequence of two reaction cascades that generate five bonds, five rings, and four stereocenters. In addition, a common synthetic intermediate is identified which allows for the rapid, asymmetric formal or complete total syntheses of eight monomeric members of the class.

Ionic interaction of myosin loop 2 with residues located beyond the N-terminal part of actin probed by chemical cross-linking.

PubMed

Pliszka, Barbara; Martin, Brian M; Karczewska, Emilia

2008-02-01

To probe ionic contacts of skeletal muscle myosin with negatively charged residues located beyond the N-terminal part of actin, myosin subfragment 1 (S1) and actin split by ECP32 protease (ECP-actin) were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). We have found that unmodified S1 can be cross-linked not only to the N-terminal part, but also to the C-terminal 36 kDa fragment of ECP-actin. Subsequent experiments performed on S1 cleaved by elastase or trypsin indicate that the cross-linking site in S1 is located within loop 2. This site is composed of Lys-636 and Lys-637 and can interact with negatively charged residues of the 36 kDa actin fragment, most probably with Glu-99 and Glu-100. Cross-links are formed both in the absence and presence of MgATP.P(i) analog, although the addition of nucleotide decreases the efficiency of the cross-linking reaction.

α-SNAP regulates dynamic, on-site assembly and calcium selectivity of Orai1 channels

PubMed Central

Li, Peiyao; Miao, Yong; Dani, Adish; Vig, Monika

2016-01-01

Orai1 forms a highly calcium-selective pore of the calcium release activated channel, and α-SNAP is necessary for its function. Here we show that α-SNAP regulates on-site assembly of Orai1 dimers into calcium-selective multimers. We find that Orai1 is a dimer in resting primary mouse embryonic fibroblasts but displays variable stoichiometry in the plasma membrane of store-depleted cells. Remarkably, α-SNAP depletion induces formation of higher-order Orai1 oligomers, which permeate significant levels of sodium via Orai1 channels. Sodium permeation in α-SNAP–deficient cells cannot be corrected by tethering multiple Stim1 domains to Orai1 C-terminal tail, demonstrating that α-SNAP regulates functional assembly and calcium selectivity of Orai1 multimers independently of Stim1 levels. Fluorescence nanoscopy reveals sustained coassociation of α-SNAP with Stim1 and Orai1, and α-SNAP–depleted cells show faster and less constrained mobility of Orai1 within ER-PM junctions, suggesting Orai1 and Stim1 coentrapment without stable contacts. Furthermore, α-SNAP depletion significantly reduces fluorescence resonance energy transfer between Stim1 and Orai1 N-terminus but not C-terminus. Taken together, these data reveal a unique role of α-SNAP in the on-site functional assembly of Orai1 subunits and suggest that this process may, in part, involve enabling crucial low-affinity interactions between Orai1 N-terminus and Stim1. PMID:27335124

Functional identification of a novel 14-3-3 epsilon splicing variant suggests dimerization is not necessary for 14-3-3 epsilon to inhibit UV-induced apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Dingding; Ye, Guangming; Liu, Tingting

2010-05-28

14-3-3 proteins function as a dimer and have been identified to involve in diverse signaling pathways. Here we reported the identification of a novel splicing variant of human 14-3-3 epsilon (14-3-3 epsilon sv), which is derived from a novel exon 1' insertion. The insertion contains a stop codon and leads to a truncated splicing variant of 14-3-3 epsilon. The splicing variant is translated from the exon 2 and results in the deletion of an N-terminal {alpha}-helix which is crucial for the dimerization. Therefore, the 14-3-3 epsilon sv could not form a dimer with 14-3-3 zeta. However, after UV irradiation 14-3-3more » epsilon sv could also support cell survival, suggesting monomer of 14-3-3 epsilon is sufficient to protect cell from apoptosis.« less

Dimerization of BTas is required for the transactivational activity of bovine foamy virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan Juan; Qiao Wentao; Xu Fengwen

2008-06-20

The BTas protein of bovine foamy virus (BFV) is a 249-amino-acid nuclear regulatory protein which transactivates viral gene expression directed by the long terminal repeat promoter (LTR) and the internal promoter (IP). Here, we demonstrate the BTas protein forms a dimeric complex in mammalian cells by using mammalian two hybrid systems and cross-linking assay. Functional analyses with deletion mutants reveal that the region of 46-62aa is essential for dimer formation. Furthermore, our results show that deleting the dimerization region of BTas did not affect the localization of BTas, but that it did result in the loss of its transactivational activitymore » on the LTR and IP. Furthermore, BTas ({delta}46-62aa) retained binding ability to the LTR and IP similar to that of the wild-type BTas. These data suggest the dimerization region is necessary for the transactivational function of BTas and is crucial to the replication of BFV.« less

Photochemical pathways of the dimeric, mixed dimer, and monomeric sulfophthalocyanines of cobalt(III) and iron(II)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ferraudi, G.

1979-04-01

The photochemical reactivity of the dimeric, mixed dimer, and monomeric sulfophthalocyanines of cobalt (III) and iron (II) was investigated by steady-state and flash irradiations. The dimeric species photodissociated into sulfophthalocyanine radicals which were coordinated to either Co(III) or Fe(II) metal centers. Reactions of such intermediates were investigated by interception with alcohols and O/sub 2/. Also, photoredox reactions were detected with monomeric acidocobalt(III) sulfophtahlocyanines. These processes produce the oxidation of the acido ligands (Cl/sup -/, Br/sup -/, N/sub 3//sup -/, I/sup -/) and the reduction of the metal center. The photoredox dissociation was also investigated by using mixed dimers of themore » cobalt sulfophthalocyanines with Cr(bpy)/sub 3//sup 3 +/ and Ru(bpy)/sub 3//sup 2 +/. The photogeneration of sulfophthalocyanine radicals was observed as a general reaction which was produced by excitation of either the Cr(bby)/sub 3//sup 3 +/ or Ru(bpy)/sub 3//sup 2 +/ units in the mixed dimer. The nature of the reactive excited states involved in the various photochemical reactions of the sulfophthalocyanines of Co(II), Co(III), Cu(II), and Fe(II) is discussed.« less

N-terminal acetylation modulates Bax targeting to mitochondria.

PubMed

Alves, Sara; Neiri, Leire; Chaves, Susana Rodrigues; Vieira, Selma; Trindade, Dário; Manon, Stephen; Dominguez, Veronica; Pintado, Belen; Jonckheere, Veronique; Van Damme, Petra; Silva, Rui Duarte; Aldabe, Rafael; Côrte-Real, Manuela

2018-02-01

The pro-apoptotic Bax protein is the main effector of mitochondrial permeabilization during apoptosis. Bax is controlled at several levels, including post-translational modifications such as phosphorylation and S-palmitoylation. However, little is known about the contribution of other protein modifications to Bax activity. Here, we used heterologous expression of human Bax in yeast to study the involvement of N-terminal acetylation by yNaa20p (yNatB) on Bax function. We found that human Bax is N-terminal (Nt-)acetylated by yNaa20p and that Nt-acetylation of Bax is essential to maintain Bax in an inactive conformation in the cytosol of yeast and Mouse Embryonic Fibroblast (MEF) cells. Bax accumulates in the mitochondria of yeast naa20Δ and Naa25 -/- MEF cells, but does not promote cytochrome c release, suggesting that an additional step is required for full activation of Bax. Altogether, our results show that Bax N-terminal acetylation by NatB is involved in its mitochondrial targeting. Copyright © 2017 Elsevier Ltd. All rights reserved.

Crosslinking Evidence for Motional Constraints within Chemoreceptor Trimers of Dimers

PubMed Central

Massazza, Diego A.; Parkinson, John S.; Studdert, Claudia A.

2011-01-01

Chemotactic behavior in bacteria relies on the sensing ability of large chemoreceptor clusters that are usually located at the cell pole. In E. coli, chemoreceptors show higher order interactions within those clusters based on a trimer-of-dimers organization. This architecture is conserved in a variety of other bacteria and archaea, implying that receptors in many microorganisms form trimer of dimer signaling teams. To gain further insight into the assembly and dynamic behavior of receptor trimers of dimers, we used in vivo crosslinking targeted to cysteine residues at various positions that define six different levels along the cytoplasmic signaling domains of the aspartate and serine chemoreceptors, Tar and Tsr. We found that the cytoplasmic domains of these receptors are close to each other near the trimer contact region at the cytoplasmic tip and lie farther apart as the receptor dimers approach the cytoplasmic membrane. Tar and Tsr reporter sites within the same or closely adjacent levels readily formed mixed crosslinks, whereas reporters lying at different distances from the tip did not. These findings indicate that there are no significant vertical displacements of one dimer with respect to the others within the trimer unit. Attractant stimuli had no discernable effect on the crosslinking efficiency of any of the reporters tested, but a strong osmotic stimulus reproducibly enhanced crosslinking at most of the reporter sites, indicating that individual dimers may move closer together under this condition. PMID:21174433

Conformational Heterogeneity of Bax Helix 9 Dimer for Apoptotic Pore Formation

NASA Astrophysics Data System (ADS)

Liao, Chenyi; Zhang, Zhi; Kale, Justin; Andrews, David W.; Lin, Jialing; Li, Jianing

2016-07-01

Helix α9 of Bax protein can dimerize in the mitochondrial outer membrane (MOM) and lead to apoptotic pores. However, it remains unclear how different conformations of the dimer contribute to the pore formation on the molecular level. Thus we have investigated various conformational states of the α9 dimer in a MOM model — using computer simulations supplemented with site-specific mutagenesis and crosslinking of the α9 helices. Our data not only confirmed the critical membrane environment for the α9 stability and dimerization, but also revealed the distinct lipid-binding preference of the dimer in different conformational states. In our proposed pathway, a crucial iso-parallel dimer that mediates the conformational transition was discovered computationally and validated experimentally. The corroborating evidence from simulations and experiments suggests that, helix α9 assists Bax activation via the dimer heterogeneity and interactions with specific MOM lipids, which eventually facilitate proteolipidic pore formation in apoptosis regulation.

Synthesis and cure mechanism characterization of phenylethynyl-terminated imide oligomers

NASA Astrophysics Data System (ADS)

Back, Susanna Branion

Polymer matrix composites (PMC) are being investigated by the Air Force for use in engine applications, which are comprised of an intermediate carbon fiber-reinforced high temperature phenylethynyl-terminated fluorinated polyimide resin. The high-temperature phenylethynyl-terminated fluorinated polyimide resin is prepared from 4-(phenylethynyl)phthalic anhydride (4-PEPA), p-phenylenediamine (p-PDA), and hexafluoroisopropylidene bisphthalic dianhydride (6FDA). In order for these materials to be exploited to their full potential, many fundamental aspects of the polymer system need to be better understood. For this study, the high-temperature cure mechanism, the chemical structures of cure reaction products, and the effect of physical properties of impurities in the 4-PEPA monomer were investigated. A phenylethynyl-terminated imide model compound, N-(3-phenoxybenzene)-4-phenylethynylphthalimide (N-PBPEP), was prepared using both industrial 4-PEPA and recrystallized 4-PEPA. The thermal cure of this low molecular weight compound was studied using a variety of analytical techniques including differential scanning calorimetry, Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR), and mass spectrometry (MS). N-PBPEP made from industrial 4-PEPA begins curing 25°C later than N-PBPEP made from recrystallized 4-PEPA, indicating that thermal cure is affected by the purity of the 4-PEPA starting material. Also, both versions form an 830 g/mol dimer with three possible mechanistic pathways.

Rapid Reversion from Monomer to Dimer Regenerates the Ultraviolet-B Photoreceptor UV RESISTANCE LOCUS8 in Intact Arabidopsis Plants1[W][OA

PubMed Central

Heilmann, Monika; Jenkins, Gareth I.

2013-01-01

Arabidopsis (Arabidopsis thaliana) UV RESISTANCE LOCUS8 (UVR8) is a photoreceptor that specifically mediates photomorphogenic responses to ultraviolet (UV)-B in plants. UV-B photoreception induces the conversion of the UVR8 dimer into a monomer that interacts with the CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) protein to regulate gene expression. However, it is not known how the dimeric photoreceptor is regenerated in plants. Here, we show, by using inhibitors of protein synthesis and degradation via the proteasome, that the UVR8 dimer is not regenerated by rapid de novo synthesis following destruction of the monomer. Rather, regeneration occurs by reversion from the monomer to the dimer. However, regeneration of dimeric UVR8 in darkness following UV-B exposure occurs much more rapidly in vivo than in vitro with illuminated plant extracts or purified UVR8, indicating that rapid regeneration requires intact cells. Rapid dimer regeneration in vivo requires protein synthesis, the presence of a carboxyl-terminal 27-amino acid region of UVR8, and the presence of COP1, which is known to interact with the carboxyl-terminal region. However, none of these factors can account fully for the difference in regeneration kinetics in vivo and in vitro, indicating that additional proteins or processes are involved in UVR8 dimer regeneration in vivo. PMID:23129206

Roles of N-Terminal Fatty Acid Acylations in Membrane Compartment Partitioning: Arabidopsis h-Type Thioredoxins as a Case Study[C][W

PubMed Central

Traverso, José A.; Micalella, Chiara; Martinez, Aude; Brown, Spencer C.; Satiat-Jeunemaître, Béatrice; Meinnel, Thierry; Giglione, Carmela

2013-01-01

N-terminal fatty acylations (N-myristoylation [MYR] and S-palmitoylation [PAL]) are crucial modifications affecting 2 to 4% of eukaryotic proteins. The role of these modifications is to target proteins to membranes. Predictive tools have revealed unexpected targets of these acylations in Arabidopsis thaliana and other plants. However, little is known about how N-terminal lipidation governs membrane compartmentalization of proteins in plants. We show here that h-type thioredoxins (h-TRXs) cluster in four evolutionary subgroups displaying strictly conserved N-terminal modifications. It was predicted that one subgroup undergoes only MYR and another undergoes both MYR and PAL. We used plant TRXs as a model protein family to explore the effect of MYR alone or MYR and PAL in the same family of proteins. We used a high-throughput biochemical strategy to assess MYR of specific TRXs. Moreover, various TRX–green fluorescent protein fusions revealed that MYR localized protein to the endomembrane system and that partitioning between this membrane compartment and the cytosol correlated with the catalytic efficiency of the N-myristoyltransferase acting at the N terminus of the TRXs. Generalization of these results was obtained using several randomly selected Arabidopsis proteins displaying a MYR site only. Finally, we demonstrated that a palmitoylatable Cys residue flanking the MYR site is crucial to localize proteins to micropatching zones of the plasma membrane. PMID:23543785

Renton's Quendall Terminals on List of EPA Superfund Sites Targeted for Immediate, Intense Attention

EPA Pesticide Factsheets

EPA released the list of Superfund sites that Administrator Pruitt has targeted for intense and immediate attention, including the Quendall Terminals Site, a former creosote facility on the shore of Lake Washington in Renton, Washington.

Dimer interface of bovine cytochrome c oxidase is influenced by local posttranslational modifications and lipid binding

PubMed Central

Liko, Idlir; Degiacomi, Matteo T.; Mohammed, Shabaz; Yoshikawa, Shinya; Schmidt, Carla; Robinson, Carol V.

2016-01-01

Bovine cytochrome c oxidase is an integral membrane protein complex comprising 13 protein subunits and associated lipids. Dimerization of the complex has been proposed; however, definitive evidence for the dimer is lacking. We used advanced mass spectrometry methods to investigate the oligomeric state of cytochrome c oxidase and the potential role of lipids and posttranslational modifications in its subunit interfaces. Mass spectrometry of the intact protein complex revealed that both the monomer and the dimer are stabilized by large lipid entities. We identified these lipid species from the purified protein complex, thus implying that they interact specifically with the enzyme. We further identified phosphorylation and acetylation sites of cytochrome c oxidase, located in the peripheral subunits and in the dimer interface, respectively. Comparing our phosphorylation and acetylation sites with those found in previous studies of bovine, mouse, rat, and human cytochrome c oxidase, we found that whereas some acetylation sites within the dimer interface are conserved, suggesting a role for regulation and stabilization of the dimer, phosphorylation sites were less conserved and more transient. Our results therefore provide insights into the locations and interactions of lipids with acetylated residues within the dimer interface of this enzyme, and thereby contribute to a better understanding of its structure in the natural membrane. Moreover dimeric cytochrome c oxidase, comprising 20 transmembrane, six extramembrane subunits, and associated lipids, represents the largest integral membrane protein complex that has been transferred via electrospray intact into the gas phase of a mass spectrometer, representing a significant technological advance. PMID:27364008

Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner

DOE Office of Scientific and Technical Information (OSTI.GOV)

Berbís, M. Álvaro; André, Sabine; Cañada, F. Javier

2014-01-03

Highlights: •Galectin-3 is composed of a carbohydrate recognition domain and an N-terminal tail. •Synthetic peptides derived from the tail are shown to interact with the CRD. •This interaction is modulated by Ser- and Tyr-phosphorylation of the peptides. -- Abstract: Galectin-3 (Gal-3) is a multi-functional effector protein that functions in the cytoplasm and the nucleus, as well as extracellularly following non-classical secretion. Structurally, Gal-3 is unique among galectins with its carbohydrate recognition domain (CRD) attached to a rather long N-terminal tail composed mostly of collagen-like repeats (nine in the human protein) and terminating in a short non-collagenous terminal peptide sequence uniquemore » in this lectin family and not yet fully explored. Although several Ser and Tyr sites within the N-terminal tail can be phosphorylated, the physiological significance of this post-translational modification remains unclear. Here, we used a series of synthetic (phospho)peptides derived from the tail to assess phosphorylation-mediated interactions with {sup 15}N-labeled Gal-3 CRD. HSQC-derived chemical shift perturbations revealed selective interactions at the backface of the CRD that were attenuated by phosphorylation of Tyr 107 and Tyr 118, while phosphorylation of Ser 6 and Ser 12 was essential. Controls with sequence scrambling underscored inherent specificity. Our studies shed light on how phosphorylation of the N-terminal tail may impact on Gal-3 function and prompt further studies using phosphorylated full-length protein.« less

SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) N-terminal tyrosine residues regulate a dynamic signaling equilibrium involving feedback of proximal T-cell receptor (TCR) signaling.

PubMed

Ji, Qinqin; Ding, Yiyuan; Salomon, Arthur R

2015-01-01

SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is a cytosolic adaptor protein that plays an important role in the T-cell receptor-mediated T-cell signaling pathway. SLP-76 links proximal receptor stimulation to downstream effectors through interaction with many signaling proteins. Previous studies showed that mutation of three tyrosine residues, Tyr(112), Tyr(128), and Tyr(145), in the N terminus of SLP-76 results in severely impaired phosphorylation and activation of Itk and PLCγ1, which leads to defective calcium mobilization, Erk activation, and NFAT activation. To expand our knowledge of the role of N-terminal phosphorylation of SLP-76 from these three tyrosine sites, we characterized nearly 1000 tyrosine phosphorylation sites via mass spectrometry in SLP-76 reconstituted wild-type cells and SLP-76 mutant cells in which three tyrosine residues were replaced with phenylalanines (Y3F mutant). Mutation of the three N-terminal tyrosine residues of SLP-76 phenocopied SLP-76-deficient cells for the majority of tyrosine phosphorylation sites observed, including feedback on proximal T-cell receptor signaling proteins. Meanwhile, reversed phosphorylation changes were observed on Tyr(192) of Lck when we compared mutants to the complete removal of SLP-76. In addition, N-terminal tyrosine sites of SLP-76 also perturbed phosphorylation of Tyr(440) of Fyn, Tyr(702) of PLCγ1, Tyr(204), Tyr(397), and Tyr(69) of ZAP-70, revealing new modes of regulation on these sites. All these findings confirmed the central role of N-terminal tyrosine sites of SLP-76 in the pathway and also shed light on novel signaling events that are uniquely regulated by SLP-76 N-terminal tyrosine residues. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

SRC Homology 2 Domain-containing Leukocyte Phosphoprotein of 76 kDa (SLP-76) N-terminal Tyrosine Residues Regulate a Dynamic Signaling Equilibrium Involving Feedback of Proximal T-cell Receptor (TCR) Signaling*

PubMed Central

Ji, Qinqin; Ding, Yiyuan; Salomon, Arthur R.

2015-01-01

SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is a cytosolic adaptor protein that plays an important role in the T-cell receptor–mediated T-cell signaling pathway. SLP-76 links proximal receptor stimulation to downstream effectors through interaction with many signaling proteins. Previous studies showed that mutation of three tyrosine residues, Tyr112, Tyr128, and Tyr145, in the N terminus of SLP-76 results in severely impaired phosphorylation and activation of Itk and PLCγ1, which leads to defective calcium mobilization, Erk activation, and NFAT activation. To expand our knowledge of the role of N-terminal phosphorylation of SLP-76 from these three tyrosine sites, we characterized nearly 1000 tyrosine phosphorylation sites via mass spectrometry in SLP-76 reconstituted wild-type cells and SLP-76 mutant cells in which three tyrosine residues were replaced with phenylalanines (Y3F mutant). Mutation of the three N-terminal tyrosine residues of SLP-76 phenocopied SLP-76-deficient cells for the majority of tyrosine phosphorylation sites observed, including feedback on proximal T-cell receptor signaling proteins. Meanwhile, reversed phosphorylation changes were observed on Tyr192 of Lck when we compared mutants to the complete removal of SLP-76. In addition, N-terminal tyrosine sites of SLP-76 also perturbed phosphorylation of Tyr440 of Fyn, Tyr702 of PLCγ1, Tyr204, Tyr397, and Tyr69 of ZAP-70, revealing new modes of regulation on these sites. All these findings confirmed the central role of N-terminal tyrosine sites of SLP-76 in the pathway and also shed light on novel signaling events that are uniquely regulated by SLP-76 N-terminal tyrosine residues. PMID:25316710

EDEM1 targets misfolded HLA-B27 dimers for endoplasmic reticulum associated degradation

PubMed Central

Guiliano, David B.; Fussell, Helen; Lenart, Izabela; Tsao, Edward; Nesbeth, Darren; Fletcher, Adam J.; Campbell, Elaine C.; Yousaf, Nasim; Williams, Sarah; Santos, Susana; Cameron, Amy; Towers, Greg J.; Kellam, Paul; Hebert, Daniel N.; Gould, Keith; Powis, Simon J.; Antoniou, Antony N.

2015-01-01

Objective HLA-B27 forms misfolded heavy chain dimers, which may predispose individuals to inflammatory arthritis by inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). We wanted to define the role of the UPR induced ER associated degradation (ERAD) pathway in the disposal of HLA-B27 dimeric conformers. Methods HeLa cell lines expressing only two copies of a carboxy terminally Sv5 tagged HLA-B27 were generated. The ER stress induced EDEM1 protein was over expressed by transfection and dimer levels monitored by immunoblotting. EDEM1, the UPR associated transcription factor XBP-1, the E3 ubiquitin ligase HRD1, the degradation associated derlin 1 and 2 proteins were inhibited by either short hairpin RNA or dominant negative mutants. The UPR associated ERAD of HLA-B27 was confirmed using ER stress inducing pharamacological agents in kinetic and pulse chase assays. Results We demonstrate that UPR induced machinery can target HLA-B27 dimers, and that dimer formation can be controlled by alterations to expression levels of components of the UPR induced ERAD pathway. HLA-B27 dimers and misfolded MHC class I monomeric molecules were detected bound to EDEM1, with overexpression of EDEM1 inhibiting HLA-B27 dimer formation. EDEM1 inhibition resulted in upregulation of HLA-B27 dimers, whilst UPR induced ERAD of dimers was prevented in the absence of EDEM1. HLA-B27 dimer formation was also enhanced in the absence of XBP-1, HRD1 and derlin1/2. Conclusion The UPR ERAD pathway as described here can dispose of HLA-B27 dimers and presents a potential novel therapeutic target for the modulation of HLA-B27 associated inflammatory disease. PMID:25132672

Properties of the two-dimensional heterogeneous Lennard-Jones dimers: An integral equation study

PubMed Central

Urbic, Tomaz

2016-01-01

Structural and thermodynamic properties of a planar heterogeneous soft dumbbell fluid are examined using Monte Carlo simulations and integral equation theory. Lennard-Jones particles of different sizes are the building blocks of the dimers. The site-site integral equation theory in two dimensions is used to calculate the site-site radial distribution functions and the thermodynamic properties. Obtained results are compared to Monte Carlo simulation data. The critical parameters for selected types of dimers were also estimated and the influence of the Lennard-Jones parameters was studied. We have also tested the correctness of the site-site integral equation theory using different closures. PMID:27875894

Non-active site mutations disturb the loop dynamics, dimerization, viral budding and egress of VP40 of the Ebola virus.

PubMed

Balmith, Marissa; Soliman, Mahmoud E S

2017-02-28

The first account of the dynamic features of the loop region of VP40 of the Ebola virus (EboV) using accelerated molecular dynamics (aMD) simulations is reported herein. Due to its major role in the Ebola life cycle, VP40 is considered a promising therapeutic target. The available experimental data on the N-terminal domain (NTD) loop indicates that mutations K127A, T129A and N130A demonstrate an unrecognized role for NTD-plasma membrane (PM) interaction for efficient VP40-PM localization, oligomerization, matrix assembly and egress. Despite experimental results, the molecular description of VP40 and the information it can provide still remain vague. Therefore, to gain further molecular insight into the effect of mutations on the loop region of VP40 and its effects on the overall protein conformation and VP40 dimerization, aMD simulations and post-dynamic analyses were employed for wildtype (WT) and mutant systems. The results showed significant variations in the presence of mutations as per RMSF, RMSD, R g , PCA and distance calculations in comparison to the WT. These results could provide researchers with insight with regards to the conformational aspects concerning VP40 and its close relation to the experimental data. We believe that the results presented in this study will ultimately provide a useful understanding of the structural landscape of the loop region of VP40, which would contribute towards the discovery of novel EboV inhibitors.

Functional characterization of the dimerization domain of the ferric uptake regulator (Fur) of Pseudomonas aeruginosa

PubMed Central

Bai, Erdeni; Rosell, Federico I.; Lige, Bao; Mauk, Marcia R.; Lelj-Garolla, Barbara; Moore, Geoffrey R.; Mauk, A. Grant

2006-01-01

The functional properties of the recombinant C-terminal dimerization domain of the Pseudomonas aeruginosa Fur (ferric uptake regulator) protein expressed in and purified from Escherichia coli have been evaluated. Sedimentation velocity measurements demonstrate that this domain is dimeric, and the UV CD spectrum is consistent with a secondary structure similar to that observed for the corresponding region of the crystallographically characterized wild-type protein. The thermal stability of the domain as determined by CD spectroscopy decreases significantly as pH is increased and increases significantly as metal ions are added. Potentiometric titrations (pH 6.5) establish that the domain possesses a high-affinity and a low-affinity binding site for metal ions. The high-affinity (sensory) binding site demonstrates association constants (KA) of 10(±7)×106, 5.7(±3)×106, 2.0(±2)×106 and 2.0(±3)×104 M−1 for Ni2+, Zn2+, Co2+ and Mn2+ respectively, while the low-affinity (structural) site exhibits association constants of 1.3(±2)×106, 3.2(±2)×104, 1.76(±1)×105 and 1.5(±2)×103 M−1 respectively for the same metal ions (pH 6.5, 300 mM NaCl, 25 °C). The stability of metal ion binding to the sensory site follows the Irving–Williams order, while metal ion binding to the partial sensory site present in the domain does not. Fluorescence experiments indicate that the quenching resulting from binding of Co2+ is reversed by subsequent titration with Zn2+. We conclude that the domain is a reasonable model for many properties of the full-length protein and is amenable to some analyses that the limited solubility of the full-length protein prevents. PMID:16928194

Dimer with gain and loss: Integrability and {P}{T}-symmetry restoration

NASA Astrophysics Data System (ADS)

Barashenkov, I. V.; Pelinovsky, D. E.; Dubard, P.

2015-08-01

A {P}{T}-symmetric nonlinear Schrödinger dimer is a two-site discrete nonlinear Schrödinger equation with one site losing and the other one gaining energy at the same rate. In this paper, two four-parameter families of cubic {P}{T}-symmetric dimers are constructed as gain-loss extensions of their conservative, Hamiltonian, counterparts. We prove that all these damped-driven equations define completely integrable Hamiltonian systems. The second aim of our study is to identify nonlinearities that give rise to the spontaneous {P}{T}-symmetry restoration. When the symmetry of the underlying linear dimer is broken and an unstable small perturbation starts to grow, the nonlinear coupling of the required type will divert an increasingly large percentage of energy from the gaining to the losing site. As a result, the exponential growth will be saturated and all trajectories remain trapped in a finite part of the phase space regardless of the value of the gain-loss coefficient.

Actin-induced dimerization of palladin promotes actin-bundling

PubMed Central

Vattepu, Ravi; Yadav, Rahul; Beck, Moriah R

2015-01-01

A subset of actin binding proteins is able to form crosslinks between two or more actin filaments, thus producing structures of parallel or networked bundles. These actin crosslinking proteins interact with actin through either bivalent binding or dimerization. We recently identified two binding sites within the actin binding domain of palladin, an actin crosslinking protein that plays an important role in normal cell adhesion and motility during wound healing and embryonic development. In this study, we show that actin induces dimerization of palladin. Furthermore, the extent of dimerization reflects earlier comparisons of actin binding and bundling between different domains of palladin. On the basis of these results we hypothesized that actin binding may promote a conformational change that results in dimerization of palladin, which in turn may drive the crosslinking of actin filaments. The proximal distance between two actin binding sites on crosslinking proteins determines the ultrastructural properties of the filament network, therefore we also explored interdomain interactions using a combination of chemical crosslinking experiments and actin cosedimentation assays. Limited proteolysis data reveals that palladin is less susceptible to enzyme digestion after actin binding. Our results suggest that domain movements in palladin are necessary for interactions with actin and are induced by interactions with actin filaments. Accordingly, we put forth a model linking the structural changes to functional dynamics. PMID:25307943

Structure of the Reston ebolavirus VP30 C-terminal domain.

PubMed

Clifton, Matthew C; Kirchdoerfer, Robert N; Atkins, Kateri; Abendroth, Jan; Raymond, Amy; Grice, Rena; Barnes, Steve; Moen, Spencer; Lorimer, Don; Edwards, Thomas E; Myler, Peter J; Saphire, Erica Ollmann

2014-04-01

The ebolaviruses can cause severe hemorrhagic fever. Essential to the ebolavirus life cycle is the protein VP30, which serves as a transcriptional cofactor. Here, the crystal structure of the C-terminal, NP-binding domain of VP30 from Reston ebolavirus is presented. Reston VP30 and Ebola VP30 both form homodimers, but the dimeric interfaces are rotated relative to each other, suggesting subtle inherent differences or flexibility in the dimeric interface.

The N-terminal strand modulates immunoglobulin light chain fibrillogenesis.

PubMed

del Pozo-Yauner, Luis; Wall, Jonathan S; González Andrade, Martín; Sánchez-López, Rosana; Rodríguez-Ambriz, Sandra L; Pérez Carreón, Julio I; Ochoa-Leyva, Adrián; Fernández-Velasco, D Alejandro

2014-01-10

It has been suggested that the N-terminal strand of the light chain variable domain (V(L)) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stability and kinetic of fibrillogenesis of the V(L) protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein. Copyright © 2013 Elsevier Inc. All rights reserved.

Open and Lys–His Hexacoordinated Closed Structures of a Globin with Swapped Proximal and Distal Sites

PubMed Central

Teh, Aik-Hong; Saito, Jennifer A.; Najimudin, Nazalan; Alam, Maqsudul

2015-01-01

Globins are haem-binding proteins with a conserved fold made up of α-helices and can possess diverse properties. A putative globin-coupled sensor from Methylacidiphilum infernorum, HGbRL, contains an N-terminal globin domain whose open and closed structures reveal an untypical dimeric architecture. Helices E and F fuse into an elongated helix, resulting in a novel site-swapped globin fold made up of helices A–E, hence the distal site, from one subunit and helices F–H, the proximal site, from another. The open structure possesses a large cavity binding an imidazole molecule, while the closed structure forms a unique Lys–His hexacoordinated species, with the first turn of helix E unravelling to allow Lys52(E10) to bind to the haem. Ligand binding induces reorganization of loop CE, which is stabilized in the closed form, and helix E, triggering a large conformational movement in the open form. These provide a mechanical insight into how a signal may be relayed between the globin domain and the C-terminal domain of HGbRL, a Roadblock/LC7 domain. Comparison with HGbI, a closely related globin, further underlines the high degree of structural versatility that the globin fold is capable of, enabling it to perform a diversity of functions. PMID:26094577

Cloning and characterization of full-length mouse thymidine kinase 2: the N-terminal sequence directs import of the precursor protein into mitochondria.

PubMed Central

Wang, L; Eriksson, S

2000-01-01

The subcellular localization of mitochondrial thymidine kinase (TK2) has been questioned, since no mitochondrial targeting sequences have been found in cloned human TK2 cDNAs. Here we report the cloning of mouse TK2 cDNA from a mouse full-length enriched cDNA library. The mouse TK2 cDNA codes for a protein of 270 amino acids, with a 40-amino-acid presumed N-terminal mitochondrial targeting signal. In vitro translation and translocation experiments with purified rat mitochondria confirmed that the N-terminal sequence directed import of the precursor TK2 into the mitochondrial matrix. A single 2.4 kb mRNA transcript was detected in most tissues examined, except in liver, where an additional shorter (1.0 kb) transcript was also observed. There was no correlation between the tissue distribution of TK2 activity and the expression of TK2 mRNA. Full-length mouse TK2 protein and two N-terminally truncated forms, one of which corresponds to the mitochondrial form of TK2 and a shorter form corresponding to the previously characterized recombinant human TK2, were expressed in Escherichia coli and affinity purified. All three forms of TK2 phosphorylated thymidine, deoxycytidine and 2'-deoxyuridine, but with different kinetic efficiencies. A number of cytostatic pyrimidine nucleoside analogues were also tested and shown to be good substrates for the various forms of TK2. The active form of full-length mouse TK2 was a dimer, as judged by Superdex 200 chromatography. These results enhance our understanding of the structure and function of TK2, and may help to explain the mitochondrial disorder, mitochondrial neurogastrointestinal encephalomyopathy. PMID:11023833

Naturally occurring disulfide-bound dimers of three-fingered toxins: a paradigm for biological activity diversification.

PubMed

Osipov, Alexey V; Kasheverov, Igor E; Makarova, Yana V; Starkov, Vladislav G; Vorontsova, Olga V; Ziganshin, Rustam Kh; Andreeva, Tatyana V; Serebryakova, Marina V; Benoit, Audrey; Hogg, Ronald C; Bertrand, Daniel; Tsetlin, Victor I; Utkin, Yuri N

2008-05-23

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of alpha-cobratoxin (a long-chain alpha-neurotoxin) and heterodimers formed by alpha-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26-30 in polypeptide loop II of alpha-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the alpha-cobratoxin capacity to compete with alpha-bungarotoxin for binding to Torpedo and alpha7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that alpha-cobratoxin dimer not only interacts with alpha7 nAChR but, in contrast to alpha-cobratoxin monomer, also blocks alpha3beta2 nAChR. In the latter activity it resembles kappa-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric alpha3beta2 nAChRs.

NMR assignments of the N-terminal domain of Nephila clavipes spidroin 1

PubMed Central

Parnham, Stuart; Gaines, William A.; Duggan, Brendan M.; Marcotte, William R.

2011-01-01

The building blocks of spider dragline silk are two fibrous proteins secreted from the major ampullate gland named spidroins 1 and 2 (MaSp1, MaSp2). These proteins consist of a large central domain composed of approximately 100 tandem copies of a 35–40 amino acid repeat sequence. Non-repetitive N and C-terminal domains, of which the C-terminal domain has been implicated to transition from soluble and insoluble states during spinning, flank the repetitive core. The N-terminal domain until recently has been largely unknown due to difficulties in cloning and expression. Here, we report nearly complete assignment for all 1H, 13C, and 15N resonances in the 14 kDa N-terminal domain of major ampullate spidroin 1 (MaSp1-N) of the golden orb-web spider Nephila clavipes. PMID:21152998

Asymptotics of the monomer-dimer model on two-dimensional semi-infinite lattices

NASA Astrophysics Data System (ADS)

Kong, Yong

2007-05-01

By using the asymptotic theory of Pemantle and Wilson [R. Pemantle and M. C. Wilson, J. Comb. Theory, Ser. AJCBTA70097-316510.1006/jcta.2001.3201 97, 129 (2002)], asymptotic expansions of the free energy of the monomer-dimer model on two-dimensional semi-infinite ∞×n lattices in terms of dimer density are obtained for small values of n , at both high- and low-dimer-density limits. In the high-dimer-density limit, the theoretical results confirm the dependence of the free energy on the parity of n , a result obtained previously by computational methods by Y. Kong [Y. Kong, Phys. Rev. EPLEEE81063-651X10.1103/PhysRevE.74.061102 74, 061102 (2006); Phys. Rev. EPLEEE81063-651X10.1103/PhysRevE.73.016106 73, 016106 (2006);Phys. Rev. EPLEEE81063-651X10.1103/PhysRevE.74.011102 74, 011102 (2006)]. In the low-dimer-density limit, the free energy on a cylinder ∞×n lattice strip has exactly the same first n terms in the series expansion as that of an infinite ∞×∞ lattice.

The Escherichia coli thioredoxin homolog YbbN/Trxsc is a chaperone and a weak protein oxidoreductase.

PubMed

Caldas, Thérèse; Malki, Abderrahim; Kern, Renée; Abdallah, Jad; Richarme, Gilbert

2006-05-12

Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, which presents a strong homology in its N-terminal part with thioredoxin 1 and 2. YbbN, however, does not possess the canonical Cys-x-x-Cys active site of thioredoxins, but instead a Ser-x-x-Cys site. In addition to Cys-38, located in the SxxC site, it contains a second cysteine, Cys-63, close to Cys-38 in the 3D model. Cys-38 and Cys-63 undergo an oxidoreduction process, suggesting that YbbN functions with two redox cysteines. Accordingly, YbbN catalyzes the oxidation of reduced RNase and the isomerization of scrambled RNase. Moreover, upon oxidation, its oligomeric state changes from dimers to tetramers and higher oligomers. YbbN also possesses chaperone properties, promoting protein folding after urea denaturation and forming complexes with unfolded proteins. This is the first biochemical characterization of a member of the YbbN class of bacterial thioredoxin-like proteins, and in vivo experiments will allow to determine the importance of its redox and chaperone properties in the cellular physiology.

The Staphylococcus aureus pSK41 plasmid-encoded ArtA protein is a master regulator of plasmid transmission genes and contains a RHH motif used in alternate DNA-binding modes.

PubMed

Ni, Lisheng; Jensen, Slade O; Ky Tonthat, Nam; Berg, Tracey; Kwong, Stephen M; Guan, Fiona H X; Brown, Melissa H; Skurray, Ronald A; Firth, Neville; Schumacher, Maria A

2009-11-01

Plasmids harbored by Staphylococcus aureus are a major contributor to the spread of bacterial multi-drug resistance. Plasmid conjugation and partition are critical to the dissemination and inheritance of such plasmids. Here, we demonstrate that the ArtA protein encoded by the S. aureus multi-resistance plasmid pSK41 is a global transcriptional regulator of pSK41 genes, including those involved in conjugation and segregation. ArtA shows no sequence homology to any structurally characterized DNA-binding protein. To elucidate the mechanism by which it specifically recognizes its DNA site, we obtained the structure of ArtA bound to its cognate operator, ACATGACATG. The structure reveals that ArtA is representative of a new family of ribbon-helix-helix (RHH) DNA-binding proteins that contain extended, N-terminal basic motifs. Strikingly, unlike most well-studied RHH proteins ArtA binds its cognate operators as a dimer. However, we demonstrate that it is also able to recognize an atypical operator site by binding as a dimer-of-dimers and the extended N-terminal regions of ArtA were shown to be essential for this dimer-of-dimer binding mode. Thus, these data indicate that ArtA is a master regulator of genes critical for both horizontal and vertical transmission of pSK41 and that it can recognize DNA utilizing alternate binding modes.

The Staphylococcus aureus pSK41 plasmid-encoded ArtA protein is a master regulator of plasmid transmission genes and contains a RHH motif used in alternate DNA-binding modes

PubMed Central

Ni, Lisheng; Jensen, Slade O.; Ky Tonthat, Nam; Berg, Tracey; Kwong, Stephen M.; Guan, Fiona H. X.; Brown, Melissa H.; Skurray, Ronald A.; Firth, Neville; Schumacher, Maria A.

2009-01-01

Plasmids harbored by Staphylococcus aureus are a major contributor to the spread of bacterial multi-drug resistance. Plasmid conjugation and partition are critical to the dissemination and inheritance of such plasmids. Here, we demonstrate that the ArtA protein encoded by the S. aureus multi-resistance plasmid pSK41 is a global transcriptional regulator of pSK41 genes, including those involved in conjugation and segregation. ArtA shows no sequence homology to any structurally characterized DNA-binding protein. To elucidate the mechanism by which it specifically recognizes its DNA site, we obtained the structure of ArtA bound to its cognate operator, ACATGACATG. The structure reveals that ArtA is representative of a new family of ribbon–helix–helix (RHH) DNA-binding proteins that contain extended, N-terminal basic motifs. Strikingly, unlike most well-studied RHH proteins ArtA binds its cognate operators as a dimer. However, we demonstrate that it is also able to recognize an atypical operator site by binding as a dimer-of-dimers and the extended N-terminal regions of ArtA were shown to be essential for this dimer-of-dimer binding mode. Thus, these data indicate that ArtA is a master regulator of genes critical for both horizontal and vertical transmission of pSK41 and that it can recognize DNA utilizing alternate binding modes. PMID:19759211

Pancake π–π Bonding Goes Double: Unexpected 4e/All-Sites Bonding in Boron- and Nitrogen-Doped Phenalenyls

DOE PAGES

Tian, Yong-Hui; Sumpter, Bobby G.; Du, Shiyu; ...

2015-06-03

Phenalenyl is an important neutral pi-radical due to its capability to form unconventional pancake pi-pi bonding interactions, whereas its analogues with graphitic boron (B) or nitrogen (N)-doping have been regarded as closed-shell systems and therefore received much less attention. By using high-level quantum chemistry calculations, we also show that the B- and N-doped closed-shell phenalenyls unexpectedly form open-shell singlet pi-dimers with diradicaloid character featuring 2e/all-sites double pi-pi bonding. Moreover, by proper substitutions, the doped phenalenyl derivatives can be made open-shell species that form closed shell singlet pi-dimers bound by stronger 4e/all-sites double pi-pi bonding. Moreover, covalent pi-pi bonding overlap ismore » distributed on all of the atomic sites giving robust and genuine pancake-shaped pi-dimers which, depending on the number of electrons available in the bonding interactions, are equally or more stable than the pi-dimers of the pristine phenalenyl.« less

Pancake π–π Bonding Goes Double: Unexpected 4e/All-Sites Bonding in Boron- and Nitrogen-Doped Phenalenyls

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tian, Yong-Hui; Sumpter, Bobby G.; Du, Shiyu

Phenalenyl is an important neutral pi-radical due to its capability to form unconventional pancake pi-pi bonding interactions, whereas its analogues with graphitic boron (B) or nitrogen (N)-doping have been regarded as closed-shell systems and therefore received much less attention. By using high-level quantum chemistry calculations, we also show that the B- and N-doped closed-shell phenalenyls unexpectedly form open-shell singlet pi-dimers with diradicaloid character featuring 2e/all-sites double pi-pi bonding. Moreover, by proper substitutions, the doped phenalenyl derivatives can be made open-shell species that form closed shell singlet pi-dimers bound by stronger 4e/all-sites double pi-pi bonding. Moreover, covalent pi-pi bonding overlap ismore » distributed on all of the atomic sites giving robust and genuine pancake-shaped pi-dimers which, depending on the number of electrons available in the bonding interactions, are equally or more stable than the pi-dimers of the pristine phenalenyl.« less

Dimeric [Mo₂S₁₂]²⁻ Cluster: A Molecular Analogue of MoS₂ Edges for Superior Hydrogen-Evolution Electrocatalysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Zhongjie; Luo, Wenjia; Ma, Lu

2015-12-07

Proton reduction is one of the most fundamental and important reactions in nature. MoS2 edges have been identified as the active sites for hydrogen evolution reaction (HER) electrocatalysis. Designing molecular mimics of MoS2 edge sites is an attractive strategy to understand the underlying catalytic mechanism of different edge sites and improve their activities. Herein we report a dimeric molecular analogue [Mo₂S₁₂]²⁻, as the smallest unit possessing both the terminal and bridging disulfide ligands. Our electrochemical tests show that [Mo₂S₁₂]²⁻ is a superior heterogeneous HER catalyst under acidic conditions. Computations suggest that the bridging disulfide ligand of [Mo₂S₁₂]²⁻ exhibits a hydrogenmore » adsorption free energy near zero (-0.05eV). This work helps shed light on the rational design of HER catalysts and biomimetics of hydrogen-evolving enzymes.« less

The insulin and IGF1 receptor kinase domains are functional dimers in the activated state

NASA Astrophysics Data System (ADS)

Cabail, M. Zulema; Li, Shiqing; Lemmon, Eric; Bowen, Mark E.; Hubbard, Stevan R.; Miller, W. Todd

2015-03-01

The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) are highly related receptor tyrosine kinases with a disulfide-linked homodimeric architecture. Ligand binding to the receptor ectodomain triggers tyrosine autophosphorylation of the cytoplasmic domains, which stimulates catalytic activity and creates recruitment sites for downstream signalling proteins. Whether the two phosphorylated tyrosine kinase domains within the receptor dimer function independently or cooperatively to phosphorylate protein substrates is not known. Here we provide crystallographic, biophysical and biochemical evidence demonstrating that the phosphorylated kinase domains of IR and IGF1R form a specific dimeric arrangement involving an exchange of the juxtamembrane region proximal to the kinase domain. In this dimer, the active position of α-helix C in the kinase N lobe is stabilized, which promotes downstream substrate phosphorylation. These studies afford a novel strategy for the design of small-molecule IR agonists as potential therapeutic agents for type 2 diabetes.

The N-terminal domain of the mammalian nucleoporin p62 interacts with other nucleoporins of the FXFG family during interphase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stochaj, Ursula; Banski, Piotr; Kodiha, Mohamed

2006-08-01

Nuclear pore complexes (NPCs) provide the only sites for macromolecular transport between nucleus and cytoplasm. The nucleoporin p62, a component of higher eukaryotic NPCs, is located at the central gated channel and involved in nuclear trafficking of various cargos. p62 is organized into an N-terminal segment that contains FXFG repeats and binds the soluble transport factor NTF2, whereas the C-terminal portion associates with other nucleoporins and importin-{beta}1. We have now identified new components that interact specifically with the p62 N-terminal domain. Using the p62 N-terminal segment as bait, we affinity-purified nucleoporins Nup358, Nup214 and Nup153 from crude cell extracts. Inmore » ligand binding assays, the N-terminal p62 segment associated with Nup358 and p62, suggesting their direct binding to the p62 N-terminal portion. Furthermore, p62 was isolated in complex with Nup358, Nup214 and Nup153 from growing HeLa cells, indicating that the interactions Nup358/p62, Nup214/p62 and p62/Nup153 also occur in vivo. The formation of Nup358/p62 and p62/Nup153 complexes was restricted to interphase cells, whereas Nup214/p62 binding was detected in interphase as well as during mitosis. Our results support a model of complex interactions between FXFG containing nucleoporins, and we propose that some of these interactions may contribute to the movement of cargo across the NPC.« less

The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain.

PubMed Central

Lorentsen, R H; Graversen, J H; Caterer, N R; Thogersen, H C; Etzerodt, M

2000-01-01

Tetranectin is a homotrimeric plasma and extracellular-matrix protein that binds plasminogen and complex sulphated polysaccharides including heparin. In terms of primary and tertiary structure, tetranectin is related to the collectin family of Ca(2+)-binding C-type lectins. Tetranectin is encoded in three exons. Exon 3 encodes the carbohydrate recognition domain, which binds to kringle 4 in plasminogen at low levels of Ca(2+). Exon 2 encodes an alpha-helix, which is necessary and sufficient to govern the trimerization of tetranectin by assembling into a triple-helical coiled-coil structural element. Here we show that the heparin-binding site in tetranectin resides not in the carbohydrate recognition domain but within the N-terminal region, comprising the 16 amino acid residues encoded by exon 1. In particular, the lysine residues in the decapeptide segment KPKKIVNAKK (tetranectin residues 6-15) are shown to be of primary importance in heparin binding. PMID:10727405

The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain.

PubMed

Lorentsen, R H; Graversen, J H; Caterer, N R; Thogersen, H C; Etzerodt, M

2000-04-01

Tetranectin is a homotrimeric plasma and extracellular-matrix protein that binds plasminogen and complex sulphated polysaccharides including heparin. In terms of primary and tertiary structure, tetranectin is related to the collectin family of Ca(2+)-binding C-type lectins. Tetranectin is encoded in three exons. Exon 3 encodes the carbohydrate recognition domain, which binds to kringle 4 in plasminogen at low levels of Ca(2+). Exon 2 encodes an alpha-helix, which is necessary and sufficient to govern the trimerization of tetranectin by assembling into a triple-helical coiled-coil structural element. Here we show that the heparin-binding site in tetranectin resides not in the carbohydrate recognition domain but within the N-terminal region, comprising the 16 amino acid residues encoded by exon 1. In particular, the lysine residues in the decapeptide segment KPKKIVNAKK (tetranectin residues 6-15) are shown to be of primary importance in heparin binding.

Intersubunit distances in full-length, dimeric, bacterial phytochrome Agp1, as measured by pulsed electron-electron double resonance (PELDOR) between different spin label positions, remain unchanged upon photoconversion.

PubMed

Kacprzak, Sylwia; Njimona, Ibrahim; Renz, Anja; Feng, Juan; Reijerse, Edward; Lubitz, Wolfgang; Krauss, Norbert; Scheerer, Patrick; Nagano, Soshichiro; Lamparter, Tilman; Weber, Stefan

2017-05-05

Bacterial phytochromes are dimeric light-regulated histidine kinases that convert red light into signaling events. Light absorption by the N-terminal photosensory core module (PCM) causes the proteins to switch between two spectrally distinct forms, Pr and Pfr, thus resulting in a conformational change that modulates the C-terminal histidine kinase region. To provide further insights into structural details of photoactivation, we investigated the full-length Agp1 bacteriophytochrome from the soil bacterium Agrobacterium fabrum using a combined spectroscopic and modeling approach. We generated seven mutants suitable for spin labeling to enable application of pulsed EPR techniques. The distances between attached spin labels were measured using pulsed electron-electron double resonance spectroscopy to probe the arrangement of the subunits within the dimer. We found very good agreement of experimental and calculated distances for the histidine-kinase region when both subunits are in a parallel orientation. However, experimental distance distributions surprisingly showed only limited agreement with either parallel- or antiparallel-arranged dimer structures when spin labels were placed into the PCM region. This observation indicates that the arrangements of the PCM subunits in the full-length protein dimer in solution differ significantly from that in the PCM crystals. The pulsed electron-electron double resonance data presented here revealed either no or only minor changes of distance distributions upon Pr-to-Pfr photoconversion. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Arabidopsis Microtubule-Associated Protein MAP65-3 Cross-Links Antiparallel Microtubules toward Their Plus Ends in the Phragmoplast via Its Distinct C-Terminal Microtubule Binding Domain[W

PubMed Central

Ho, Chin-Min Kimmy; Lee, Yuh-Ru Julie; Kiyama, Lindsay D.; Dinesh-Kumar, Savithramma P.; Liu, Bo

2012-01-01

Plant cytokinesis is brought about by the phragmoplast, which contains an antiparallel microtubule (MT) array. The MT-associated protein MAP65-3 acts as an MT-bundling factor that specifically cross-links antiparallel MTs near their plus ends. MAP65 family proteins contain an N-terminal dimerization domain and C-terminal MT interaction domain. Compared with other MAP65 isoforms, MAP65-3 contains an extended C terminus. A MT binding site was discovered in the region between amino acids 496 and 588 and found to be essential for the organization of phragmoplast MTs. The frequent cytokinetic failure caused by loss of MAP65-3 was not rescued by ectopic expression of MAP65-1 under the control of the MAP65-3 promoter, indicating nonoverlapping functions between the two isoforms. In the presence of MAP65-3, however, ectopic MAP65-1 appeared in the phragmoplast midline. We show that MAP65-1 could acquire the function of MAP65-3 when the C terminus of MAP65-3, which contains the MT binding site, was grafted to it. Our results also show that MAP65-1 and MAP65-3 may share redundant functions in MT stabilization. Such a stabilization effect was likely brought about by MT binding and bundling. We conclude that MAP65-3 contains a distinct C-terminal MT binding site with a specific role in cross-linking antiparallel MTs toward their plus ends in the phragmoplast. PMID:22570443

Controlled n-Type Doping of Carbon Nanotube Transistors by an Organorhodium Dimer.

PubMed

Geier, Michael L; Moudgil, Karttikay; Barlow, Stephen; Marder, Seth R; Hersam, Mark C

2016-07-13

Single-walled carbon nanotube (SWCNT) transistors are among the most developed nanoelectronic devices for high-performance computing applications. While p-type SWCNT transistors are easily achieved through adventitious adsorption of atmospheric oxygen, n-type SWCNT transistors require extrinsic doping schemes. Existing n-type doping strategies for SWCNT transistors suffer from one or more issues including environmental instability, limited carrier concentration modulation, undesirable threshold voltage control, and/or poor morphology. In particular, commonly employed benzyl viologen n-type doping layers possess large thicknesses, which preclude top-gate transistor designs that underlie high-density integrated circuit layouts. To overcome these limitations, we report here the controlled n-type doping of SWCNT thin-film transistors with a solution-processed pentamethylrhodocene dimer. The charge transport properties of organorhodium-treated SWCNT thin films show consistent n-type behavior when characterized in both Hall effect and thin-film transistor geometries. Due to the molecular-scale thickness of the organorhodium adlayer, large-area arrays of top-gated, n-type SWCNT transistors are fabricated with high yield. This work will thus facilitate ongoing efforts to realize high-density SWCNT integrated circuits.

Adsorption of ethylene on Sn and In terminated Si(001) surface studied by photoelectron spectroscopy and scanning tunneling microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zimmermann, Petr, E-mail: petr.zimmermann@mff.cuni.cz; Sobotík, Pavel; Kocán, Pavel

2016-09-07

Interaction of ethylene (C{sub 2}H{sub 4}) with Si(001)-Sn-2 × 2 and Si(001)-In-2 × 2 at room temperature has been studied using core level (C 1s) X-ray photoelectron spectroscopy with synchrotron radiation and scanning tunneling microscopy. Sn and In form similar dimer chains on Si(001)2 × 1, but exhibit different interaction with ethylene. While ethylene adsorbs on top of Sn dimers of the Si(001)-Sn-2 × 2 surface, the Si(001)-In-2 × 2 surface turned out to be inert. Furthermore, the reactivity of the Sn terminated surface is found to be considerably decreased in comparison with Si(001)2 × 1. According to the proposedmore » adsorption model ethylene bonds to Sn dimers via [2 + 2] cycloaddition by interacting with their π dimer bonds. In contrast, indium dimers do not contain π bonds, which renders the In terminated Si(001) surface inert for ethylene adsorption.« less

Insight into the evolution of nidovirus endoribonuclease based on the finding that Nsp15 from porcine deltacoronavirus functions as a dimer.

PubMed

Zheng, Anjun; Shi, Yuejun; Shen, Zhou; Wang, Gang; Shi, Jiale; Xiong, Qiqi; Fang, Liurong; Xiao, Shaobo; Fu, Zhen F; Peng, Guiqing

2018-06-10

Nidovirus endoribonucleases (NendoUs) include Nsp15 from coronaviruses and Nsp11 from arteriviruses, both of which have been reported to participate in the viral replication process and in the evasion of the host immune system. Results from a previous study of coronaviruses SARS-CoV, HCoV-229E and MHV Nsp15 indicate that it mainly forms a functional hexamer, whereas Nsp11 from the arterivirus PRRSV is a dimer. Here, we found that porcine deltacoronavirus (PDCoV) Nsp15 primarily exists as dimers and monomers in vitro. Biological experiments reveal that a PDCoV Nsp15 mutant lacking the first 27 amino acids of the N-terminal domain (NTD, Asn-1-Asn-27) forms more monomers and displays decreased enzymatic activity, indicating that this region is important for its dimerization. Moreover, multiple sequence alignments and three-dimensional structural analysis indicated that the C-terminal region (His-251-Val-261) of PDCoV Nsp15 is 10 amino acids shorter and forms a shorter loop than that formed by the equivalent sequence (Gln-259-Phe-279) of SARS-CoV Nsp15. This result may explain why PDCoV Nsp15 failed to form hexamers. We speculate that NendoUs may have originated from XendoU endoribonucleases (XendoUs) forming monomers in eukaryotic cells and that NendoU from arterivirus gained ability to form dimers and that the coronavirus variants then evolved the capacity to assemble into hexamers. We further propose that PDCoV Nsp15 may be an intermediate in this evolutionary process. Our findings provide a theoretical basis for improving our understanding of NendoU evolution and offer useful clues for designing drugs and vaccines against nidoviruses. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Crystal structure of a dimeric β-diketiminate magnesium complex

PubMed Central

MacNeil, Connor S.; Johnson, Kevin R. D.; Hayes, Paul G.; Boeré, René T.

2016-01-01

The solid-state structure of a dimeric β-diketiminate magnesium(II) complex is discussed. The compound, di-μ-iodido-bis­[(­{4-amino-1,5-bis­[2,6-bis­(propan-2-yl)phen­yl]pent-3-en-2-yl­idene}aza­nido-κ2 N,N′)magnesium(II)] toluene sesquisolvate, [Mg2(C29H41N2)2I2]·1.5C7H8, crystallizes as two independent mol­ecules, each with 2/m crystallographic site symmetry, located at Wyckoff sites 2c and 2d. These have symmetry-equivalent magnesium atoms bridged by μ-iodide ligands with very similar Mg—I distances. The two Mg atoms are located slightly below (∼0.5 Å) the least-squares plane defined by N–C—C–N atoms in the ligand scaffold, and are approximately tetra­hedrally coordinated. One and one-half toluene solvent mol­ecules are disordered with respect to mirror-site symmetry at Wyckoff sites 4i and 2a, respectively. In the former case, two toluene mol­ecules inter­act in an off-center parallel stacking arrangement; the shortest C to C′ (π–π) distance of 3.72 (1) Å was measured for this inter­action. PMID:27980823

Mechanism for controlling the monomer-dimer conversion of SARS coronavirus main protease.

PubMed

Wu, Cheng Guo; Cheng, Shu Chun; Chen, Shiang Chuan; Li, Juo Yan; Fang, Yi Hsuan; Chen, Yau Hung; Chou, Chi Yuan

2013-05-01

The Severe acute respiratory syndrome coronavirus (SARS-CoV) main protease (M(pro)) cleaves two virion polyproteins (pp1a and pp1ab); this essential process represents an attractive target for the development of anti-SARS drugs. The functional unit of M(pro) is a homodimer and each subunit contains a His41/Cys145 catalytic dyad. Large amounts of biochemical and structural information are available on M(pro); nevertheless, the mechanism by which monomeric M(pro) is converted into a dimer during maturation still remains poorly understood. Previous studies have suggested that a C-terminal residue, Arg298, interacts with Ser123 of the other monomer in the dimer, and mutation of Arg298 results in a monomeric structure with a collapsed substrate-binding pocket. Interestingly, the R298A mutant of M(pro) shows a reversible substrate-induced dimerization that is essential for catalysis. Here, the conformational change that occurs during substrate-induced dimerization is delineated by X-ray crystallography. A dimer with a mutual orientation of the monomers that differs from that of the wild-type protease is present in the asymmetric unit. The presence of a complete substrate-binding pocket and oxyanion hole in both protomers suggests that they are both catalytically active, while the two domain IIIs show minor reorganization. This structural information offers valuable insights into the molecular mechanism associated with substrate-induced dimerization and has important implications with respect to the maturation of the enzyme.

Analysis of the Intrinsically Disordered N-Terminus of the DNA Junction-Resolving Enzyme T7 Endonuclease I: Identification of Structure Formed upon DNA Binding

PubMed Central

2016-01-01

The four-way (Holliday) DNA junction of homologous recombination is processed by the symmetrical cleavage of two strands by a nuclease. These junction-resolving enzymes bind to four-way junctions in dimeric form, distorting the structure of the junction in the process. Crystal structures of T7 endonuclease I have been determined as free protein, and the complex with a DNA junction. In neither crystal structure was the N-terminal 16-amino acid peptide visible, yet deletion of this peptide has a marked effect on the resolution process. Here we have investigated the N-terminal peptide by inclusion of spin-label probes at unique sites within this region, studied by electron paramagnetic resonance. Continuous wave experiments show that these labels are mobile in the free protein but become constrained on binding a DNA junction, with the main interaction occurring for residues 7–10 and 12. Distance measurements between equivalent positions within the two peptides of a dimer using PELDOR showed that the intermonomeric distances for residues 2–12 are long and broadly distributed in the free protein but are significantly shortened and become more defined on binding to DNA. These results suggest that the N-terminal peptides become more organized on binding to the DNA junction and nestle into the minor grooves at the branchpoint, consistent with the biochemical data indicating an important role in the resolution process. This study demonstrates the presence of structure within a protein region that cannot be viewed by crystallography. PMID:27387136

Participation of the extracellular domain in (pro)renin receptor dimerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suzuki-Nakagawa, Chiharu; Nishimura, Misa; Tsukamoto, Tomoko

Highlights: • The (pro)renin receptor [(P)RR] is a regulator of the renin–angiotensin system. • The region responsible for (P)RR dimerization was investigated. • (P)RR extracellular domain constructs were retained intracellularly. • The extracellular domain of (P)RR is responsible for its dimerization. • Novel insight into the regulatory mechanism of soluble (P)RR secretion is provided. - Abstract: The (pro)renin receptor [(P)RR] induces the catalytic activation of prorenin, as well as the activation of the mitogen-activated protein kinase (MAPK) signaling pathway; as such, it plays an important regulatory role in the renin–angiotensin system. (P)RR is known to form a homodimer, but themore » region participating in its dimerization is unknown. Using glutathione S-transferase (GST) as a carrier protein and a GST pull-down assay, we investigated the interaction of several (P)RR constructs with full-length (FL) (P)RR in mammalian cells. GST fusion proteins with FL (P)RR (GST-FL), the C-terminal M8-9 fragment (GST-M8-9), the extracellular domain (ECD) of (P)RR (GST-ECD), and the (P)RR ECD with a deletion of 32 amino acids encoded by exon 4 (GST-ECDd4) were retained intracellularly, whereas GST alone was efficiently secreted into the culture medium when transiently expressed in COS-7 cells. Immunofluorescence microscopy showed prominent localization of GST-ECD to the endoplasmic reticulum. The GST pull-down analysis revealed that GST-FL, GST-ECD, and GST-ECDd4 bound FLAG-tagged FL (P)RR, whereas GST-M8-9 showed little or no binding when transiently co-expressed in HEK293T cells. Furthermore, pull-down analysis using His-tag affinity resin showed co-precipitation of soluble (P)RR with FL (P)RR from a stable CHO cell line expressing FL h(P)RR with a C-terminal decahistidine tag. These results indicate that the (P)RR ECD participates in dimerization.« less

Tubulin Dimer Reversible Dissociation

PubMed Central

Schuck, Peter; Sackett, Dan L.

2016-01-01

Tubulins are evolutionarily conserved proteins that reversibly polymerize and direct intracellular traffic. Of the tubulin family only αβ-tubulin forms stable dimers. We investigated the monomer-dimer equilibrium of rat brain αβ-tubulin using analytical ultracentrifugation and fluorescence anisotropy, observing tubulin in virtually fully monomeric and dimeric states. Monomeric tubulin was stable for a few hours and exchanged into preformed dimers, demonstrating reversibility of dimer dissociation. Global analysis combining sedimentation velocity and fluorescence anisotropy yielded Kd = 84 (54–123) nm. Dimer dissociation kinetics were measured by analyzing the shape of the sedimentation boundary and by the relaxation of fluorescence anisotropy following rapid dilution of labeled tubulin, yielding koff in the range 10−3–10−2 s−1. Thus, tubulin dimers reversibly dissociate with moderately fast kinetics. Monomer-monomer association is much less sensitive than dimer-dimer association to solution changes (GTP/GDP, urea, and trimethylamine oxide). PMID:26934918

How different is the borazine-acetylene dimer from the benzene-acetylene dimer? A matrix isolation infrared and ab initio quantum chemical study

NASA Astrophysics Data System (ADS)

Verma, Kanupriya; Viswanathan, K. S.; Majumder, Moumita; Sathyamurthy, N.

2017-11-01

The 1:1 dimer of borazine-acetylene has been studied for the first time, both experimentally and computationally. The borazine-acetylene dimer was trapped in Ar and N2 matrices, and studied using infrared spectroscopy. Our experiments clearly revealed two isomers of the borazine-acetylene complex, one in which the N-H of borazine interacted with the carbon of acetylene, and another in which the C-H of acetylene formed a hydrogen bond with a nitrogen atom of borazine. The formation of both isomers in the matrix was evidenced by shifts in the vibrational frequencies of the appropriate modes. Reassuringly, the experimental observations were corroborated by our computations using the second-order Møller-Plesset perturbation theoretic method and coupled-cluster singles, doubles and perturbative triples method in conjunction with different Dunning basis sets, which indicated both these isomers to be stable minima, with the N-HṡṡṡC complex being the global minimum. Atoms-in-molecules and energy decomposition analysis were also carried out for the different isomers of the dimer. These studies reveal that replacing the three C-C linkages in benzene with three B-N linkages in borazine modifies the interaction in the dimer sufficiently, to result in a different potential energy landscape for the borazine-acetylene system when compared with the benzene-acetylene system.

Integrability and conformal data of the dimer model

NASA Astrophysics Data System (ADS)

Morin-Duchesne, Alexi; Rasmussen, Jørgen; Ruelle, Philippe

2016-04-01

The central charge of the dimer model on the square lattice is still being debated in the literature. In this paper, we provide evidence supporting the consistency of a c=-2 description. Using Lieb’s transfer matrix and its description in terms of the Temperley-Lieb algebra {{TL}}n at β =0, we provide a new solution of the dimer model in terms of the model of critical dense polymers on a tilted lattice and offer an understanding of the lattice integrability of the dimer model. The dimer transfer matrix is analyzed in the scaling limit, and the result for {L}0-\\frac{c}{24} is expressed in terms of fermions. Higher Virasoro modes are likewise constructed as limits of elements of {{TL}}n and are found to yield a c=-2 realization of the Virasoro algebra, familiar from fermionic bc ghost systems. In this realization, the dimer Fock spaces are shown to decompose, as Virasoro modules, into direct sums of Feigin-Fuchs modules, themselves exhibiting reducible yet indecomposable structures. In the scaling limit, the eigenvalues of the lattice integrals of motion are found to agree exactly with those of the c=-2 conformal integrals of motion. Consistent with the expression for {L}0-\\frac{c}{24} obtained from the transfer matrix, we also construct higher Virasoro modes with c = 1 and find that the dimer Fock space is completely reducible under their action. However, the transfer matrix is found not to be a generating function for the c = 1 integrals of motion. Although this indicates that Lieb’s transfer matrix description is incompatible with the c = 1 interpretation, it does not rule out the existence of an alternative, c = 1 compatible, transfer matrix description of the dimer model.

N-terminal galanin-(1-16) fragment is an agonist at the hippocampal galanin receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fisone, G.; Berthold, M.; Bedecs, K.

1989-12-01

The galanin N-terminal fragment (galanin-(1-16)) has been prepared by solid-phase synthesis and by enzymic cleavage of galanin by endoproteinase Asp-N. This peptide fragment displaced {sup 125}I-labeled galanin in receptor autoradiography experiments on rat forebrain and spinal cord and in equilibrium binding experiments from high-affinity binding sites in the ventral hippocampus with an IC50 of approximately 3 nM. In tissue slices of the same brain area, galanin-(1-16), similarly to galanin, inhibited the muscarinic agonist-stimulated breakdown of inositol phospholipids. Upon intracerebroventricular administration, galanin-(1-16) (10 micrograms/15 microliters) also inhibited the scopolamine (0.3 mg/kg, s.c.)-evoked release of acetylcholine, as studied in vivo by microdialysis.more » Substitution of (L-Trp2) for (D-Trp2) resulted in a 500-fold loss in affinity as compared with galanin-(1-16). It is concluded that, in the ventral hippocampus, the N-terminal galanin fragment (galanin-(1-16)) is recognized by the galanin receptors controlling acetylcholine release and muscarinic agonist-stimulated inositol phospholipid breakdown as a high-affinity agonist and that amino acid residue (Trp2) plays an important role in the receptor-ligand interactions.« less

Site-specific O-glycosylation of N-terminal serine residues by polypeptide GalNAc-transferase 2 modulates human δ-opioid receptor turnover at the plasma membrane.

PubMed

Lackman, Jarkko J; Goth, Christoffer K; Halim, Adnan; Vakhrushev, Sergey Y; Clausen, Henrik; Petäjä-Repo, Ulla E

2018-01-01

G protein-coupled receptors (GPCRs) are an important protein family of signalling receptors that govern a wide variety of physiological functions. The capacity to transmit extracellular signals and the extent of cellular response are largely determined by the amount of functional receptors at the cell surface that is subject to complex and fine-tuned regulation. Here, we demonstrate that the cell surface expression level of an inhibitory GPCR, the human δ-opioid receptor (hδOR) involved in pain and mood regulation, is modulated by site-specific N-acetylgalactosamine (GalNAc) -type O-glycosylation. Importantly, we identified one out of the 20 polypeptide GalNAc-transferase isoforms, GalNAc-T2, as the specific regulator of O-glycosylation of Ser6, Ser25 and Ser29 in the N-terminal ectodomain of the receptor. This was demonstrated by in vitro glycosylation assays using peptides corresponding to the hδOR N-terminus, Vicia villosa lectin affinity purification of receptors expressed in HEK293 SimpleCells capable of synthesizing only truncated O-glycans, GalNAc-T edited cell line model systems, and site-directed mutagenesis of the putative O-glycosylation sites. Interestingly, a single-nucleotide polymorphism, at residue 27 (F27C), was found to alter O-glycosylation of the receptor in efficiency as well as in glycosite usage. Furthermore, flow cytometry and cell surface biotinylation assays using O-glycan deficient CHO-ldlD cells revealed that the absence of O-glycans results in decreased receptor levels at the plasma membrane due to enhanced turnover. In addition, mutation of the identified O-glycosylation sites led to a decrease in the number of ligand-binding competent receptors and impaired agonist-mediated inhibition of cyclic AMP accumulation in HEK293 cells. Thus, site-specific O-glycosylation by a selected GalNAc-T isoform can increase the stability of a GPCR, in a process that modulates the constitutive turnover and steady-state levels of functional receptors

Novel pppGpp binding site at the C-terminal region of the Rel enzyme from Mycobacterium smegmatis.

PubMed

Syal, Kirtimaan; Joshi, Himanshu; Chatterji, Dipankar; Jain, Vikas

2015-10-01

Mycobacterium tuberculosis elicits the stringent response under unfavorable growth conditions, such as those encountered by the pathogen inside the host. The hallmark of this response is production of guanosine tetra- and pentaphosphates, collectively termed (p)ppGpp, which have pleiotropic effects on the bacterial physiology. As the stringent response is connected to survival under stress, it is now being targeted for developing inhibitors against bacterial persistence. The Rel enzyme in mycobacteria has two catalytic domains at its N-terminus that are involved in the synthesis and hydrolysis of (p)ppGpp, respectively. However, the function of the C-terminal region of the protein remained unknown. Here, we have identified a binding site for pppGpp in the C-terminal region of Rel. The binding affinity of pppGpp was quantified by isothermal titration calorimetry. The binding site was determined by crosslinking using the nucleotide analog azido-pppGpp, and examining the crosslink product by mass spectrometry. Additionally, mutations in the Rel protein were created to confirm the site of pppGpp binding by isothermal titration calorimetry. These mutants showed increased pppGpp synthesis and reduced hydrolytic activity. We believe that binding of pppGpp to Rel provides a feedback mechanism that allows the protein to detect and adjust the (p)ppGpp level in the cell. Our work suggests that such sites should also be considered while designing inhibitors to target the stringent response. © 2015 FEBS.

Comparative experimental/theoretical studies on the EGFR dimerization under the effect of EGF/EGF analogues binding: Highlighting the importance of EGF/EGFR interactions at site III interface.

PubMed

Mehrabi, Masomeh; Mahdiuni, Hamid; Rasouli, Hassan; Mansouri, Kamran; Shahlaei, Mohsen; Khodarahmi, Reza

2018-04-14

Epidermal growth factor receptors (EGFRs) and their cytoplasmic tyrosine kinases play significant roles in cell proliferation and signaling. All the members of the EGFR/ErbB family are primary goals for cancer therapy, particularly for tumors of breast, cervix, ovaries, kidney, esophagus, prostate and non-small-cell lung carcinoma and head and neck tumors. However, the therapeutic ability of accessible anti-ErbB agents is limited. Therefore, recognizing EGF analogues or small organic molecules with high affinity for the extracellular domain of the EGFR is a critical target on cancer research. An effective EGF analogue should have a comparable binding affinity for EGFR in order to create an effective ligand competitive inhibition against circulating wild EGF while fails to transduce appropriate downstream signaling into the cancer cell. In our earlier study we have developed a mutant form of human EGF (mEGF, lacking the four critical amino acid residues; Gln 43 , Tyr 44 , Arg 45 and Asp 46 at the C-terminal of the protein) and its binding properties and mitogenic activity were assessed. The mEGF showed high affinity for EGFR binding domains but caused poor EGFR dimerization and phosphorylation and especially, mEGF induced EGFR internalization. However, underlying mechanism of action of EGF analogues is still unclear and thus considered to be worthwhile for further study. With regard to different effects of the EGF analogue on EGFR activating process, computational analysis of wild EGF/EGFR and mEGF/EGFR complexes (along with EGFt/EGFR complex) were done. Results of the protein dissection identified several interactions within "ligand/EGFR" that are common among EGF and EGFt/mEGF. These results disclose that while several interactions are conserved within EGF/EGFR interfaces, EGF/EGFR interactions on site III interface controls the affinity, EGFR dimerization and subsequent downstream signaling through a heterogeneous set of non-covalent interactions. These findings

Linked Production of Pyroglutamate-Modified Proteins via Self-Cleavage of Fusion Tags with TEV Protease and Autonomous N-Terminal Cyclization with Glutaminyl Cyclase In Vivo

PubMed Central

Shih, Yan-Ping; Chou, Chi-Chi; Chen, Yi-Ling; Huang, Kai-Fa; Wang, Andrew H.- J.

2014-01-01

Overproduction of N-terminal pyroglutamate (pGlu)-modified proteins utilizing Escherichia coli or eukaryotic cells is a challenging work owing to the fact that the recombinant proteins need to be recovered by proteolytic removal of fusion tags to expose the N-terminal glutaminyl or glutamyl residue, which is then converted into pGlu catalyzed by the enzyme glutaminyl cyclase. Herein we describe a new method for production of N-terminal pGlu-containing proteins in vivo via intracellular self-cleavage of fusion tags by tobacco etch virus (TEV) protease and then immediate N-terminal cyclization of passenger target proteins by a bacterial glutaminyl cyclase. To combine with the sticky-end PCR cloning strategy, this design allows the gene of target proteins to be efficiently inserted into the expression vector using two unique cloning sites (i.e., SnaB I and Xho I), and the soluble and N-terminal pGlu-containing proteins are then produced in vivo. Our method has been successfully applied to the production of pGlu-modified enhanced green fluorescence protein and monocyte chemoattractant proteins. This design will facilitate the production of protein drugs and drug target proteins that possess an N-terminal pGlu residue required for their physiological activities. PMID:24733552

Dimerization of the keto tautomer of acetohydroxamic acid—infrared matrix isolation and theoretical study

NASA Astrophysics Data System (ADS)

Sałdyka, Magdalena; Mielke, Zofia

2005-05-01

Dimerization of the keto tautomer of acetohydroxamic acid has been studied using FTIR matrix isolation spectroscopy and DFT(B3LYP)/6-31+G(d,p) calculations. Analysis of CH 3CONHOH/Ar matrix spectra indicates formation of two dimers in which two intramolecular CO···H sbnd ON bonds within two interacting acetohydroxamic acid molecules are retained. A chain dimer I is stabilized by the intermolecular CO···H sbnd N hydrogen bond, whereas the cyclic dimer II is stabilized by two intermolecular N sbnd H···O(H)N bonds. Twelve vibrations were identified for dimer I and six vibrations for dimer II; the observed frequency shifts show a good agreement with the calculated ones for the structures I and II. Both dimers have comparable binding energies ( ΔEZPECPI, II = -7.02, -6.34 kcal mol -1) being less stable than calculated structures III and IV ( ΔEZPECPIII, IV = -9.50, -8.87 kcal mol -1) in which one or two intramolecular hydrogen bonds are disrupted. In the most stable 10-membered cyclic dimer III, two intermolecular CO···H sbnd ON hydrogen bonds are formed at expense of intramolecular hydrogen bonds of the same type. The formation of the less stable (AHA) 2 dimers in the studied matrixes indicates that the formation of (AHA) 2 is kinetically and not thermodynamically controlled.

Development of the sigma-1 receptor in C-terminals of motoneurons and colocalization with the N,N'-dimethyltryptamine forming enzyme, indole-N-methyl transferase.

PubMed

Mavlyutov, T A; Epstein, M L; Liu, P; Verbny, Y I; Ziskind-Conhaim, L; Ruoho, A E

2012-03-29

The function of the sigma-1 receptor (S1R) has been linked to modulating the activities of ion channels and G-protein-coupled receptors (GPCR). In the CNS, the S1R is expressed ubiquitously but is enriched in mouse motoneurons (MN), where it is localized to subsurface cisternae of cholinergic postsynaptic densities, also known as C-terminals. We found that S1R is enriched in mouse spinal MN at late stages of embryonic development when it is first visualized in the endoplasmic reticulum. S1Rs appear to concentrate at C-terminals of mouse MN only on the second week of postnatal development. We found that indole-N-methyl transferase (INMT), an enzyme that converts tryptamine into the sigma-1 ligand dimethyltryptamine (DMT), is also localized to postsynaptic sites of C-terminals in close proximity to the S1R. This close association of INMT and S1Rs suggest that DMT is synthesized locally to effectively activate S1R in MN. Published by Elsevier Ltd.

Interaction between the C-terminal domains of measles virus nucleoprotein and phosphoprotein: a tight complex implying one binding site.

PubMed

Blocquel, David; Habchi, Johnny; Costanzo, Stéphanie; Doizy, Anthony; Oglesbee, Michael; Longhi, Sonia

2012-10-01

The intrinsically disordered C-terminal domain (N(TAIL) ) of the measles virus (MeV) nucleoprotein undergoes α-helical folding upon binding to the C-terminal X domain (XD) of the phosphoprotein. The N(TAIL) region involved in binding coupled to folding has been mapped to a conserved region (Box2) encompassing residues 489-506. In the previous studies published in this journal, we obtained experimental evidence supporting a K(D) for the N(TAIL) -XD binding reaction in the nM range and also showed that an additional N(TAIL) region (Box3, aa 517-525) plays a role in binding to XD. In striking contrast with these data, studies published in this journal by Kingston and coworkers pointed out a much less stable complex (K(D) in the μM range) and supported lack of involvement of Box3 in complex formation. The objective of this study was to critically re-evaluate the role of Box3 in N(TAIL) -XD binding. Since our previous studies relied on N(TAIL) -truncated forms possessing an irrelevant Flag sequence appended at their C-terminus, we, herein, generated an N(TAIL) devoid of Box3 and any additional C-terminal residues, as well as a form encompassing only residues 482-525. We then used isothermal titration calorimetry to characterize the binding reactions between XD and these N(TAIL) forms. Results effectively argue for the presence of a single XD-binding site located within Box2, in agreement with the results by Kingston et al., while providing clear experimental support for a high-affinity complex. Altogether, the present data provide mechanistic insights into the replicative machinery of MeV and clarify a hitherto highly debated point. Copyright © 2012 The Protein Society.

Disruption of the HIV-1 protease dimer with interface peptides: structural studies using NMR spectroscopy combined with [2-(13)C]-Trp selective labeling.

PubMed

Frutos, Silvia; Rodriguez-Mias, Ricard A; Madurga, Sergio; Collinet, Bruno; Reboud-Ravaux, Michèle; Ludevid, Dolors; Giralt, Ernest

2007-01-01

HIV-1 protease (HIV-1 PR), which is encoded by retroviruses, is required for the processing of gag and pol polyprotein precursors, hence it is essential for the production of infectious viral particles. In vitro inhibition of the enzyme results in the production of progeny virions that are immature and noninfectious, suggesting its potential as a therapeutic target for AIDS. Although a number of potent protease inhibitor drugs are now available, the onset of resistance to these agents due to mutations in HIV-1 PR has created an urgent need for new means of HIV-1 PR inhibition. Whereas enzymes are usually inactivated by blocking of the active site, the structure of dimeric HIV-1 PR allows an alternative inhibitory mechanism. Since the active site is formed by two half-enzymes, which are connected by a four-stranded antiparallel beta-sheet involving the N- and C- termini of both monomers, enzyme activity can be abolished by reagents targeting the dimer interface in a region relatively free of mutations would interfere with formation or stability of the functional HIV-1 PR dimer. This strategy has been explored by several groups who targeted the four-stranded antiparallel beta-sheet that contributes close to 75% of the dimerization energy. Interface peptides corresponding to native monomer N- or C-termini of several of their mimetics demonstrated, mainly on the basis of kinetic analyses, to act as dimerization inhibitors. However, to the best of our knowledge, neither X-ray crystallography nor NMR structural studies of the enzyme-inhibitor complex have been performed to date. In this article we report a structural study of the dimerization inhibition of HIV-1 PR by NMR using selective Trp side chain labeling.

Constitutive activation and uncoupling of the atrial natriuretic peptide receptor by mutations at the dimer interface. Role of the dimer structure in signalling.

PubMed

Qiu, Yue; Ogawa, Haruo; Miyagi, Masaru; Misono, Kunio S

2004-02-13

The crystal packing of the extracellular hormone binding domain of the atrial natriuretic peptide (ANP) receptor contains two possible dimer pairs, the head-to-head (hh) and tail-to-tail (tt) dimer pairs associated through the membrane-distal and membrane-proximal subdomains, respectively. The tt-dimer structure has been proposed previously (van den Akker, F., Zhang, X., Miyagi, M., Huo, X., Misono, K. S., and Yee, V. C. (2000) Nature 406, 101-104). However, no direct evidence is available to identify the physiological dimer form. Here we report site-directed mutagenesis studies of residues at the two alternative dimer interfaces in the full-length receptor expressed on COS cells. The Trp74 to Arg mutation (W74R) or D71R at the hh-dimer interface caused partial constitutive guanylate cyclase activation, whereas mutation F96D or H99D caused receptor uncoupling. In contrast, mutation Y196D or L225D at the tt-interface had no such effect. His99 modification at the hh-dimer interface by ethoxyformic anhydride abolished ANP binding. These results suggest that the hh-dimer represents the physiological structure. Recently, we determined the crystal structure of ANPR complexed with ANP and proposed a hormone-induced rotation mechanism mediating transmembrane signaling (H. Ogawa, Y. Qiu, C. M. Ogata, and K. S. Misono, submitted for publication). The observed effects of mutations are consistent with the ANP-induced structural change identified from the crystal structures with and without ANP and support the proposed rotation mechanism for ANP receptor signaling.

Structural and Kinetic Analyses of Macrophage Migration Inhibitory Factor Active Site Interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crichlow, G.; Lubetsky, J; Leng, L

Macrophage migration inhibitory factor (MIF) is a secreted protein expressed in numerous cell types that counters the antiinflammatory effects of glucocorticoids and has been implicated in sepsis, cancer, and certain autoimmune diseases. Interestingly, the structure of MIF contains a catalytic site resembling the tautomerase/isomerase sites of microbial enzymes. While bona fide physiological substrates remain unknown, model substrates have been identified. Selected compounds that bind in the tautomerase active site also inhibit biological functions of MIF. It had previously been shown that the acetaminophen metabolite, N-acetyl-p-benzoquinone imine (NAPQI), covalently binds to the active site of MIF. In this study, kinetic datamore » indicate that NAPQI inhibits MIF both covalently and noncovalently. The structure of MIF cocrystallized with NAPQI reveals that the NAPQI has undergone a chemical alteration forming an acetaminophen dimer (bi-APAP) and binds noncovalently to MIF at the mouth of the active site. We also find that the commonly used protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), forms a covalent complex with MIF and inhibits the tautomerase activity. Crystallographic analysis reveals the formation of a stable, novel covalent bond for PMSF between the catalytic nitrogen of the N-terminal proline and the sulfur of PMSF with complete, well-defined electron density in all three active sites of the MIF homotrimer. Conclusions are drawn from the structures of these two MIF-inhibitor complexes regarding the design of novel compounds that may provide more potent reversible and irreversible inhibition of MIF.« less

Saccharomyces cerevisiae RNA Polymerase I Terminates Transcription at the Reb1 Terminator In Vivo

PubMed Central

Reeder, Ronald H.; Guevara, Palmira; Roan, Judith G.

1999-01-01

We have mapped transcription termination sites for RNA polymerase I in the yeast Saccharomyces cerevisiae. S1 nuclease mapping shows that the primary terminator is the Reb1p terminator located at +93 downstream of the 3′ end of 25S rRNA. Reverse transcription coupled with quantitative PCR shows that approximately 90% of all transcripts terminate at this site. Transcripts which read through the +93 site quantitatively terminate at a fail-safe terminator located further downstream at +250. Inactivation of Rnt1p (an RNase III involved in processing the 3′ end of 25S rRNA) greatly stabilizes transcripts extending to both sites and increases readthrough at the +93 site. In vivo assay of mutants of the Reb1p terminator shows that this site operates in vivo by the same mechanism as has previously been delineated through in vitro studies. PMID:10523625

Cold-active alkaline phosphatase is irreversibly transformed into an inactive dimer by low urea concentrations.

PubMed

Hjörleifsson, Jens Guðmundur; Ásgeirsson, Bjarni

2016-07-01

Alkaline phosphatase is a homodimeric metallo-hydrolase where both Zn(2+) and Mg(2+) are important for catalysis and stability. Cold-adapted alkaline phosphatase variants have high activity at low temperatures and lower thermal stability compared with variants from mesophilic hosts. The instability, and thus inactivation, could be due to loose association of the dimers and/or loosely bound Mg(2)(+) in the active site, but this has not been studied in detail for the cold-adapted variants. Here, we focus on using the intrinsic fluorescence of Trp in alkaline phosphatase from the marine bacterium Vibrio splendidus (VAP) to probe for dimerization. Trp→Phe substitutions showed that two out of the five native Trp residues contributed mostly to the fluorescence emission. One residue, 15Å away from the active site (W460) and highly solvent excluded, was phosphorescent and had a distant role in substrate binding. An additional Trp residue was introduced to the dimer interface to act as a possible probe for dimerization. Urea denaturation curves indicated that an inactive dimer intermediate, structurally equivalent to the native state, was formed before dimer dissociation took place. This is the first example of the transition of a native dimer to an inactive dimer intermediate for alkaline phosphatase without using mutagenesis, ligands, or competitive inhibition. Copyright © 2016 Elsevier B.V. All rights reserved.

Large enhancement of functional activity of active site-inhibited factor VIIa due to protein dimerization: insights into mechanism of assembly/disassembly from tissue factor.

PubMed

Stone, Matthew D; Harvey, Stephen B; Martinez, Michael B; Bach, Ronald R; Nelsestuen, Gary L

2005-04-26

Active site-inhibited blood clotting factor VIIa (fVIIai) binds to tissue factor (TF), a cell surface receptor that is exposed upon injury and initiates the blood clotting cascade. FVIIai blocks binding of the corresponding enzyme (fVIIa) or zymogen (fVII) forms of factor VII and inhibits coagulation. Although several studies have suggested that fVIIai may have superior anticoagulation effects in vivo, a challenge for use of fVIIai is cost of production. This study reports the properties of dimeric forms of fVIIai that are cross-linked through their active sites. Dimeric wild-type fVIIai was at least 75-fold more effective than monomeric fVIIai in blocking fVIIa association with TF. The dimer of a mutant fVIIai with higher membrane affinity was 1600-fold more effective. Anticoagulation by any form of fVIIai differed substantially from agents such as heparin and showed a delayed mode of action. Coagulation proceeded normally for the first minutes, and inhibition increased as equilibrium binding was established. It is suggested that association of fVIIa(i) with TF in a collision-dependent reaction gives equal access of inhibitor and enzyme to TF. Assembly was not influenced by the higher affinity and slower dissociation of the dimer. As a result, anticoagulation was delayed until the reaction reached equilibrium. Properties of different dissociation experiments suggested that dissociation of fVIIai from TF occurred by a two-step mechanism. The first step was separation of TF-fVIIa(i) while both proteins remained bound to the membrane, and the second step was dissociation of the fVIIa(i) from the membrane. These results suggest novel actions of fVIIai that distinguish it from most of the anticoagulants that block later steps of the coagulation cascade.

Specific binding of the WASP N-terminal domain to Btk is critical for TLR2 signaling in macrophages.

PubMed

Sakuma, Chisato; Sato, Mitsuru; Takenouchi, Takato; Kitani, Hiroshi

2015-02-01

Wiskott-Aldrich syndrome protein (WASP) is an adaptor molecule in immune cells. Recently, we revealed that WASP is involved in lipopolysaccharide-TLR4 signaling in macrophages by association of Bruton's tyrosine kinase (Btk) with the WASP N-terminal domain. Btk has been shown to play important roles in the signaling of several TLRs and to modulate the inflammatory response in macrophages. In this study, we evaluated the importance of the interaction between Btk and WASP in TLR2 signaling by using bone marrow-derived macrophage cell lines from transgenic (Tg) mice expressing anti-WASP N-terminal domain single-chain variable fragment (scFv) or VL single-domain intrabodies. In this Tg bone marrow-derived macrophages, specific interaction between WASP and Btk were strongly inhibited by masking of the binding site in the WASP N-terminal domain. There was impairment of gene expression of TNF-α, IL-6, and IL-1β and phosphorylation of inhibitor of κB α/β (IKKα/β) and nuclear factor (NF)-κB upon stimulation with TLR2 ligands. Furthermore, tyrosine phosphorylation of WASP following TLR2-ligand stimulation was severely inhibited in the Tg bone marrow-derived macrophages, as shown by the impairment in WASP tyrosine phosphorylation following lipopolysaccharide stimulation. These results strongly suggest that the association between the WASP N-terminal domain and Btk plays an important role in the TLR2-signaling pathway in macrophages. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identification of the cleavage sites of the RNA2-encoded polyproteins for two members of the genus Torradovirus by N-terminal sequencing of the virion capsid proteins.

PubMed

Ferriol, I; Silva Junior, D M; Nigg, J C; Zamora-Macorra, E J; Falk, B W

2016-11-01

Torradoviruses, family Secoviridae, are emergent bipartite RNA plant viruses. RNA1 is ca. 7kb and has one open reading frame (ORF) encoding for the protease, helicase and RNA-dependent RNA polymerase (RdRp). RNA2 is ca. 5kb and has two ORFs. RNA2-ORF1 encodes for a putative protein with unknown function(s). RNA2-ORF2 encodes for a putative movement protein and three capsid proteins. Little is known about the replication and polyprotein processing strategies of torradoviruses. Here, the cleavage sites in the RNA2-ORF2-encoded polyproteins of two torradoviruses, Tomato marchitez virus isolate M (ToMarV-M) and tomato chocolate spot virus, were determined by N-terminal sequencing, revealing that the amino acid (aa) at the -1 position of the cleavage sites is a glutamine. Multiple aa sequence comparison confirmed that this glutamine is conserved among other torradoviruses. Finally, site-directed mutagenesis of conserved aas in the ToMarV-M RdRp and protease prevented substantial accumulation of viral coat proteins or RNAs. Copyright © 2016 Elsevier Inc. All rights reserved.

Theoretical investigation of structures and energetics of sodium adatom and its dimer on graphene: DFT study

NASA Astrophysics Data System (ADS)

Kaur, Gagandeep; Gupta, Shuchi; Rani, Pooja; Dharamvir, Keya

2015-11-01

Extensive ab initio calculations have been performed to study the energetics of a sodium (Na) atom and its dimer adsorbed on graphene using the SIESTA package Soler et al. (2002) [1] which works within a DFT(density functional theory)-GGA (generalized gradient approximation) pseudopotential framework. The adsorption energy, geometry, charge transfer, ionization potential and density of states (DOS), partial density states (PDOS) of adatom/dimer-graphene system have been calculated. After considering various sites for adsorption of Na on graphene, the center of a hexagonal ring of carbon atoms is found to be the preferred site of adsorption while the Na2 dimer prefers to rest parallel to the graphene sheet. We find insignificant energy differences among adsorption configurations involving different possible sites in parallel orientation, which implies high mobility of the dimer on the graphene sheet. We also notice only a slight distortion of the graphene sheet perpendicular to its plane upon adatom adsorption. However, some lateral displacements seen are more perceptible. Summary The adsorption energy, geometry, charge transfer, ionization potential and density of states (DOS) and PDOS of adatom/dimer-graphene system have been calculated using SIESTA package Soler et al. (2002) [1] which works within a DFT(density functional theory)-GGA (generalized gradient approximation) pseudopotential framework. Preferred site for adsorption of a sodium atom on graphene is the hollow site. For the Na dimer adsorption, we found that horizontal orientation is favored over the vertical one. From DOS plots, it is clear that graphene's states are nearly unaffected by the adsorption of Na adatom and Interaction between sodium and graphene is predominantly ionic

The S-Layer Glycoprotein of the Crenarchaeote Sulfolobus acidocaldarius Is Glycosylated at Multiple Sites with Chitobiose-Linked N-Glycans

PubMed Central

Peyfoon, Elham; Meyer, Benjamin; Hitchen, Paul G.; Panico, Maria; Morris, Howard R.; Haslam, Stuart M.; Albers, Sonja-Verena; Dell, Anne

2010-01-01

Glycosylation of the S-layer of the crenarchaea Sulfolobus acidocaldarius has been investigated using glycoproteomic methodologies. The mature protein is predicted to contain 31 N-glycosylation consensus sites with approximately one third being found in the C-terminal domain spanning residues L1004-Q1395. Since this domain is rich in Lys and Arg and therefore relatively tractable to glycoproteomic analysis, this study has focused on mapping its N-glycosylation. Our analysis identified nine of the 11 consensus sequence sites, and all were found to be glycosylated. This constitutes a remarkably high glycosylation density in the C-terminal domain averaging one site for each stretch of 30–40 residues. Each of the glycosylation sites observed was shown to be modified with a heterogeneous family of glycans, with the largest having a composition Glc1Man2GlcNAc2 plus 6-sulfoquinovose (QuiS), consistent with the tribranched hexasaccharide previously reported in the cytochrome b558/566 of S. acidocaldarius. S. acidocaldarius is the only archaeal species whose N-glycans are known to be linked via the chitobiose core disaccharide that characterises the N-linked glycans of Eukarya. PMID:20936123

Structural model of the dimeric Parkinson’s protein LRRK2 reveals a compact architecture involving distant interdomain contacts

PubMed Central

Guaitoli, Giambattista; Raimondi, Francesco; Gilsbach, Bernd K.; Gómez-Llorente, Yacob; Deyaert, Egon; Renzi, Fabiana; Li, Xianting; Schaffner, Adam; Jagtap, Pravin Kumar Ankush; Boldt, Karsten; von Zweydorf, Felix; Gotthardt, Katja; Lorimer, Donald D.; Yue, Zhenyu; Burgin, Alex; Janjic, Nebojsa; Sattler, Michael; Versées, Wim; Ueffing, Marius; Ubarretxena-Belandia, Iban; Kortholt, Arjan; Gloeckner, Christian Johannes

2016-01-01

Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein containing two catalytic domains: a Ras of complex proteins (Roc) G-domain and a kinase domain. Mutations associated with familial and sporadic Parkinson’s disease (PD) have been identified in both catalytic domains, as well as in several of its multiple putative regulatory domains. Several of these mutations have been linked to increased kinase activity. Despite the role of LRRK2 in the pathogenesis of PD, little is known about its overall architecture and how PD-linked mutations alter its function and enzymatic activities. Here, we have modeled the 3D structure of dimeric, full-length LRRK2 by combining domain-based homology models with multiple experimental constraints provided by chemical cross-linking combined with mass spectrometry, negative-stain EM, and small-angle X-ray scattering. Our model reveals dimeric LRRK2 has a compact overall architecture with a tight, multidomain organization. Close contacts between the N-terminal ankyrin and C-terminal WD40 domains, and their proximity—together with the LRR domain—to the kinase domain suggest an intramolecular mechanism for LRRK2 kinase activity regulation. Overall, our studies provide, to our knowledge, the first structural framework for understanding the role of the different domains of full-length LRRK2 in the pathogenesis of PD. PMID:27357661

Periodic table of 3d-metal dimers and their ions.

PubMed

Gutsev, G L; Mochena, M D; Jena, P; Bauschlicher, C W; Partridge, H

2004-10-08

The ground states of the mixed 3d-metal dimers TiV, TiCr, TiMn, TiFe, TiCo, TiNi, TiCu, TiZn, VCr, VMn, VFe, VCo, VNi, VCu, VZn, CrMn, CrFe, CrCo, CrNi, CrCu, CrZn, MnFe, MnCo, MnNi, MnCu, MnZn, FeCo, FeNi, FeCu, FeZn, CoNi, CoCu, CoZn, NiCu, NiZn, and CuZn along with their singly negatively and positively charged ions are assigned based on the results of computations using density functional theory with generalized gradient approximation for the exchange-correlation functional. Except for TiCo and CrMn, our assignment agrees with experiment. Computed spectroscopic constants (r(e),omega(e),D(o)) are in fair agreement with experiment. The ground-state spin multiplicities of all the ions are found to differ from the spin multiplicities of the corresponding neutral parents by +/-1. Except for TiV, MnFe, and MnCu, the number of unpaired electrons, N, in a neutral ground-state dimer is either N(1)+N(2) or mid R:N(1)-N(2)mid R:, where N(1) and N(2) are the numbers of unpaired 3d electrons in the 3d(n)4s(1) occupation of the constituent atoms. Combining the present and previous results obtained at the same level of theory for homonuclear 3d-metal and ScX (X=Ti-Zn) dimers allows one to construct "periodic" tables of all 3d-metal dimers along with their singly charged ions.

Evidence for dimer formation by an amphiphilic heptapeptide that mediates chloride and carboxyfluorescein release from liposomes

PubMed Central

Pajewski, Robert; Ferdani, Riccardo; Pajewska, Jolanta; Djedovič, Natasha; Schlesinger, Paul H.; Gokel, George W.

2008-01-01

Heptapeptides having dioctadecyl, N-terminal hydrocarbon chains insert in phospholipid bilayer membranes and form pores through which at least chloride ions pass. Although amphiphilic, these compounds do not typically form vesicles themselves. They insert in the bilayers of phospholipid vesicles and mediate the release of carboxyfluorescein. Hill analysis indicates that at least two molecules of the amphiphile are involved in pore formation. In CD2Cl2, dimer formation is detected by NMR chemical shift changes. The anion release activity of individual anion transporters is increased by linking them covalently at the C-terminus or, even more, by linking them at the N-terminus. Evidence is presented that either linked molecule releases chloride from liposomes more effectively and rapidly than the individual transporter molecule at a comparable concentration. PMID:15703797

Targeted mass spectrometric analysis of N-terminally truncated isoforms generated via alternative translation initiation.

PubMed

Kobayashi, Ryuji; Patenia, Rebecca; Ashizawa, Satoshi; Vykoukal, Jody

2009-07-21

Alternative translation initiation is a mechanism whereby functionally altered proteins are produced from a single mRNA. Internal initiation of translation generates N-terminally truncated protein isoforms, but such isoforms observed in immunoblot analysis are often overlooked or dismissed as degradation products. We identified an N-terminally truncated isoform of human Dok-1 with N-terminal acetylation as seen in the wild-type. This Dok-1 isoform exhibited distinct perinuclear localization whereas the wild-type protein was distributed throughout the cytoplasm. Targeted analysis of blocked N-terminal peptides provides rapid identification of protein isoforms and could be widely applied for the general evaluation of perplexing immunoblot bands.

Preferential assembly of heteromeric kainate and AMPA receptor amino terminal domains

PubMed Central

Lomash, Suvendu; Chittori, Sagar; Glasser, Carla

2017-01-01

Ion conductivity and the gating characteristics of tetrameric glutamate receptor ion channels are determined by their subunit composition. Competitive homo- and hetero-dimerization of their amino-terminal domains (ATDs) is a key step controlling assembly. Here we measured systematically the thermodynamic stabilities of homodimers and heterodimers of kainate and AMPA receptors using fluorescence-detected sedimentation velocity analytical ultracentrifugation. Measured affinities span many orders of magnitude, and complexes show large differences in kinetic stabilities. The association of kainate receptor ATD dimers is generally weaker than the association of AMPA receptor ATD dimers, but both show a general pattern of increased heterodimer stability as compared to the homodimers of their constituents, matching well physiologically observed receptor combinations. The free energy maps of AMPA and kainate receptor ATD dimers provide a framework for the interpretation of observed receptor subtype combinations and possible assembly pathways. PMID:29058671

Preferential assembly of heteromeric kainate and AMPA receptor amino terminal domains.

PubMed

Zhao, Huaying; Lomash, Suvendu; Chittori, Sagar; Glasser, Carla; Mayer, Mark L; Schuck, Peter

2017-10-23

Ion conductivity and the gating characteristics of tetrameric glutamate receptor ion channels are determined by their subunit composition. Competitive homo- and hetero-dimerization of their amino-terminal domains (ATDs) is a key step controlling assembly. Here we measured systematically the thermodynamic stabilities of homodimers and heterodimers of kainate and AMPA receptors using fluorescence-detected sedimentation velocity analytical ultracentrifugation. Measured affinities span many orders of magnitude, and complexes show large differences in kinetic stabilities. The association of kainate receptor ATD dimers is generally weaker than the association of AMPA receptor ATD dimers, but both show a general pattern of increased heterodimer stability as compared to the homodimers of their constituents, matching well physiologically observed receptor combinations. The free energy maps of AMPA and kainate receptor ATD dimers provide a framework for the interpretation of observed receptor subtype combinations and possible assembly pathways.

The localization of a vitamin K-induced modification in an N-terminal fragment of human prothrombin

PubMed Central

Skotland, Tore; Holm, Turid; Østerud, Bjarne; Flengsrud, Ragnar; Prydz, Hans

1974-01-01

1. The N-terminal fragment (PF-I) split off from prothrombin during coagulation was purified to homogeneity from human serum. 2. The apparent molecular weight is 27000±2000 in sodium dodecyl sulphate–polyacrylamide-gel electrophoresis, whereas a value of about 19600 is obtained by calculation based on amino acid and carbohydrate analyses. The N-terminal sequence is an Ala-Asx bond. The fragment contains about 16% carbohydrate, binds phospholipids in the presence of Ca2+ and is adsorbed to BaSO4. The pKa of its BaSO4-binding group(s) is 3.1–3.5. 3. By CNBr cleavage of fragment PF-I two peptides (C-1 and C-2) were obtained with molecular weights of about 5900 (C-2) and 12400 (C-1) on the basis of amino acid and carbohydrate analyses. Only the smaller (N-terminal) peptide is adsorbed to BaSO4 and, since the ability of the whole protein to bind to BaSO4 is known to be absent in samples obtained from patients treated with vitamin K antagonists, this peptide probably contains the site of a modification to the structure of the protein which occurs during biosynthesis and depends on vitamin K. This peptide does not contain hexosamine or sialic acid. ImagesFig. 2. PMID:4219283

Role of an RNase III Binding Site in Transcription Termination at λ nutL by HK022 Nun Protein

PubMed Central

Washburn, Robert S.; Court, Donald L.; Gottesman, Max E.

2006-01-01

The phage HK022 Nun protein excludes phage λ by binding nascent λ pL and pR transcripts at nutL and nutR, respectively, and inducing transcription termination just downstream of these sites. Termination is more efficient at nutL than at nutR. One difference between nutL and nutR is the presence of RNase III processing sites (rIII) located immediately promoter distal to λ nutL. We found that deletion of rIII dramatically reduced Nun transcription arrest in vitro but had little effect on termination in vivo. However, consistent with the in vitro results, overexpression of a transcript carrying nutL and rIII efficiently titrated Nun, allowing λ to grow on a strain that expressed Nun, whereas a transcript carrying only nutL or nutL-rIII with nucleotides 97 to 141 deleted was ineffective. Rnc70, an RNase III mutant that binds but does not cleave rIII, also prevented Nun-mediated λ exclusion. We propose that rIII enhances the on-rate of Nun at nutL, stimulating Nun-mediated arrest in vitro. We have shown that a specific element in rIII, i.e., box C (G89GUGUGUG), strongly enhances arrest on rIII+ templates. Nun-rIII interactions do not stimulate Nun termination in vivo, presumably because formation of the Nun-nutL complex is normally not rate-limiting in the cell. In contrast to Nun, N is not occluded by Rnc70 and is not efficiently titrated by a nutL-rIII transcript. PMID:16980485

CO adsorption on small Au{sub n} (n = 1–4) structures supported on hematite. II. Adsorption on the O-rich termination of α-Fe{sub 2}O{sub 3}(0001) surface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pabisiak, Tomasz; Kiejna, Adam, E-mail: kiejna@ifd.uni.wroc.pl; Winiarski, Maciej J.

2016-01-28

The adsorption of small Au{sub n} (n = 1–4) nanostructures on oxygen terminated α-Fe{sub 2}O{sub 3}(0001) surface was investigated using density functional theory in the generalized gradient approximation of Perdew-Burke-Ernzerhof (PBE) form with Hubbard correction U, accounting for strong electron correlations (PBE+U). The structural, energetic, and electronic properties were examined for two classes of the adsorbed Au{sub n} nanostructures with vertical and flattened configurations. Similarly to the Fe-terminated α-Fe{sub 2}O{sub 3}(0001) surface considered in Part I, the flattened configurations were found energetically more favored than vertical ones. The binding of Au{sub n} to the O-terminated surface is much stronger thanmore » to the Fe-termination. The adsorption bonding energy of Au{sub n} and the work function of the Au{sub n}/α-Fe{sub 2}O{sub 3}(0001) systems decrease with the increased number of Au atoms in a structure. All of the adsorbed Au{sub n} structures are positively charged. The bonding of CO molecules to the Au{sub n} structures is distinctly stronger than on the Fe-terminated surface; however, it is weaker than the binding to the bare O-terminated surface. The CO molecule binds to the Au{sub n}/α-Fe{sub 2}O{sub 3}(0001) system through a peripheral Au atom partly detached from the Au{sub n} structure. The results of this work indicate that the most energetically favored sites for adsorption of a CO molecule on the Au{sub n}/α-Fe{sub 2}O{sub 3}(0001) systems are atoms in the Au{sup 0.5+} oxidation state.« less

OVERVIEW OF STAMP MILL SITE,LOOKING SOUTHWEST. THE LOWER TRAM TERMINAL ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

OVERVIEW OF STAMP MILL SITE,LOOKING SOUTHWEST. THE LOWER TRAM TERMINAL IS OUT OF FRAME, JUST TO THE RIGHT. WATER TANK, LOADING PLATFORM, AND TRAM TRESTLE LEADING UP TO THE TRAM TERMINAL ARE AT RIGHT. THE STRUCTURE AT EXTREME RIGHT BELOW THE TRESTLE, ARE REMAINS OF A SECONDARY ORE BIN, WITH BALL MILL FOUNDATIONS AND WOOD DEBRIS JUST BELOW ON THE SECOND LEVEL. AT CENTER IS A BOILER AND THE FRAME WORK OF A FILTER PRESS. THE SMALL STRUCTURE AT CENTER LEFT IS AN INTERPRETIVE SIGN PLACED BY THE PARK SERVICE. AT LOWER LEFT, THIRD LEVEL OF THE MILL, ARE THE REMAINS OF A BLACKSMITH'S FORGE. - Keane Wonder Mine, Park Route 4 (Daylight Pass Cutoff), Death Valley Junction, Inyo County, CA

Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor

PubMed Central

Lübker, Carolin; Dove, Stefan; Tang, Wei-Jen; Urbauer, Ramona J. Bieber; Moskovitz, Jackob; Urbauer, Jeffrey L.; Seifert, Roland

2015-01-01

Bacillus anthracis adenylyl cyclase toxin edema factor (EF) is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca2+-sensor calmodulin (CaM) leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, thus reducing production of reactive oxygen species (ROS) used for host defense in activated neutrophils and thereby facilitating bacterial growth. Methionine (Met) residues in CaM, important for interactions between CaM and its binding partners, can be oxidized by ROS. We investigated the impact of site-specific oxidation of Met in CaM on EF activation using thirteen CaM-mutants (CaM-mut) with Met to leucine (Leu) substitutions. EF activation shows high resistance to oxidative modifications in CaM. An intact structure in the C-terminal region of oxidized CaM is sufficient for major EF activation despite altered secondary structure in the N-terminal region associated with Met oxidation. The secondary structures of CaM-mut were determined and described in previous studies from our group. Thus, excess cAMP production and the associated impairment of host defence may be afforded even under oxidative conditions in activated neutrophils. PMID:26184312

Plasma D-dimer as a predictor of the progression of abdominal aortic aneurysm.

PubMed

Vele, E; Kurtcehajic, A; Zerem, E; Maskovic, J; Alibegovic, E; Hujdurovic, A

2016-11-01

Essentials D-dimer could provide important information about abdominal aortic aneurysm (AAA) progression. The greatest diameter of the infrarenal aorta and the value of plasma D-dimer were determined. AAA progression is correlated with increasing plasma D-dimer levels. The increasing value of plasma D-dimer could be a predictor of aneurysm progression. Background The natural course of abdominal aortic aneurysm (AAA) is mostly asymptomatic and unpredictable. D-dimer could provide potentially important information about subsequent AAA progression. Objectives The aims of this study were to establish the relationship between the progression of an abdominal aortic aneurysm (AAA) and plasma D-dimer concentration over a 12-month period and determine the value of plasma D-dimer in patients with sub-aneurysmal aortic dilatation. Patients/Methods This was a prospective observational study that involved 33 patients with an AAA, 30 patients with sub-aneurysmal aortic dilatation and 30 control subjects. The greatest diameter of the infrarenal aorta, which was assessed by ultrasound, and the value of plasma D-dimer were determined for all subjects at baseline assessment, as well as after 12 months for those with an AAA. Results A positive correlation was found between the diameter of an AAA and plasma D-dimer concentration at the baseline and the control measurement stages. There was a strong positive correlation between AAA progression and increasing plasma D-dimer concentration over a 12-month period. Among patients with sub-aneurysmal aortic dilatation (n = 30), the value of plasma D-dimer was higher compared with matched controls (n = 30). Conclusions There is a strongly positive correlation between AAA progression and increasing plasma D-dimer concentration. The value of plasma D-dimer is higher in patients with sub-aneurysmal aortic dilatation than in control subjects. © 2016 International Society on Thrombosis and Haemostasis.

Structure of the N-terminal fragment of Escherichia coli Lon protease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Mi; Gustchina, Alla; Rasulova, Fatima S.

2010-10-22

The structure of a recombinant construct consisting of residues 1-245 of Escherichia coli Lon protease, the prototypical member of the A-type Lon family, is reported. This construct encompasses all or most of the N-terminal domain of the enzyme. The structure was solved by SeMet SAD to 2.6 {angstrom} resolution utilizing trigonal crystals that contained one molecule in the asymmetric unit. The molecule consists of two compact subdomains and a very long C-terminal {alpha}-helix. The structure of the first subdomain (residues 1-117), which consists mostly of {beta}-strands, is similar to that of the shorter fragment previously expressed and crystallized, whereas themore » second subdomain is almost entirely helical. The fold and spatial relationship of the two subdomains, with the exception of the C-terminal helix, closely resemble the structure of BPP1347, a 203-amino-acid protein of unknown function from Bordetella parapertussis, and more distantly several other proteins. It was not possible to refine the structure to satisfactory convergence; however, since almost all of the Se atoms could be located on the basis of their anomalous scattering the correctness of the overall structure is not in question. The structure reported here was also compared with the structures of the putative substrate-binding domains of several proteins, showing topological similarities that should help in defining the binding sites used by Lon substrates.« less

Hsa-miR-1587 G-quadruplex formation and dimerization induced by NH4+, molecular crowding environment and jatrorrhizine derivatives.

PubMed

Tan, Wei; Yi, Long; Zhu, Zhentao; Zhang, Lulu; Zhou, Jiang; Yuan, Gu

2018-03-01

A guanine-rich human mature microRNA, miR-1587, was discovered to form stable intramolecular G-quadruplexes in the presence of K + , Na + and low concentration of NH 4 + (25mM) by electrospray ionization mass spectrometry (ESI-MS) combined with circular dichroism (CD) spectroscopy. Furthermore, under high concentration of NH 4 + (100mM) or molecular crowding environments, miR-1587 formed a dimeric G-quadruplex through 3'-to-3' stacking of two monomeric G-quadruplex subunits with one ammonium ion sandwiched between the interfaces. Specifically, two synthesized jatrorrhizine derivatives with terminal amine groups could also induce the dimerization of miR-1587 G-quadruplex and formed 1:1 and 2:1 complexes with the dimeric G-quadruplex. In contrast, jatrorrhizine could bind with the dimeric miR-1587 G-quadruplex, but could not induce dimerization of miR-1587 G-quadruplex. These results provide a new strategy to regulate the functions of miR-1587 through induction of G-quadruplex formation and dimerization. Copyright © 2017 Elsevier B.V. All rights reserved.

Thermodynamic characterization of binding Oxytricha nova single strand telomere DNA with the alpha protein N-terminal domain.

PubMed

Buczek, Pawel; Horvath, Martin P

2006-06-23

The Oxytricha nova telemere binding protein alpha subunit binds single strand DNA and participates in a nucleoprotein complex that protects the very ends of chromosomes. To understand how the N-terminal, DNA binding domain of alpha interacts with DNA we measured the stoichiometry, enthalpy (DeltaH), entropy (DeltaS), and dissociation constant (K(D-DNA)) for binding telomere DNA fragments at different temperatures and salt concentrations using native gel electrophoresis and isothermal titration calorimetry (ITC). About 85% of the total free energy of binding corresponded with non-electrostatic interactions for all DNAs. Telomere DNA fragments d(T(2)G(4)), d(T(4)G(4)), d(G(3)T(4)G(4)), and d(G(4)T(4)G(4)) each formed monovalent protein complexes. In the case of d(T(4)G(4)T(4)G(4)), which has two tandemly repeated d(TTTTTGGGG) telomere motifs, two binding sites were observed. The high-affinity "A site" has a dissociation constant, K(D-DNA(A)) = 13(+/-4) nM, while the low-affinity "B site" is characterized by K(D-DNA(B)) = 5600(+/-600) nM at 25 degrees C. Nucleotide substitution variants verified that the A site corresponds principally with the 3'-terminal portion of d(T(4)G(4)T(4)G(4)). The relative contributions of entropy (DeltaS) and enthalpy (DeltaH) for binding reactions were DNA length-dependent as was heat capacity (DeltaCp). These trends with respect to DNA length likely reflect structural transitions in the DNA molecule that are coupled with DNA-protein association. Results presented here are important for understanding early intermediates and subsequent stages in the assembly of the full telomere nucleoprotein complex and how binding events can prepare the telomere DNA for extension by telomerase, a critical event in telomere biology.

N-TERMINALLY ELONGATED SpliInx2 AND SpliInx3 REDUCE BACULOVIRUS-TRIGGERED APOPTOSIS VIA HEMICHANNEL CLOSURE.

PubMed

Chen, Ya-Bin; Xiao, Wei; Li, Ming; Zhang, Yan; Yang, Yang; Hu, Jian-Sheng; Luo, Kai-Jun

2016-05-01

The hemichannel and gap junction channel are major portals for the release of factors responsible for the effects of apoptotic cells on the spread of apoptosis to neighboring cells and apoptotic corpse clearance, typically by phagocytes. The N-terminal cytoplasmic domain in the connexins, gap junction proteins in vertebrate, has been implicated in regulating channel closure. However, little is known about how the hemichannel close responds to apoptotic signaling transduction leading to the reduction of neighboring cellular apoptosis in an invertebrate. An insect Bac-to-Bac expression system, pFastBac(TM) HT A, allows us to construct an N-terminally elongated SpliInx2 (Nte-Inx2) and SpliInx3 (Nte-Inx3). Here, we demonstrated that recombinant baculovirus Bac-Nte-Inx2 (reBac-Net-Inx2) and Bac-Nte-Inx3 (reBac-Nte-Inx3) closed the endogenous hemichannel on the Sf9 cell surface. Importantly, primary baculovirus infections significantly caused early apoptosis, and this apoptosis was reduced by hemichannel-closed Sf9 cells at 24-h post-infection (PI). Although N-terminal-elongated residue led to the increase in the phosphorylated sites in both Nte-Inx2 and Nte-Inx3 and an additional transmembrane domain in Nte-Inx3, both the proteins localized on the cell surface, suggesting Nte-Inxs proteins could mediate hemichannel closure. Further supporting evidence showed that hemichannel closure was dependent on N-Inxs expressed by baculovirus polyhedrin promoter, which began to express at 18-24 h PI. These results identify an unconventional function of N-terminal-elongated innexins that could act as a plug to manipulate hemichannel closure and provide a mechanism connecting the effect of hemichannel closure directly to apoptotic signaling transduction from intracellular to extracellular compartment. © 2016 Wiley Periodicals, Inc.

Synthesis and Characterization of an f-Block Terminal Parent Imido [U=NH] Complex: A Masked Uranium(IV) Nitride

PubMed Central

2014-01-01

Deprotonation of [U(TrenTIPS)(NH2)] (1) [TrenTIPS = N(CH2CH2NSiPri3)3] with organoalkali metal reagents MR (M = Li, R = But; M = Na–Cs, R = CH2C6H5) afforded the imido-bridged dimers [{U(TrenTIPS)(μ-N[H]M)}2] [M = Li–Cs (2a–e)]. Treatment of 2c (M = K) with 2 equiv of 15-crown-5 ether (15C5) afforded the uranium terminal parent imido complex [U(TrenTIPS)(NH)][K(15C5)2] (3c), which can also be viewed as a masked uranium(IV) nitride. The uranium–imido linkage was found to be essentially linear, and theoretical calculations suggested σ2π4 polarized U–N multiple bonding. Attempts to oxidize 3c to afford the neutral uranium terminal parent imido complex [U(TrenTIPS)(NH)] (4) resulted in spontaneous disproportionation to give 1 and the uranium–nitride complex [U(TrenTIPS)(N)] (5); this reaction is a new way to prepare the terminal uranium–nitride linkage and was calculated to be exothermic by −3.25 kcal mol–1. PMID:24697157

Optoelectronic properties and structural effects of the incremental addition of pyridyl moieties on a rhodium dimer.

PubMed

Chartrand, Daniel; Hanan, Garry S

2014-11-13

The synthesis and characterization of five C-C coupling products obtained from the reaction of a paddlewheel tetrakis 4-bromo-N,N'-diphenylbenzamidinate dirhodium dimer with 4-pyridineboronic acid pinacol ester are reported. The coupling reactions occur on one to four amidinate ligands, leading to rhodium dimers containing [tetrakis, tris, cis-bis, trans-bis, or mono]-N,N'-diphenyl-4-(pyridin-4-yl)benzamidinate ligands, effectively creating new binding sites on the metal complexes. The new compounds were isolated by column chromatography, and the exact conformations were verified by X-ray crystallography. Redox processes showed only a small variation within the coupling products and included two oxidations (1.30 ± 0.02 V, 0.27 ± 0.01 V vs SCE) and one reduction (-1.55 ± 0.02 V vs SCE), all centered on the Rh-Rh core. Time-dependent density functional theory (TD-DFT) was used to analyze this series with four other fully characterized N,N'-diphenyl-aryl-amidinate rhodium dimers that were found in the literature. The two main absorption bands of these nine rhodium dimers were compared to TD-DFT calculations, both giving excellent correlation. The first, a metal-to-metal (MM) transition around 11800 cm(-1) (845 nm) was blue-shifted in the calculation, with an average difference of 1378 cm(-1) but had only a 15 cm(-1) standard deviation, showing a strong correlation despite the energy difference. The second, a metal-to-ligand charge transfer (MLCT) transition around 18900 cm(-1) (530 nm) was a near perfect match with only a 64 cm(-1) average difference and a 35 cm(-1) standard deviation. The electronic transition, redox potentials, and HOMO and LUMO energies of all dimers were plotted versus the Hammett parameter (σ) of the aryl group and Taft's model with 2 components: field effects (σF) and resonance (σR). The properties involving only the Rh-Rh core (MM band, all oxidation potentials, HOMO and LUMO) were fit with a single set of σF and σR contributions (73

Effects of site-directed mutagenesis of Asn116 in the β-hairpin of the N-terminal domain of thermolysin on its activity and stability.

PubMed

Menach, Evans; Yasukawa, Kiyoshi; Inouye, Kuniyo

2012-09-01

In the N-terminal domain of thermolysin, two anti-parallel β-strands, Asn112-Ala113-Phe114-Trp115 and Ser118-Gln119-Met120-Val121-Tyr122 are connected by an Asn116-Gly117 turn to form a β-hairpin structure. In this study, we examined the role of Asn116 in the activity and stability of thermolysin by site-directed mutagenesis. Of the 19 Asn116 variants, four (N116A, N116D, N116T and N116Q) were produced in Escherichia coli, by co-expressing the mature and pro domains separately, while the other 15 were not. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide (FAGLA) at 25°C, the intrinsic k(cat)/K(m) value of N116D was 320% of that of the wild-type thermolysin (WT), and in the hydrolysis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (ZDFM) at pH 7.5 at 25°C, the k(cat)/K(m) value of N116D was 140% of that of WT, indicating that N116D exhibited higher activity than WT. N116Q exhibited similar activity as WT, and N116A and N116T exhibited reduced activities. The first-order rate constants, k(obs), of the thermal inactivation at 80°C were in the order N116A, N116D, N116T > N116Q > WT at all CaCl(2) concentrations examined (1-100 mM), indicating that all variants exhibited reduced stabilities. These results suggest that Asn116 plays an important role in the activity and stability of thermolysin presumably by stabilizing this β-hairpin structure.

Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor.

PubMed

Kourouniotis, George; Wang, Yi; Pennock, Steven; Chen, Xinmei; Wang, Zhixiang

2016-07-25

The binding of epidermal growth factor (EGF) to EGF receptor (EGFR) stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internalization is still very controversial. In this study, we constructed a chimeric EGFR by replacing its extracellular domain with leucine zipper (LZ) and tagged a green fluorescent protein (GFP) at its C-terminus. We showed that the chimeric LZ-EGFR-GFP was constitutively dimerized. The LZ-EGFR-GFP dimer autophosphorylated each of its five well-defined C-terminal tyrosine residues as the ligand-induced EGFR dimer does. Phosphorylated LZ-EGFR-GFP was localized to both the plasma membrane and endosomes, suggesting it is capable of endocytosis. We also showed that LZ-EGFR-GFP activated major signalling proteins including Src homology collagen-like (Shc), extracellular signal-regulated kinase (ERK) and Akt. Moreover, LZ-EGFR-GFP was able to stimulate cell proliferation. These results indicate that non-ligand induced dimerization is sufficient to activate EGFR and initiate cell signalling and EGFR endocytosis. We conclude that receptor dimerization is a critical event in EGF-induced cell signalling and EGFR endocytosis.

Paralogs of the C-Terminal Domain of the Cyanobacterial Orange Carotenoid Protein Are Carotenoid Donors to Helical Carotenoid Proteins1[OPEN

PubMed Central

Muzzopappa, Fernando; Wilson, Adjélé; Yogarajah, Vinosa; Cot, Sandrine; Perreau, François; Montigny, Cédric; Bourcier de Carbon, Céline

2017-01-01

The photoactive Orange Carotenoid Protein (OCP) photoprotects cyanobacteria cells by quenching singlet oxygen and excess excitation energy. Its N-terminal domain is the active part of the protein, and the C-terminal domain regulates the activity. Recently, the characteristics of a family of soluble carotenoid-binding proteins (Helical Carotenoid Proteins [HCPs]), paralogs of the N-terminal domain of OCP, were described. Bioinformatics studies also revealed the existence of genes coding for homologs of CTD. Here, we show that the latter genes encode carotenoid proteins (CTDHs). This family of proteins contains two subgroups with distinct characteristics. One CTDH of each clade was further characterized, and they proved to be very good singlet oxygen quenchers. When synthesized in Escherichia coli or Synechocystis PCC 6803, CTDHs formed dimers that share a carotenoid molecule and are able to transfer their carotenoid to apo-HCPs and apo-OCP. The CTDHs from clade 2 have a cysteine in position 103. A disulfide bond is easily formed between the monomers of the dimer preventing carotenoid transfer. This suggests that the transfer of the carotenoid could be redox regulated in clade 2 CTDH. We also demonstrate here that apo-OCPs and apo-CTDHs are able to take the carotenoid directly from membranes, while HCPs are unable to do so. HCPs need the presence of CTDH to become holo-proteins. We propose that, in cyanobacteria, the CTDHs are carotenoid donors to HCPs. PMID:28935842

Probing Intermolecular Electron Delocalization in Dimer Radical Anions by Vibrational Spectroscopy

DOE PAGES

Mani, Tomoyasu; Grills, David C.

2017-07-05

Delocalization of charges is one of the factors controlling charge transport in conjugated molecules. It is considered to play an important role in the performance of a wide range of molecular technologies, including organic solar cells and organic electronics. Dimerization reactions are well-suited as a model to investigate intermolecular spatial delocalization of charges. And while dimerization reactions of radical cations are well investigated, studies on radical anions are still scarce. Upon dimerization of radical anions with neutral counterparts, an electron is considered to delocalize over the two molecules. By using time-resolved infrared (TRIR) detection coupled with pulse radiolysis, we showmore » that radical anions of 4-n-hexyl-4'-cyanobiphenyl (6CB) undergo such dimerization reactions, with an electron equally delocalized over the two molecules. We have recently demonstrated that nitrile ν(C≡N) vibrations respond to the degree of electron localization of nitrile-substituted anions: we can quantify the changes in the electronic charges from the neutral to the anion states in the nitriles by monitoring the ν(C≡N) IR shifts. In the first part of this article, we show that the sensitivity of the ν(C≡N) IR shifts does not depend on solvent polarity. In the second part, we describe how probing the shifts of the nitrile IR vibrational band unambiguously confirms the formation of dimer radical anions, with K dim = 3 × 10 4 M –1. IR findings are corroborated by electronic absorption spectroscopy and electronic structure calculations. We find that the presence of a hexyl chain and the formation of π–π interactions are both crucial for dimerization of radical anions of 6CB with neutral 6CB. Our study provides clear evidence of spatial delocalization of electrons over two molecular fragments.« less

Dimerization of human immunodeficiency virus (type 1) RNA: stimulation by cations and possible mechanism.

PubMed

Marquet, R; Baudin, F; Gabus, C; Darlix, J L; Mougel, M; Ehresmann, C; Ehresmann, B

1991-05-11

The retroviral genome consists of two identical RNA molecules joined close to their 5' ends by the dimer linkage structure. Recent findings indicated that retroviral RNA dimerization and encapsidation are probably related events during virion assembly. We studied the cation-induced dimerization of HIV-1 RNA and results indicate that all in vitro generated HIV-1 RNAs containing a 100 nucleotide domain downstream from the 5' splice site are able to dimerize. RNA dimerization depends on the concentration of RNA, mono- and multivalent cations, the size of the monovalent cation, temperature, and pH. Up to 75% of HIV-1 RNA is dimeric in the presence of spermidine. HIV-1 RNA dimer is fairly resistant to denaturing agents and unaffected by intercalating drugs. Antisense HIV-1 RNA does not dimerize but heterodimers can be formed between HIV-1 RNA and either MoMuLV or RSV RNA. Therefore retroviral RNA dimerization probably does not simply proceed through mechanisms involving Watson-Crick base-pairing. Neither adenine and cytosine protonation, nor quartets containing only guanines appear to determine the stability of the HIV-1 RNA dimer, while quartets involving both adenine(s) and guanine(s) could account for our results. A consensus sequence PuGGAPuA found in the putative dimerization-encapsidation region of all retroviral genomes examined may participate in the dimerization process.

Dimerization of human immunodeficiency virus (type 1) RNA: stimulation by cations and possible mechanism.

PubMed Central

Marquet, R; Baudin, F; Gabus, C; Darlix, J L; Mougel, M; Ehresmann, C; Ehresmann, B

1991-01-01

The retroviral genome consists of two identical RNA molecules joined close to their 5' ends by the dimer linkage structure. Recent findings indicated that retroviral RNA dimerization and encapsidation are probably related events during virion assembly. We studied the cation-induced dimerization of HIV-1 RNA and results indicate that all in vitro generated HIV-1 RNAs containing a 100 nucleotide domain downstream from the 5' splice site are able to dimerize. RNA dimerization depends on the concentration of RNA, mono- and multivalent cations, the size of the monovalent cation, temperature, and pH. Up to 75% of HIV-1 RNA is dimeric in the presence of spermidine. HIV-1 RNA dimer is fairly resistant to denaturing agents and unaffected by intercalating drugs. Antisense HIV-1 RNA does not dimerize but heterodimers can be formed between HIV-1 RNA and either MoMuLV or RSV RNA. Therefore retroviral RNA dimerization probably does not simply proceed through mechanisms involving Watson-Crick base-pairing. Neither adenine and cytosine protonation, nor quartets containing only guanines appear to determine the stability of the HIV-1 RNA dimer, while quartets involving both adenine(s) and guanine(s) could account for our results. A consensus sequence PuGGAPuA found in the putative dimerization-encapsidation region of all retroviral genomes examined may participate in the dimerization process. Images PMID:1645868

Structural Rearrangement in an RsmA/CsrA Ortholog of Pseudomonas aeruginosa Creates a Dimeric RNA-Binding Protein, RsmN

PubMed Central

Morris, Elizabeth R.; Hall, Gareth; Li, Chan; Heeb, Stephan; Kulkarni, Rahul V.; Lovelock, Laura; Silistre, Hazel; Messina, Marco; Cámara, Miguel; Emsley, Jonas; Williams, Paul; Searle, Mark S.

2013-01-01

Summary In bacteria, the highly conserved RsmA/CsrA family of RNA-binding proteins functions as global posttranscriptional regulators acting on mRNA translation and stability. Through phenotypic complementation of an rsmA mutant in Pseudomonas aeruginosa, we discovered a family member, termed RsmN. Elucidation of the RsmN crystal structure and that of the complex with a hairpin from the sRNA, RsmZ, reveals a uniquely inserted α helix, which redirects the polypeptide chain to form a distinctly different protein fold to the domain-swapped dimeric structure of RsmA homologs. The overall β sheet structure required for RNA recognition is, however, preserved with compensatory sequence and structure differences, allowing the RsmN dimer to target binding motifs in both structured hairpin loops and flexible disordered RNAs. Phylogenetic analysis indicates that, although RsmN appears unique to P. aeruginosa, homologous proteins with the inserted α helix are more widespread and arose as a consequence of a gene duplication event. PMID:23954502

Global Structure of a Three-Way Junction in a Phi29 Packaging RNA Dimer Determined Using Site-Directed Spin Labeling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Xiaojun; Tung, Chang-Shung; Sowa, Glenna

2012-02-08

The condensation of bacteriophage phi29 genomic DNA into its preformed procapsid requires the DNA packaging motor, which is the strongest known biological motor. The packaging motor is an intricate ring-shaped protein/RNA complex, and its function requires an RNA component called packaging RNA (pRNA). Current structural information on pRNA is limited, which hinders studies of motor function. Here, we used site-directed spin labeling to map the conformation of a pRNA three-way junction that bridges binding sites for the motor ATPase and the procapsid. The studies were carried out on a pRNA dimer, which is the simplest ring-shaped pRNA complex and servesmore » as a functional intermediate during motor assembly. Using a nucleotide-independent labeling scheme, stable nitroxide radicals were attached to eight specific pRNA sites without perturbing RNA folding and dimer formation, and a total of 17 internitroxide distances spanning the three-way junction were measured using Double Electron-Electron Resonance spectroscopy. The measured distances, together with steric chemical constraints, were used to select 3662 viable three-way junction models from a pool of 65 billion. The results reveal a similar conformation among the viable models, with two of the helices (HT and HL) adopting an acute bend. This is in contrast to a recently reported pRNA tetramer crystal structure, in which HT and HL stack onto each other linearly. The studies establish a new method for mapping global structures of complex RNA molecules, and provide information on pRNA conformation that aids investigations of phi29 packaging motor and developments of pRNA-based nanomedicine and nanomaterial.« less

Mycobacterium tuberculosis NmtR harbors a nickel sensing site with parallels to Escherichia coli RcnR†

PubMed Central

Reyes-Caballero, Hermes; Lee, Chul Won; Giedroc, David P.

2011-01-01

Mycobacterium tuberculosis NmtR is a Ni(II)/Co(II)-sensing metalloregulatory protein from the extensively studied ArsR/SmtB family. Two Ni(II) ions bind to the NmtR dimer to form octahedral coordination complexes with stepwise binding affinities of KNi1=1.2 (±0.1) × 1010 and KNi2=0.7 (±0.4) × 1010 M-1 (pH 7.0). A glutamine scanning mutagenesis approach reveals that Asp91, His93, His104 and His107, all contained within the C-terminal α5 helix, and His3 as part of the conserved α-NH2-Gly2-His3-Gly4 motif at the N-terminus make significant contributions to the magnitude of KNi. In contrast, substitution of residues from the C-terminal region, His109, Asp114 and His116, previously implicated in Ni(II) binding and metalloregulation in cells, gives rise to wild-type KNi and Ni(II)-dependent allosteric coupling free energies. Interestingly, deletion of residues 112-120 in the C-terminal region (Δ111 NmtR) reduces the Ni(II) binding stoichiometry to one per dimer and greatly reduces Ni(II) responsiveness. H3Q and Δ111 NmtRs also show clear perturbations in the rank order of metal responsiveness to Ni(II), Co(II) and Zn(II) that is distinct from wild-type NmtR. 15N relaxation experiments with apo-NmtR reveal that both N-terminal (residues 2-14) and C-terminal (residues 110-120) regions are unstructured in solution, and this property likely dictates the metal specificity profile characteristic of the Ni(II)-sensor NmtR relative to other ArsR family regulators. PMID:21819125

A Convenient Approach to Synthesizing Peptide C-Terminal N-Alkyl Amides

PubMed Central

Fang, Wei-Jie; Yakovleva, Tatyana; Aldrich, Jane V.

2014-01-01

Peptide C-terminal N-alkyl amides have gained more attention over the past decade due to their biological properties, including improved pharmacokinetic and pharmacodynamic profiles. However, the synthesis of this type of peptide on solid phase by current available methods can be challenging. Here we report a convenient method to synthesize peptide C-terminal N-alkyl amides using the well-known Fukuyama N-alkylation reaction on a standard resin commonly used for the synthesis of peptide C-terminal primary amides, the PAL-PEG-PS (Peptide Amide Linker-polyethylene glycol-polystyrene) resin. The alkylation and oNBS deprotection were conducted under basic conditions and were therefore compatible with this acid labile resin. The alkylation reaction was very efficient on this resin with a number of different alkyl iodides or bromides, and the synthesis of model enkephalin N-alkyl amide analogs using this method gave consistently high yields and purities, demonstrating the applicability of this methodology. The synthesis of N-alkyl amides was more difficult on a Rink amide resin, especially the coupling of the first amino acid to the N-alkyl amine, resulting in lower yields for loading the first amino acid onto the resin. This method can be widely applied in the synthesis of peptide N-alkyl amides. PMID:22252422

Study of DNA Origami Dimerization and Dimer Dissociation Dynamics and of the Factors that Limit Dimerization.

PubMed

Liber, Miran; Tomov, Toma E; Tsukanov, Roman; Berger, Yaron; Popov, Mary; Khara, Dinesh C; Nir, Eyal

2018-06-01

Organizing DNA origami building blocks into higher order structures is essential for fabrication of large structurally and functionally diverse devices and molecular machines. Unfortunately, the yields of origami building block attachment reactions are typically not sufficient to allow programed assembly of DNA devices made from more than a few origami building blocks. To investigate possible reasons for these low yields, a detailed single-molecule fluorescence study of the dynamics of rectangular origami dimerization and origami dimer dissociation reactions is conducted. Reactions kinetics and yields are investigated at different origami and ion concentrations, for different ion types, for different lengths of bridging strands, and for the "sticky end" and "weaving welding" attachment techniques. Dimerization yields are never higher than 86%, which is typical for such systems. Analysis of the dynamic data shows that the low yield cannot be explained by thermodynamic instability or structural imperfections of the origami constructs. Atomic force microscopy and gel electrophoresis evidence reveal self-dimerization of the origami monomers, likely via blunt-end interactions made possible by the presence of bridging strands. It is suggested that this mechanism is the major factor that inhibits correct dimerization and means to overcome it are discussed. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The C- and N-Terminal Residues of Synthetic Heptapeptide Ion Channels Influence Transport Efficacy Through Phospholipid Bilayers

PubMed Central

Djedovič, Natasha; Ferdani, Riccardo; Harder, Egan; Pajewska, Jolanta; Pajewski, Robert; Weber, Michelle E.; Schlesinger, Paul H.; Gokel, George W.

2008-01-01

The synthetic peptide, R2N-COCH2OCH2CO-Gly-Gly-Gly-Pro-Gly-Gly-Gly-OR’, was shown to be selective for Cl- over K+ when R is n-octadecyl and R’ is benzyl. Nineteen heptapeptides have now been prepared in which the N-terminal and C-terminal residues have been varied. All of the N-terminal residues are dialkyl but the C-terminal chains are esters, 2° amides, or 3° amides. The compounds having varied N-terminal anchors and C-terminal benzyl groups are as follows: 1, R = n-propyl; 2, R = n-hexyl; 3, R = n-octyl; 4, R = n-decyl; 5, R = n-dodecyl; 6, R = n-tetradecyl; 7, R = n-hexadecyl; 8, R = n-octadecyl. Compounds 9-19 have R = n-octadecyl and C-terminal residues as follows: 9, OR’ = OCH2CH3; 10, OR’ = OCH(CH3)2; 11, OR’ = O(CH2)6CH3; 12, OR’ = OCH2-c-C6H11; 13, OR’ = O(CH2)9CH3; 14, OR’ = O (CH2)17CH3; 15, NR’2 = N[(CH2)6CH3]2; 16, NHR’ = NH(CH2)9CH3; 17, NR’2 = N[(CH2)9CH3]2; 18, NHR’ = NH(CH2)17CH3; 19, NR’2 = N[(CH2)17CH3]2. The highest anion transport activities were observed as follows. For the benzyl esters whose N-terminal residues were varied, i.e. 1-8, compound 3 was most active. For the C18 anchored esters 10-14, n-heptyl ester 11 was most active. For the C18 anchored, C-terminal amides 15-19, di-n-decylamide 17 was most active. It was concluded that both the C- and N-terminal anchors were important for channel function in the bilayer but that activity was lost unless only one of the two anchoring groups was dominant. PMID:19633728

The Cytoplasmic Adaptor Protein Dok7 Activates the Receptor Tyrosine Kinase MuSK via Dimerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bergamin, E.; Hallock, P; Burden, S

Formation of the vertebrate neuromuscular junction requires, among others proteins, Agrin, a neuronally derived ligand, and the following muscle proteins: LRP4, the receptor for Agrin; MuSK, a receptor tyrosine kinase (RTK); and Dok7 (or Dok-7), a cytoplasmic adaptor protein. Dok7 comprises a pleckstrin-homology (PH) domain, a phosphotyrosine-binding (PTB) domain, and C-terminal sites of tyrosine phosphorylation. Unique among adaptor proteins recruited to RTKs, Dok7 is not only a substrate of MuSK, but also an activator of MuSK's kinase activity. Here, we present the crystal structure of the Dok7 PH-PTB domains in complex with a phosphopeptide representing the Dok7-binding site on MuSK.more » The structure and biochemical data reveal a dimeric arrangement of Dok7 PH-PTB that facilitates trans-autophosphorylation of the kinase activation loop. The structure provides the molecular basis for MuSK activation by Dok7 and for rationalizing several Dok7 loss-of-function mutations found in patients with congenital myasthenic syndromes.« less

Specificity of cell–cell adhesion by classical cadherins: Critical role for low-affinity dimerization through β-strand swapping

PubMed Central

Chen, Chien Peter; Posy, Shoshana; Ben-Shaul, Avinoam; Shapiro, Lawrence; Honig, Barry H.

2005-01-01

Cadherins constitute a family of cell-surface proteins that mediate intercellular adhesion through the association of protomers presented from juxtaposed cells. Differential cadherin expression leads to highly specific intercellular interactions in vivo. This cell–cell specificity is difficult to understand at the molecular level because individual cadherins within a given subfamily are highly similar to each other both in sequence and structure, and they dimerize with remarkably low binding affinities. Here, we provide a molecular model that accounts for these apparently contradictory observations. The model is based in part on the fact that cadherins bind to one another by “swapping” the N-terminal β-strands of their adhesive domains. An inherent feature of strand swapping (or, more generally, the domain swapping phenomenon) is that “closed” monomeric conformations act as competitive inhibitors of dimer formation, thus lowering affinities even when the dimer interface has the characteristics of high-affinity complexes. The model describes quantitatively how small affinity differences between low-affinity cadherin dimers are amplified by multiple cadherin interactions to establish large specificity effects at the cellular level. It is shown that cellular specificity would not be observed if cadherins bound with high affinities, thus emphasizing the crucial role of strand swapping in cell–cell adhesion. Numerical estimates demonstrate that the strength of cellular adhesion is extremely sensitive to the concentration of cadherins expressed at the cell surface. We suggest that the domain swapping mechanism is used by a variety of cell-adhesion proteins and that related mechanisms to control affinity and specificity are exploited in other systems. PMID:15937105

Her4 and Her2/neu tyrosine kinase domains dimerize and activate in a reconstituted in vitro system.

PubMed

Monsey, John; Shen, Wei; Schlesinger, Paul; Bose, Ron

2010-03-05

Her4 (ErbB-4) and Her2/neu (ErbB-2) are receptor-tyrosine kinases belonging to the epidermal growth factor receptor (EGFR) family. Crystal structures of EGFR and Her4 kinase domains demonstrate kinase dimerization and activation through an allosteric mechanism. The kinase domains form an asymmetric dimer, where the C-lobe surface of one monomer contacts the N-lobe of the other monomer. EGFR kinase dimerization and activation in vitro was previously reported using a nickel-chelating lipid-liposome system, and we now apply this system to all other members of the EGFR family. Polyhistidine-tagged Her4, Her2/neu, and Her3 kinase domains are bound to these nickel-liposomes and are brought to high local concentration, mimicking what happens to full-length receptors in vivo following ligand binding. Addition of nickel-liposomes to Her4 kinase domain results in 40-fold activation in kinase activity and marked enhancement of C-terminal tail autophosphorylation. Activation of Her4 shows a sigmoidal dependence on kinase concentration, consistent with a cooperative process requiring kinase dimerization. Her2/neu kinase activity is also activated by nickel-liposomes, and is increased further by heterodimerization with Her3 or Her4. The ability of Her3 and Her4 to heterodimerize and activate other family members is studied in vitro. Her3 kinase domain readily activates Her2/neu but is a poor activator of Her4, which differs from the prediction made by the asymmetric dimer model. Mutation of Her3 residues (952)ENI(954) to the corresponding sequence in Her4 enhanced the ability of Her3 to activate Her4, demonstrating that sequence differences on the C-lobe surface influence the heterodimerization and activation of ErbB kinase domains.

Involvement of the N-terminal region in alpha-crystallin-lens membrane recognition

NASA Technical Reports Server (NTRS)

Ifeanyi, F.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

1991-01-01

Previous studies have demonstrated that alpha-crystallin binds specifically, in a saturable manner, to lens membrane. To determine the region of the alpha-crystallin molecule that might be involved in this binding, native alpha-crystallin from the bovine lens has been treated by limited digestion with trypsin, to produce alpha-A molecules with an intact C-terminal region, and a nicked N-terminal region. Compared to intact alpha-crystallin, trypsin-treated alpha-crystallin binds less avidly to lens membrane, suggesting that the N-terminal region of the alpha-A molecule may play a key role in the recognition between lens membrane and crystallin.

In-Operando Spatial Imaging of Edge Termination Electric Fields in GaN Vertical p-n Junction Diodes

DOE PAGES

Leonard, Francois; Dickerson, J. R.; King, M. P.; ...

2016-05-03

Control of electric fields with edge terminations is critical to maximize the performance of high-power electronic devices. We proposed a variety of edge termination designs which makes the optimization of such designs challenging due to many parameters that impact their effectiveness. And while modeling has recently allowed new insight into the detailed workings of edge terminations, the experimental verification of the design effectiveness is usually done through indirect means, such as the impact on breakdown voltages. In this letter, we use scanning photocurrent microscopy to spatially map the electric fields in vertical GaN p-n junction diodes in operando. We alsomore » reveal the complex behavior of seemingly simple edge termination designs, and show how the device breakdown voltage correlates with the electric field behavior. Modeling suggests that an incomplete compensation of the p-type layer in the edge termination creates a bilayer structure that leads to these effects, with variations that significantly impact the breakdown voltage.« less

Site-specific O-Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates β1-Adrenergic Receptor N-terminal Cleavage.

PubMed

Goth, Christoffer K; Tuhkanen, Hanna E; Khan, Hamayun; Lackman, Jarkko J; Wang, Shengjun; Narimatsu, Yoshiki; Hansen, Lasse H; Overall, Christopher M; Clausen, Henrik; Schjoldager, Katrine T; Petäjä-Repo, Ulla E

2017-03-17

The β 1 -adrenergic receptor (β 1 AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for β-adrenergic antagonists, such as β-blockers, relatively little is yet known about its regulation. We have shown previously that β 1 AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation and, moreover, that the receptor is modified by O -glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specifically O -glycosylates β 1 AR at five residues in the extracellular N terminus, including the Ser-49 residue at the location of the common S49G single-nucleotide polymorphism. Using in vitro O -glycosylation and proteolytic cleavage assays, a cell line deficient in O -glycosylation, GalNAc-T-edited cell line model systems, and a GalNAc-T2 knock-out rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of β 1 AR. Furthermore, we demonstrate that impaired O -glycosylation and enhanced proteolysis lead to attenuated receptor signaling, because the maximal response elicited by the βAR agonist isoproterenol and its potency in a cAMP accumulation assay were decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, a GPCR as a target for co-regulatory functions of site-specific O -glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of β 1 AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Site-specific O-Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates β1-Adrenergic Receptor N-terminal Cleavage*

PubMed Central

Goth, Christoffer K.; Tuhkanen, Hanna E.; Khan, Hamayun; Lackman, Jarkko J.; Wang, Shengjun; Narimatsu, Yoshiki; Hansen, Lasse H.; Overall, Christopher M.; Clausen, Henrik; Schjoldager, Katrine T.; Petäjä-Repo, Ulla E.

2017-01-01

The β1-adrenergic receptor (β1AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for β-adrenergic antagonists, such as β-blockers, relatively little is yet known about its regulation. We have shown previously that β1AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation and, moreover, that the receptor is modified by O-glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specifically O-glycosylates β1AR at five residues in the extracellular N terminus, including the Ser-49 residue at the location of the common S49G single-nucleotide polymorphism. Using in vitro O-glycosylation and proteolytic cleavage assays, a cell line deficient in O-glycosylation, GalNAc-T-edited cell line model systems, and a GalNAc-T2 knock-out rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of β1AR. Furthermore, we demonstrate that impaired O-glycosylation and enhanced proteolysis lead to attenuated receptor signaling, because the maximal response elicited by the βAR agonist isoproterenol and its potency in a cAMP accumulation assay were decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, a GPCR as a target for co-regulatory functions of site-specific O-glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of β1AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases. PMID:28167537

Theoretical study on the ring-opening hydrolysis reactions of N-alkylmaleimide dimers

NASA Astrophysics Data System (ADS)

Tan, Xue-Jie; Wang, Chao; Guo, Xian-Kun

2018-01-01

On the basis of our previous experimental results, the ring-opening hydrolysis reaction mechanisms of two kinds of N-alkylmaleimide dimers without or with the assistance of one and two water molecules have been theoretically investigated in detail. All possible geometries were optimized using the B3LYP/6-311+G(d,p)//B3LYP/6-31+G(d) method in the gas phase and ethanol solution. Calculated results show that every pathway is a four-step hydrolytic degradation process and every step could occur in a concerted way, instead of previously suggested asynchronous stepwise mechanism. Extra H2O or ethanol could act as carriers of proton. The results are consistent with experimental observations.

Detectability of Noble Gases in Jovian Atmospheres Utilizing Dimer Spectral Structures

NASA Astrophysics Data System (ADS)

Kim, S. J.; Min, Y.; Kim, Y.; Lee, Y.; Trafton, L.; Miller, S.; McKellar, A. R. W.

1997-07-01

The detection of jovian hydrogen-hydrogen dimers through the clear telluric 2-micron window (Kim et al. 1995; Trafton et al. 1997) suggests possibility to detect noble gases in the form of dimer with hydrogen in jovian atmospheres. Since noble gases do not have spectral structures in the infrared, it has been difficult to derive their abundances in the atmospheres of jovian planets. If there is a significant component of noble gases other than helium in the jovian atmospheres, it might be detected through its dimer spectrum with hydrogen molecule. The relatively sharp spectral structures of hydrogen-argon and hydrogen-neon dimers compared with those of hydrogen-hydrogen dimers are useful for the detection, if adequate S/N is obtained. However, these dimer structures should be much weaker than the nearby hydrogen-hydrogen features because noble gases are expected to be minor constituents of these atmospheres. We will discuss the detectability of these dimers based on laboratory measurements (McKellar, 1994; 1996), and current technology of infrared observations.

Engineering of a novel Ca{sup 2+}-regulated kinesin molecular motor using a calmodulin dimer linker

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shishido, Hideki; Maruta, Shinsaku, E-mail: maruta@soka.ac.jp

Highlights: Black-Right-Pointing-Pointer Engineered kinesin-M13 and calmodulin involving single cysteine were prepared. Black-Right-Pointing-Pointer CaM mutant was cross-linked to dimer by bifunctional thiol reactive reagent. Black-Right-Pointing-Pointer Kinesin-M13 was dimerized via CaM dimer in the presence of calcium. Black-Right-Pointing-Pointer Function of the engineered kinesin was regulated by a Ca{sup 2+}-calmodulin dimer linker. -- Abstract: The kinesin-microtubule system holds great promise as a molecular shuttle device within biochips. However, one current barrier is that such shuttles do not have 'on-off' control of their movement. Here we report the development of a novel molecular motor powered by an accelerator and brake system, using a kinesinmore » monomer and a calmodulin (CaM) dimer. The kinesin monomer, K355, was fused with a CaM target peptide (M13 peptide) at the C-terminal part of the neck region (K355-M13). We also prepared CaM dimers using CaM mutants (Q3C), (R86C), or (A147C) and crosslinkers that react with cysteine residues. Following induction of K355-M13 dimerization with CaM dimers, we measured K355-M13 motility and found that it can be reversibly regulated in a Ca{sup 2+}-dependent manner. We also found that velocities of K355-M13 varied depending on the type and crosslink position of the CaM dimer used; crosslink length also had a moderate effect on motility. These results suggest Ca{sup 2+}-dependent dimerization of K355-M13 could be used as a novel molecular shuttle, equipped with an accelerator and brake system, for biochip applications.« less

Determination of structure of the MinD-ATP complex reveals the orientation of MinD on the membrane and the relative location of the binding sites for MinE and MinC

PubMed Central

Wu, Wei; Park, Kyung-Tae; Holyoak, Todd; Lutkenhaus, Joe

2011-01-01

Summary The three Min proteins spatially regulate Z ring positioning in E. coli and are dynamically associated with the membrane. MinD binds to vesicles in the presence of ATP and can recruit MinC or MinE. Biochemical and genetic evidence indicate the binding sites for these two proteins on MinD overlap. Here we solved the structure of a hydrolytic-deficient mutant of MinD truncated for the C-terminal amphipathic helix involved in binding to the membrane. The structure solved in the presence of ATP is a dimer and reveals the face of MinD abutting the membrane. Using a combination of random and extensive site-directed mutagenesis additional residues important for MinE and MinC binding were identified. The location of these residues on the MinD structure confirms that the binding sites overlap and reveals that the binding sites are at the dimer interface and exposed to the cytosol. The location of the binding sites at the dimer interface offers a simple explanation for the ATP-dependency of MinC and MinE binding to MinD. PMID:21231967

10 CFR 40.42 - Expiration and termination of licenses and decommissioning of sites and separate buildings or...

Code of Federal Regulations, 2010 CFR

2010-01-01

... 10 Energy 1 2010-01-01 2010-01-01 false Expiration and termination of licenses and decommissioning of sites and separate buildings or outdoor areas. 40.42 Section 40.42 Energy NUCLEAR REGULATORY COMMISSION DOMESTIC LICENSING OF SOURCE MATERIAL Licenses § 40.42 Expiration and termination of licenses and...

10 CFR 40.42 - Expiration and termination of licenses and decommissioning of sites and separate buildings or...

Code of Federal Regulations, 2014 CFR

2014-01-01

... 10 Energy 1 2014-01-01 2014-01-01 false Expiration and termination of licenses and decommissioning of sites and separate buildings or outdoor areas. 40.42 Section 40.42 Energy NUCLEAR REGULATORY COMMISSION DOMESTIC LICENSING OF SOURCE MATERIAL Licenses § 40.42 Expiration and termination of licenses and...

10 CFR 40.42 - Expiration and termination of licenses and decommissioning of sites and separate buildings or...

Code of Federal Regulations, 2012 CFR

2012-01-01

... 10 Energy 1 2012-01-01 2012-01-01 false Expiration and termination of licenses and decommissioning of sites and separate buildings or outdoor areas. 40.42 Section 40.42 Energy NUCLEAR REGULATORY COMMISSION DOMESTIC LICENSING OF SOURCE MATERIAL Licenses § 40.42 Expiration and termination of licenses and...

10 CFR 40.42 - Expiration and termination of licenses and decommissioning of sites and separate buildings or...

Code of Federal Regulations, 2013 CFR

2013-01-01

... 10 Energy 1 2013-01-01 2013-01-01 false Expiration and termination of licenses and decommissioning of sites and separate buildings or outdoor areas. 40.42 Section 40.42 Energy NUCLEAR REGULATORY COMMISSION DOMESTIC LICENSING OF SOURCE MATERIAL Licenses § 40.42 Expiration and termination of licenses and...

10 CFR 40.42 - Expiration and termination of licenses and decommissioning of sites and separate buildings or...

Code of Federal Regulations, 2011 CFR

2011-01-01

... 10 Energy 1 2011-01-01 2011-01-01 false Expiration and termination of licenses and decommissioning of sites and separate buildings or outdoor areas. 40.42 Section 40.42 Energy NUCLEAR REGULATORY COMMISSION DOMESTIC LICENSING OF SOURCE MATERIAL Licenses § 40.42 Expiration and termination of licenses and...

The R35 residue of the influenza A virus NS1 protein has minimal effects on nuclear localization but alters virus replication through disrupting protein dimerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lalime, Erin N.; Pekosz, Andrew, E-mail: apekosz@jhsph.edu

The influenza A virus NS1 protein has a nuclear localization sequence (NLS) in the amino terminal region. This NLS overlaps sequences that are important for RNA binding as well as protein dimerization. To assess the significance of the NS1 NLS on influenza virus replication, the NLS amino acids were individually mutated to alanines and recombinant viruses encoding these mutations were rescued. Viruses containing NS1 proteins with mutations at R37, R38 and K41 displayed minimal changes in replication or NS1 protein nuclear localization. Recombinant viruses encoding NS1 R35A were not recovered but viruses containing second site mutations at position D39 inmore » addition to the R35A mutation were isolated. The mutations at position 39 were shown to partially restore NS1 protein dimerization but had minimal effects on nuclear localization. These data indicate that the amino acids in the NS1 NLS region play a more important role in protein dimerization compared to nuclear localization. - Highlights: • Mutations were introduced into influenza NS1 NLS1. • NS1 R37A, R38A, K41A viruses had minimal changes in replication and NS1 localization. • Viruses from NS1 R35A rescue all contained additional mutations at D39. • NS1 R35A D39X mutations recover dimerization lost in NS1 R35A mutations. • These results reaffirm the importance of dimerization for NS1 protein function.« less

Dimerization of the docking/adaptor protein HEF1 via a carboxy-terminal helix-loop-helix domain.

PubMed

Law, S F; Zhang, Y Z; Fashena, S J; Toby, G; Estojak, J; Golemis, E A

1999-10-10

HEF1, p130(Cas), and Efs define a family of multidomain docking proteins which plays a central coordinating role for tyrosine-kinase-based signaling related to cell adhesion. HEF1 function has been specifically implicated in signaling pathways important for cell adhesion and differentiation in lymphoid and epithelial cells. While the SH3 domains and SH2-binding site domains (substrate domains) of HEF1 family proteins are well characterized and binding partners known, to date the highly conserved carboxy-terminal domains of the three proteins have lacked functional definition. In this study, we have determined that the carboxy-terminal domain of HEF1 contains a divergent helix-loop-helix (HLH) motif. This motif mediates HEF1 homodimerization and HEF1 heterodimerization with a recognition specificity similar to that of the transcriptional regulatory HLH proteins Id2, E12, and E47. We had previously demonstrated that the HEF1 carboxy-terminus expressed as a separate domain in yeast reprograms cell division patterns, inducing constitutive pseudohyphal growth. Here we show that pseudohyphal induction by HEF1 requires an intact HLH, further supporting the idea that this motif has an effector activity for HEF1, and implying that HEF1 pseudohyphal activity derives in part from interactions with yeast helix-loop-helix proteins. These combined results provide initial insight into the mode of function of the HEF1 carboxy-terminal domain and suggest that the HEF1 protein may interact with cellular proteins which control differentiation. Copyright 1999 Academic Press.

Comparison of effect of gamma ray irradiation on wild-type and N-terminal mutants of αA-crystallin.

PubMed

Ramkumar, Srinivasagan; Fujii, Noriko; Fujii, Norihiko; Thankappan, Bency; Sakaue, Hiroaki; Ingu, Kim; Natarajaseenivasan, Kalimuthusamy; Anbarasu, Kumarasamy

2014-01-01

To study the comparative structural and functional changes between wild-type (wt) and N-terminal congenital cataract causing αA-crystallin mutants (R12C, R21L, R49C, and R54C) upon exposure to different dosages of gamma rays. Alpha A crystallin N-terminal mutants were created with the site-directed mutagenesis method. The recombinantly overexpressed and purified wt and mutant proteins were used for further studies. A (60)Co source was used to generate gamma rays to irradiate wild and mutant proteins at dosages of 0.5, 1.0, and 2.0 kGy. The biophysical property of the gamma irradiated (GI) and non-gamma irradiated (NGI) αA-crystallin wt and N-terminal mutants were determined. Oligomeric size was determined by size exclusion high-performance liquid chromatography (HPLC), the secondary structure with circular dichroism (CD) spectrometry, conformation of proteins with surface hydrophobicity, and the functional characterization were determined regarding chaperone activity using the alcohol dehydrogenase (ADH) aggregation assay. αA-crystallin N-terminal mutants formed high molecular weight (HMW) cross-linked products as well as aggregates when exposed to GI compared to the NGI wt counterparts. Furthermore, all mutants exhibited changed β-sheet and random coil structure. The GI mutants demonstrated decreased surface hydrophobicity when compared to αA-crystallin wt at 0, 1.0, and 1.5 kGy; however, at 2.0 kGy a drastic increase in hydrophobicity was observed only in the mutant R54C, not the wt. In contrast, chaperone activity toward ADH was gradually elevated at the minimum level in all GI mutants, and significant elevation was observed in the R12C mutant. Our findings suggest that the N-terminal mutants of αA-crystallin are structurally and functionally more sensitive to GI when compared to their NGI counterparts and wt. Protein oxidation as a result of gamma irradiation drives the protein to cross-link and aggregate culminating in cataract formation.

Deglacial hydrography and IRD inputs: A comparison of Terminations I and II in the N.E. Atlantic

NASA Astrophysics Data System (ADS)

Hibbert, Fiona; Chapman, Mark; Austin, William; Rohling, Eelco

2015-04-01

We present a high resolution marine record (MD04-2822) from the N.E. Atlantic. This record captures the demise of the penultimate glaciation (Termination II) in high resolution. The record of co-registered proxies offers the opportunity to investigate the evolution of the last two deglacial events in the North Atlantic. The deglacial evolution of Termination II is much less well documented than the last deglaciation (Termination I). A striking feature of Termination II in the MD04-2822 record, are several large (~1 ‰) oscillations in benthic δ18O, reflecting oscillations in sea level (e.g. Grant et al., 2012, Thomas et al., 2009) and/or deep sea temperatures (cf. Skinner and Shackleton, 2006). Also notable is the markedly different pattern of surface and deep water evolution for the two deglaciations. Termination I is characterised by a short offset between benthic δ18O decrease and δ13C increase (and northwards migration of the polar front) whereas during Termination II, benthic δ13C 'improvement' (and inferred resumption in overturning) occurs only during the Marine Isotope Stage (MIS) 5e plateau, giving the marine record it's characteristic 'drawn-out' appearance. The most conspicuous feature of the penultimate deglacial in most marine cores is Heinrich event 11 (H11), an extensive episode of ice rafted debris (IRD) discharge that spread across the North Atlantic to the margin of what is now the subtropical gyre (Chapman et al., 2000). H11 generally manifests in marine records as one large and long (~ 2.5 ka) event throughout the Termination. In MD04-2822 however, there are multiple IRD events within the Termination. The continued influence of the disintegrating N. hemisphere ice sheets is also evident within the benthic δ13C and surface conditions (the polar front migrates north of the core site early within MIS 5e following a brief SST reversal).

The Folding of de Novo Designed Protein DS119 via Molecular Dynamics Simulations.

PubMed

Wang, Moye; Hu, Jie; Zhang, Zhuqing

2016-04-26

As they are not subjected to natural selection process, de novo designed proteins usually fold in a manner different from natural proteins. Recently, a de novo designed mini-protein DS119, with a βαβ motif and 36 amino acids, has folded unusually slowly in experiments, and transient dimers have been detected in the folding process. Here, by means of all-atom replica exchange molecular dynamics (REMD) simulations, several comparably stable intermediate states were observed on the folding free-energy landscape of DS119. Conventional molecular dynamics (CMD) simulations showed that when two unfolded DS119 proteins bound together, most binding sites of dimeric aggregates were located at the N-terminal segment, especially residues 5-10, which were supposed to form β-sheet with its own C-terminal segment. Furthermore, a large percentage of individual proteins in the dimeric aggregates adopted conformations similar to those in the intermediate states observed in REMD simulations. These results indicate that, during the folding process, DS119 can easily become trapped in intermediate states. Then, with diffusion, a transient dimer would be formed and stabilized with the binding interface located at N-terminals. This means that it could not quickly fold to the native structure. The complicated folding manner of DS119 implies the important influence of natural selection on protein-folding kinetics, and more improvement should be achieved in rational protein design.

N-terminal pro-brain natriuretic peptide in acute Kawasaki disease correlates with coronary artery involvement.

PubMed

Adjagba, Philippe M; Desjardins, Laurent; Fournier, Anne; Spigelblatt, Linda; Montigny, Martine; Dahdah, Nagib

2015-10-01

We have lately documented the importance of N-terminal pro-brain natriuretic peptide in aiding the diagnosis of Kawasaki disease. We sought to investigate the potential value of N-terminal pro-brain natriuretic peptide pertaining to the prediction of coronary artery dilatation (Z-score>2.5) and/or of resistance to intravenous immunoglobulin therapy. We hypothesised that increased serum N-terminal pro-brain natriuretic peptide level correlates with increased coronary artery dilatation and/or resistance to intravenous immunoglobulin. We carried out a prospective study involving newly diagnosed patients treated with 2 g/kg intravenous immunoglobulin within 5-10 days of onset of fever. Echocardiography was performed in all patients at onset, then weekly for 3 weeks, then at month 2, and month 3. Coronary arteries were measured at each visit, and coronary artery Z-score was calculated. All the patients had N-terminal pro-brain natriuretic peptide serum level measured at onset, and the Z-score calculated. There were 109 patients enrolled at 6.58±2.82 days of fever, age 3.79±2.92 years. High N-terminal pro-brain natriuretic peptide level was associated with coronary artery dilatation at onset in 22.2 versus 5.6% for normal N-terminal pro-brain natriuretic peptide levels (odds ratio 4.8 [95% confidence interval 1.05-22.4]; p=0.031). This was predictive of cumulative coronary artery dilatation for the first 3 months (p=0.04-0.02), but not during convalescence at 2-3 months (odds ratio 1.28 [95% confidence interval 0.23-7.3]; p=non-significant). Elevated N-terminal pro-brain natriuretic peptide levels did not predict intravenous immunoglobulin resistance, 15.3 versus 13.5% (p=1). Elevated N-terminal pro-brain natriuretic peptide level correlates with acute coronary artery dilatation in treated Kawasaki disease, but not with intravenous immunoglobulin resistance.

C-terminal oligomerization of podocin mediates interallelic interactions.

PubMed

Stráner, Pál; Balogh, Eszter; Schay, Gusztáv; Arrondel, Christelle; Mikó, Ágnes; L'Auné, Gerda; Benmerah, Alexandre; Perczel, András; K Menyhárd, Dóra; Antignac, Corinne; Mollet, Géraldine; Tory, Kálmán

2018-07-01

Interallelic interactions of membrane proteins are not taken into account while evaluating the pathogenicity of sequence variants in autosomal recessive disorders. Podocin, a membrane-anchored component of the slit diaphragm, is encoded by NPHS2, the major gene mutated in hereditary podocytopathies. We formerly showed that its R229Q variant is only pathogenic when trans-associated to specific 3' mutations and suggested the causal role of an abnormal C-terminal dimerization. Here we show by FRET analysis and size exclusion chromatography that podocin oligomerization occurs exclusively through the C-terminal tail (residues 283-382): principally through the first C-terminal helical region (H1, 283-313), which forms a coiled coil as shown by circular dichroism spectroscopy, and through the 332-348 region. We show the principal role of the oligomerization sites in mediating interallelic interactions: while the monomer-forming R286Tfs*17 podocin remains membranous irrespective of the coexpressed podocin variant identity, podocin variants with an intact H1 significantly influence each other's localization (r 2  = 0.68, P = 9.2 × 10 -32 ). The dominant negative effect resulting in intracellular retention of the pathogenic F344Lfs*4-R229Q heterooligomer occurs in parallel with a reduction in the FRET efficiency, suggesting the causal role of a conformational rearrangement. On the other hand, oligomerization can also promote the membrane localization: it can prevent the endocytosis of F344Lfs*4 or F344* podocin mutants induced by C-terminal truncation. In conclusion, C-terminal oligomerization of podocin can mediate both a dominant negative effect and interallelic complementation. Interallelic interactions of NPHS2 are not restricted to the R229Q variant and have to be considered in compound heterozygous individuals. Copyright © 2018 Elsevier B.V. All rights reserved.

DEVELOPMENT OF THE SIGMA-1 RECEPTOR IN C-TERMINALS OF MOTONEURONS AND COLOCALIZATION WITH THE N,N’-DIMETHYLTRYPTAMINE FORMING ENZYME, INDOLE-N-METHYL TRANSFERASE

PubMed Central

Mavlyutov, Timur A.; Epstein, Miles L.; Liu, Patricia; Verbny, Yakov I.; Ziskind-Conhaim, Lea; Ruoho, Arnold E.

2012-01-01

The function of the sigma-1 receptor (S1R) has been linked to modulating the activities of ion channels and G-protein coupled receptors (GPCR). In the CNS the S1R is expressed ubiquitously but is enriched in mouse motoneurons (MN), where it is localized to subsurface cisternae of cholinergic postsynaptic densities, also known as C-terminals. We found that S1R is enriched in mouse spinal MN at late stages of embryonic development when it is first visualized in the endoplasmic reticulum. S1Rs appear to concentrate at C-terminals of mouse MN only on the second week of postnatal development. We found that Indole-N-methyl transferase (INMT), an enzyme that converts tryptamine into the sigma-1 ligand dimethyltryptamine (DMT), is also localized to postsynaptic sites of C-terminals in close proximity to the S1R. This close association of INMT and SIRs suggest that DMT is synthesized locally to effectively activate S1R in MN. PMID:22265729

Photodissociation pathways and lifetimes of protonated peptides and their dimers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aravind, G.; Klaerke, B.; Rajput, J.

2012-01-07

Photodissociation lifetimes and fragment channels of gas-phase, protonated YA{sub n} (n = 1,2) peptides and their dimers were measured with 266 nm photons. The protonated monomers were found to have a fast dissociation channel with an exponential lifetime of {approx}200 ns while the protonated dimers show an additional slow dissociation component with a lifetime of {approx}2 {mu}s. Laser power dependence measurements enabled us to ascribe the fast channel in the monomer and the slow channel in the dimer to a one-photon process, whereas the fast dimer channel is from a two-photon process. The slow (1 photon) dissociation channel in themore » dimer was found to result in cleavage of the H-bonds after energy transfer through these H-bonds. In general, the dissociation of these protonated peptides is non-prompt and the decay time was found to increase with the size of the peptides. Quantum RRKM calculations of the microcanonical rate constants also confirmed a statistical nature of the photodissociation processes in the dipeptide monomers and dimers. The classical RRKM expression gives a rate constant as an analytical function of the number of active vibrational modes in the system, estimated separately on the basis of the equipartition theorem. It demonstrates encouraging results in predicting fragmentation lifetimes of protonated peptides. Finally, we present the first experimental evidence for a photo-induced conversion of tyrosine-containing peptides into monocyclic aromatic hydrocarbon along with a formamide molecule both found in space.« less

Effects of Cd{sup 2+} on cis-dimer structure of E-cadherin in living cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takeda, Hiroshi, E-mail: hirotake@sapmed.ac.jp

2014-02-21

Highlights: • The effects of Cd on the dimer of cadherin in living cells was analyzed. • Cd induced cadherin dimer formation was not detected in living cell with low Ca. • Ca mediated structural cooperativity and allostery in the native cadherin. • Ca concentration-dependent competitive displacement of Cd from cadherin is proposed. - Abstract: E-cadherin, a calcium (Ca{sup 2+})-dependent cell–cell adhesion molecule, plays a key role in the maintenance of tissue integrity. We have previously demonstrated that E-cadherin functions in vivo as a cis-dimer through chemical cross-linking reagents. Ca{sup 2+} plays an important role in the cis-dimer formation ofmore » cadherin. However, the molecular mechanisms by which Ca{sup 2+} interacts with the binding sites that regulate cis-dimer structures have not been completely elucidated. As expected for a Ca{sup 2+} antagonist, cadmium (Cd{sup 2+}) disrupts cadherin function by displacing Ca{sup 2+} from its binding sites on the cadherin molecules. We used Cd{sup 2+} as a probe for investigating the role of Ca{sup 2+} in the dynamics of the E-cadherin extracellular region that involve cis-dimer formation and adhesion. While cell–cell adhesion assembly was completely disrupted in the presence of Cd{sup 2+}, the amount of cis-dimers of E-cadherin that formed at the cell surface was not affected. In our “Cd{sup 2+}-switch” experiments, we did not find that Cd{sup 2+}-induced E-cadherin cis-dimer formation in EL cells when they were incubated in low-Ca{sup 2+} medium. In the present study, we demonstrated for the first time the effects of Cd{sup 2+} on the cis-dimer structure of E-cadherin in living cells using a chemical cross-link analysis.« less

mRNA Molecules Containing Murine Leukemia Virus Packaging Signals Are Encapsidated as Dimers

PubMed Central

Hibbert, Catherine S.; Mirro, Jane; Rein, Alan

2004-01-01

Prior work by others has shown that insertion of ψ (i.e., leader) sequences from the Moloney murine leukemia virus (MLV) genome into the 3′ untranslated region of a nonviral mRNA leads to the specific encapsidation of this RNA in MLV particles. We now report that these RNAs are, like genomic RNAs, encapsidated as dimers. These dimers have the same thermostability as MLV genomic RNA dimers; like them, these dimers are more stable if isolated from mature virions than from immature virions. We characterized encapsidated mRNAs containing deletions or truncations of MLV ψ or with ψ sequences from MLV-related acute transforming viruses. The results indicate that the dimeric linkage in genomic RNA can be completely attributed to the ψ region of the genome. While this conclusion agrees with earlier electron microscopic studies on mature MLV dimers, it is the first evidence as to the site of the linkage in immature dimers for any retrovirus. Since the Ψ+ mRNA is not encapsidated as well as genomic RNA, it is only present in a minority of virions. The fact that it is nevertheless dimeric argues strongly that two of these molecules are packaged into particles together. We also found that the kissing loop is unnecessary for this coencapsidation or for the stability of mature dimers but makes a major contribution to the stability of immature dimers. Our results are consistent with the hypothesis that the packaging signal involves a dimeric structure in which the RNAs are joined by intermolecular interactions between GACG loops. PMID:15452213

Density functional Gaussian-type-orbital approach to theoretical study of nitric oxide dimers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jursic, B.S.; Zdravkovski, Z.

Structure and total energies of the cis NO dimer, the trans NO dimer, and the NO monomer were calculated by ab initio methods (UHF, UMP2, and MP3) and density functional theory methods (LSDA and BLYP) with different basis sets [from 3-21G* to 6-311++(3df,3pd)]. The system is especially hard to model because two NO molecules are weakly associated in a dimer that has very long N-N bond. The results obtained by different methods are compared and the necessity of correlational methods for studying these systems is discussed.

Difference in dimer conformation between amyloid-β(1-42) and (1-43) proteins: Replica exchange molecular dynamics simulations in water

NASA Astrophysics Data System (ADS)

Yano, Atsushi; Okamoto, Akisumi; Nomura, Kazuya; Higai, Shin'ichi; Kurita, Noriyuki

2014-03-01

We searched stable conformations of amyloid-β (Aβ) dimers composed of Aβ(1-42) or Aβ(1-43) protein in water by replica-exchange molecular dynamics simulations and found that Thr43 of the C-terminal of Aβ(1-43) is hydrogen bonded to Arg5 of the same monomer in the Aβ(1-43) dimer, resulting in its ring-shaped conformation, while Aβ(1-42) has no such hydrogen-bond. This conformation is expected to aggregate more easily into a compact conformation of Aβ fibrils. We also investigated the binding affinity and the specific interactions between Aβ monomers by ab initio fragment molecular orbital calculations to elucidate which Aβ residues contribute to the dimerization.

Elastic and viscous properties of the nematic dimer CB7CB

NASA Astrophysics Data System (ADS)

Babakhanova, Greta; Parsouzi, Zeinab; Paladugu, Sathyanarayana; Wang, Hao; Nastishin, Yu. A.; Shiyanovskii, Sergij V.; Sprunt, Samuel; Lavrentovich, Oleg D.

2017-12-01

We present a comprehensive set of measurements of optical, dielectric, diamagnetic, elastic, and viscous properties in the nematic (N) phase formed by a liquid crystalline dimer. The studied dimer, 1,7-bis-4-(4'-cyanobiphenyl) heptane (CB7CB), is composed of two rigid rodlike cyanobiphenyl segments connected by a flexible aliphatic link with seven methyl groups. CB7CB and other nematic dimers are of interest due to their tendency to adopt bent configurations and to form two states possessing a modulated nematic director structure, namely, the twist-bend nematic, NTB, and the oblique helicoidal cholesteric, C hOH , which occurs when the achiral dimer is doped with a chiral additive and exposed to an external electric or magnetic field. We characterize the material parameters as functions of temperature in the entire temperature range of the N phase, including the pretransitional regions near the N -NTB and N-to-isotropic (I) transitions. The splay constant K11 is determined by two direct and independent techniques, namely, detection of the Frederiks transition and measurement of director fluctuation amplitudes by dynamic light scattering (DLS). The bend K33 and twist K22 constants are measured by DLS. K33, being the smallest of the three constants, shows a strong nonmonotonous temperature dependence with a negative slope in both N-I and N -NTB pretransitional regions. The measured ratio K11/K22 is larger than 2 in the entire nematic temperature range. The orientational viscosities associated with splay, twist, and bend fluctuations in the N phase are comparable to those of nematics formed by rodlike molecules. All three show strong temperature dependence, increasing sharply near the N -NTB transition.

c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis

DTIC Science & Technology

2014-10-01

AWARD NUMBER: W81XWH-12-1-0431 TITLE: “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis ” PRINCIPAL INVESTIGATOR...TITLE AND SUBTITLE “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Scelerosis” 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH

Hsp90 N- and C-terminal double inhibition synergistically suppresses Bcr-Abl-positive human leukemia cells

PubMed Central

Chen, Xianling; Chen, Xiaole; Li, Ding; Fan, Yingjuan; Xu, Jianhua; Chen, Yuanzhong; Wu, Lixian

2017-01-01

Heat shock protein 90 (Hsp90) contains amino (N)–terminal domain, carboxyl(C)-terminal domain, and middle domains, which activate Hsp90 chaperone function cooperatively in tumor cells. One terminal occupancy might influence another terminal binding with inhibitor. The Bcr-Abl kinase is one of the Hsp90 clients implicated in the pathogenesis of chronic myeloid leukemia (CML). Present studies demonstrate that double inhibition of the N- and C-terminal termini can disrupt Hsp90 chaperone function synergistically, but not antagonistically, in Bcr-Abl-positive human leukemia cells. Furthermore, both the N-terminal inhibitor 17-AAG and the C-terminal inhibitor cisplatin (CP) have the capacity to suppress progenitor cells; however, only CP is able to inhibit leukemia stem cells (LSCs) significantly, which implies that the combinational treatment is able to suppress human leukemia in different mature states. PMID:28036294

The proteolytic processing site of the precursor of lysyl oxidase.

PubMed Central

Cronshaw, A D; Fothergill-Gilmore, L A; Hulmes, D J

1995-01-01

The precise cleavage site of the N-terminal propeptide region of the precursor of lysyl oxidase has not yet been established, due to N-terminal blocking of the mature protein. Using a combination of peptide fragmentation, amino acid sequencing, time-of-flight m.s. and partial chemical unblocking procedures, it is shown that the mature form of lysyl oxidase begins at residue Asp-169 of the precursor protein (numbered according to the human sequence). The cleavage site is 28 residues to the C-terminal side of the site previously suggested on the basis of apparant molecular mass by SDS/PAGE, with the consequence that the two putative, N-linked glycosylation sites and the position of the Arg/Gln sequence polymorphism are now all in the precursor region. PMID:7864821

Pub1p C-Terminal RRM Domain Interacts with Tif4631p through a Conserved Region Neighbouring the Pab1p Binding Site

PubMed Central

Rico-Lastres, Palma; Pérez-Cañadillas, José Manuel

2011-01-01

Pub1p, a highly abundant poly(A)+ mRNA binding protein in Saccharomyces cerevisiae, influences the stability and translational control of many cellular transcripts, particularly under some types of environmental stresses. We have studied the structure, RNA and protein recognition modes of different Pub1p constructs by NMR spectroscopy. The structure of the C-terminal RRM domain (RRM3) shows a non-canonical N-terminal helix that packs against the canonical RRM fold in an original fashion. This structural trait is conserved in Pub1p metazoan homologues, the TIA-1 family, defining a new class of RRM-type domains that we propose to name TRRM (TIA-1 C-terminal domain-like RRM). Pub1p TRRM and the N-terminal RRM1-RRM2 tandem bind RNA with high selectivity for U-rich sequences, with TRRM showing additional preference for UA-rich ones. RNA-mediated chemical shift changes map to β-sheet and protein loops in the three RRMs. Additionally, NMR titration and biochemical in vitro cross-linking experiments determined that Pub1p TRRM interacts specifically with the N-terminal region (1–402) of yeast eIF4G1 (Tif4631p), very likely through the conserved Box1, a short sequence motif neighbouring the Pab1p binding site in Tif4631p. The interaction involves conserved residues of Pub1p TRRM, which define a protein interface that mirrors the Pab1p-Tif4631p binding mode. Neither protein nor RNA recognition involves the novel N-terminal helix, whose functional role remains unclear. By integrating these new results with the current knowledge about Pub1p, we proposed different mechanisms of Pub1p recruitment to the mRNPs and Pub1p-mediated mRNA stabilization in which the Pub1p/Tif4631p interaction would play an important role. PMID:21931728

Dimerization effects on coacervation property of an elastin-derived synthetic peptide (FPGVG)5.

PubMed

Suyama, Keitaro; Taniguchi, Suguru; Tatsubo, Daiki; Maeda, Iori; Nose, Takeru

2016-04-01

Elastin, a core protein of the elastic fibers, exhibits the coacervation (temperature-dependent reversible association/dissociation) under physiological conditions. Because of this characteristic, elastin and elastin-derived peptides have been considered to be useful as base materials for developing various biomedical products, skin substitutes, synthetic vascular grafts, and drug delivery systems. Although elastin-derived polypeptide (Val-Pro-Gly-Val-Gly)n also has been known to demonstrate coacervation property, a sufficiently high (VPGVG)n repetition number (n>40) is required for coacervation. In the present study, a series of elastin-derived peptide (Phe-Pro-Gly-Val-Gly)5 dimers possessing high coacervation potential were newly developed. These novel dimeric peptides exhibited coacervation at significantly lower concentrations and temperatures than the commonly used elastin-derived peptide analogs; this result suggests that the coacervation ability of the peptides is enhanced by dimerization. Circular dichroism (CD) measurements indicate that the dimers undergo similar temperature-dependent and reversible conformational changes when coacervation occurs. The molecular dynamics calculation results reveal that the sheet-turn-sheet motif involving a type II β-turn-like structure commonly observed among the dimers and caused formation of globular conformation of them. These synthesized peptide dimers may be useful not only as model peptides for structural analysis of elastin and elastin-derived peptides, but also as base materials for developing various temperature-sensitive biomedical and industrial products. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Structure and stability of the N-hydroxyurea dimer: Post-Hartree-Fock quantum mechanical study

NASA Astrophysics Data System (ADS)

Jabalameli, Ali; Venkatraman, Ramaiyer; Nowek, Andrzej; Sullivan, Richard H.

2000-10-01

The potential energy surface (PES) search of the N-hydroxyurea dimer was searched with second-order Møller-Plesset perturbation theory (MP2) and the 6-31G(d,p) basis set. Eight local minimum energy structures have been found. Four of them have relatively strong (ΔE˜-10 to -13 kcal/mol) intermolecular interactions and the others are moderately strongly interacting species (ΔE˜-3 to -7 kcal/mol). Final estimation of interaction energies was performed using the larger 6-311G(df,pd) and 6-311G(2df,2pd) basis sets. The predicted interaction energies are ΔE=-14.26 kcal/mol and -3.43 kcal/mol for the strongest and the weakest interacting forms of the studied complex, respectively, at the MP2/6-311G(2df,2pd)//MP2/6-31G(d,p) level of theory. The self-consistent field (SCF) interaction energy decomposition indicates the important influence of the deformation term magnitude on ΔE(SCF). The calculated electron correlation contribution to ΔE(MP2) depends on the geometry of the system and varies from -0.5 to -5 kcal/mol. The estimated influence of water on the stability (free energy of hydration) of N-hydroxyurea dimers using the self-consistent isodensity polarized continuum (SCI-PCM) model of solvation varies from ˜-11 kcal/mol to ˜-21 kcal/mol. The forms predicted to be more strongly interacting species in gas phase are less influenced by hydration than the more weakly interacting ones.

Influence of the C-terminus of the glycophorin A transmembrane fragment on the dimerization process.

PubMed Central

Orzáez, M.; Pérez-Payá, E.; Mingarro, I.

2000-01-01

The monomer-dimer equilibrium of the glycophorin A (GpA) transmembrane (TM) fragment has been used as a model system to investigate the amino acid sequence requirements that permit an appropriate helix-helix packing in a membrane-mimetic environment. In particular, we have focused on a region of the helix where no crucial residues for packing have been yet reported. Various deletion and replacement mutants in the C-terminal region of the TM fragment showed that the distance between the dimerization motif and the flanking charged residues from the cytoplasmic side of the protein is important for helix packing. Furthermore, selected GpA mutants have been used to illustrate the rearrangement of TM fragments that takes place when leucine repeats are introduced in such protein segments. We also show that secondary structure of GpA derivatives was independent from dimerization, in agreement with the two-stage model for membrane protein folding and oligomerization. PMID:10892817

Synchronous termination of replication of the two chromosomes is an evolutionary selected feature in Vibrionaceae

PubMed Central

Kemter, Franziska S.; Messerschmidt, Sonja J.; Schallopp, Nadine; Sobetzko, Patrick; Bunk, Boyke; Spröer, Cathrin; Teschler, Jennifer K.; Yildiz, Fitnat H.

2018-01-01

Vibrio cholerae, the causative agent of the cholera disease, is commonly used as a model organism for the study of bacteria with multipartite genomes. Its two chromosomes of different sizes initiate their DNA replication at distinct time points in the cell cycle and terminate in synchrony. In this study, the time-delayed start of Chr2 was verified in a synchronized cell population. This replication pattern suggests two possible regulation mechanisms for other Vibrio species with different sized secondary chromosomes: Either all Chr2 start DNA replication with a fixed delay after Chr1 initiation, or the timepoint at which Chr2 initiates varies such that termination of chromosomal replication occurs in synchrony. We investigated these two models and revealed that the two chromosomes of various Vibrionaceae species terminate in synchrony while Chr2-initiation timing relative to Chr1 is variable. Moreover, the sequence and function of the Chr2-triggering crtS site recently discovered in V. cholerae were found to be conserved, explaining the observed timing mechanism. Our results suggest that it is beneficial for bacterial cells with multiple chromosomes to synchronize their replication termination, potentially to optimize chromosome related processes as dimer resolution or segregation. PMID:29505558

The diagnostic value of plasma N-terminal connective tissue growth factor levels in children with heart failure.

PubMed

Li, Gang; Song, Xueqing; Xia, Jiyi; Li, Jing; Jia, Peng; Chen, Pengyuan; Zhao, Jian; Liu, Bin

2017-01-01

The aim of this study was to assess the diagnostic value of plasma N-terminal connective tissue growth factor in children with heart failure. Methods and results Plasma N-terminal connective tissue growth factor was determined in 61 children, including 41 children with heart failure, 20 children without heart failure, and 30 healthy volunteers. The correlations between plasma N-terminal connective tissue growth factor levels and clinical parameters were investigated. Moreover, the diagnostic value of N-terminal connective tissue growth factor levels was evaluated. Compared with healthy volunteers and children without heart failure, plasma N-terminal connective tissue growth factor levels were significantly elevated in those with heart failure (p0.05), but it obviously improved the ability of diagnosing heart failure in children, as demonstrated by the integrated discrimination improvement (6.2%, p=0.013) and net re-classification improvement (13.2%, p=0.017) indices. Plasma N-terminal connective tissue growth factor is a promising diagnostic biomarker for heart failure in children.

Rational Organization of Lanthanide-Based SMM Dimers into Three-Dimensional Networks.

PubMed

Yi, Xiaohui; Calvez, Guillaume; Daiguebonne, Carole; Guillou, Olivier; Bernot, Kevin

2015-06-01

Optimization of the reaction of [Ln(hfac)3]·2H2O and pyridine-N-oxide (PyNO), which is known to afford double-bridged dimers, leads to triple-bridged dimers of formula [(Ln(hfac)3)2(PyNO)3] (Ln = Gd (1), Dy (2)) from which the Dy derivative (2) behaves as a single-molecule magnet (SMM). The pseudo threefold axis symmetry of this zero-dimensional building block makes possible its extension into a tridimensional network. By changing PyNO for 4,4'-bipyridine N,N'-dioxide (4,4'BipyNO) a tridimensional compound of formula {[Ln(hfac)3]2(4,4'BipyNO)2]} (Ln = Eu (3), Gd (4), and Dy (5)) is then rationally obtained. This covalent three-dimensional (3D) network has a remarkably high cell volume (V = 24 419 A(3)) and is an arrangement of interpenetrated 3D subnetworks whose triple-bridged dimers still behave as SMMs.

Atomic resolution crystal structure of VcLMWPTP-1 from Vibrio cholerae O395: Insights into a novel mode of dimerization in the low molecular weight protein tyrosine phosphatase family

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nath, Seema; Banerjee, Ramanuj; Sen, Udayaditya, E-mail: udayaditya.sen@saha.ac.in

Highlights: • VcLMWPTP-1 forms dimer in solution. • The dimer is catalytically active unlike other reported dimeric LMWPTPs. • The formation of extended dimeric surface excludes the active site pocket. • The surface bears closer resemblance to eukaryotic LMWPTPs. - Abstract: Low molecular weight protein tyrosine phosphatase (LMWPTP) is a group of phosphotyrosine phosphatase ubiquitously found in a wide range of organisms ranging from bacteria to mammals. Dimerization in the LMWPTP family has been reported earlier which follows a common mechanism involving active site residues leading to an enzymatically inactive species. Here we report a novel form of dimerization inmore » a LMWPTP from Vibrio cholera 0395 (VcLMWPTP-1). Studies in solution reveal the existence of the dimer in solution while kinetic study depicts the active form of the enzyme. This indicates that the mode of dimerization in VcLMWPTP-1 is different from others where active site residues are not involved in the process. A high resolution (1.45 Å) crystal structure of VcLMWPTP-1 confirms a different mode of dimerization where the active site is catalytically accessible as evident by a tightly bound substrate mimicking ligand, MOPS at the active site pocket. Although being a member of a prokaryotic protein family, VcLMWPTP-1 structure resembles very closely to LMWPTP from a eukaryote, Entamoeba histolytica. It also delineates the diverse surface properties around the active site of the enzyme.« less

EGFR oligomerization organizes kinase-active dimers into competent signalling platforms

PubMed Central

Needham, Sarah R.; Roberts, Selene K.; Arkhipov, Anton; Mysore, Venkatesh P.; Tynan, Christopher J.; Zanetti-Domingues, Laura C.; Kim, Eric T.; Losasso, Valeria; Korovesis, Dimitrios; Hirsch, Michael; Rolfe, Daniel J.; Clarke, David T.; Winn, Martyn D.; Lajevardipour, Alireza; Clayton, Andrew H. A.; Pike, Linda J.; Perani, Michela; Parker, Peter J.; Shan, Yibing; Shaw, David E.; Martin-Fernandez, Marisa L.

2016-01-01

Epidermal growth factor receptor (EGFR) signalling is activated by ligand-induced receptor dimerization. Notably, ligand binding also induces EGFR oligomerization, but the structures and functions of the oligomers are poorly understood. Here, we use fluorophore localization imaging with photobleaching to probe the structure of EGFR oligomers. We find that at physiological epidermal growth factor (EGF) concentrations, EGFR assembles into oligomers, as indicated by pairwise distances of receptor-bound fluorophore-conjugated EGF ligands. The pairwise ligand distances correspond well with the predictions of our structural model of the oligomers constructed from molecular dynamics simulations. The model suggests that oligomerization is mediated extracellularly by unoccupied ligand-binding sites and that oligomerization organizes kinase-active dimers in ways optimal for auto-phosphorylation in trans between neighbouring dimers. We argue that ligand-induced oligomerization is essential to the regulation of EGFR signalling. PMID:27796308

Interaction with the Src homology (SH3-SH2) region of the Src-family kinase Hck structures the HIV-1 Nef dimer for kinase activation and effector recruitment.

PubMed

Alvarado, John Jeff; Tarafdar, Sreya; Yeh, Joanne I; Smithgall, Thomas E

2014-10-10

HIV-1 Nef supports high titer viral replication in vivo and is essential for AIDS progression. Nef function depends on interactions with multiple host cell effectors, including Hck and other Src-family kinases. Here we describe the x-ray crystal structure of Nef in complex with the Hck SH3-SH2 regulatory region to a resolution of 1.86 Å. The complex crystallized as a dimer of complexes, with the conserved Nef PXXPXR motif engaging the Hck SH3 domain. A new intercomplex contact was found between SH3 Glu-93, and Nef Arg-105. Mutagenesis of Hck SH3 Glu-93 interfered with Nef·Hck complex formation and kinase activation in cells. The Hck SH2 domains impinge on the N-terminal region of Nef to stabilize a dimer conformation that exposes Asp-123, a residue critical for Nef function. Our results suggest that in addition to serving as a kinase effector for Nef, Hck binding may reorganize the Nef dimer for functional interaction with other signaling partners. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Interaction with the Src Homology (SH3-SH2) Region of the Src-family Kinase Hck Structures the HIV-1 Nef Dimer for Kinase Activation and Effector Recruitment*

PubMed Central

Alvarado, John Jeff; Tarafdar, Sreya; Yeh, Joanne I.; Smithgall, Thomas E.

2014-01-01

HIV-1 Nef supports high titer viral replication in vivo and is essential for AIDS progression. Nef function depends on interactions with multiple host cell effectors, including Hck and other Src-family kinases. Here we describe the x-ray crystal structure of Nef in complex with the Hck SH3-SH2 regulatory region to a resolution of 1.86 Å. The complex crystallized as a dimer of complexes, with the conserved Nef PXXPXR motif engaging the Hck SH3 domain. A new intercomplex contact was found between SH3 Glu-93, and Nef Arg-105. Mutagenesis of Hck SH3 Glu-93 interfered with Nef·Hck complex formation and kinase activation in cells. The Hck SH2 domains impinge on the N-terminal region of Nef to stabilize a dimer conformation that exposes Asp-123, a residue critical for Nef function. Our results suggest that in addition to serving as a kinase effector for Nef, Hck binding may reorganize the Nef dimer for functional interaction with other signaling partners. PMID:25122770

Dimer monomer transition and dimer re-formation play important role for ATM cellular function during DNA repair

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Fengxia; Zhang, Minjie; University of Chinese Academy of Sciences, Beijing 100049

2014-10-03

Highlights: • ATM phosphorylates the opposite strand of the dimer in response to DNA damage. • The PETPVFRLT box of ATM plays a key role in its dimer dissociation in DNA repair. • The dephosphorylation of ATM is critical for dimer re-formation after DNA repair. - Abstract: The ATM protein kinase, is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks, mediates responses to ionizing radiation in mammalian cells. Here we show that ATM is held inactive in unirradiated cells as a dimer and phosphorylates the opposite strand of the dimer in response to DNA damage.more » Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity. ATM cannot phosphorylate the substrates when it could not undergo dimer monomer transition. After DNA repair, the active monomer will undergo dephosphorylation to form dimer again and dephosphorylation is critical for dimer re-formation. Our work reveals novel function of ATM dimer monomer transition and explains why ATM dimer monomer transition plays such important role for ATM cellular activity during DNA repair.« less

Equilibrium unfolding studies of the rat liver methionine adenosyltransferase III, a dimeric enzyme with intersubunit active sites.

PubMed Central

Gasset, María; Alfonso, Carlos; Neira, José L; Rivas, Germán; Pajares, María A

2002-01-01

The reversible unfolding of rat liver methionine adenosyltransferase dimer by urea under equilibrium conditions has been monitored by fluorescence spectroscopy, CD, size-exclusion chromatography, analytical ultracentrifugation and enzyme activity measurements. The results obtained indicate that unfolding takes place through a three-state mechanism, involving an inactive monomeric intermediate. This intermediate has a 70% native secondary structure, binds less 8-anilinonaphthalene-1-sulphonic acid than the native dimer and has a sedimentation coefficient of 4.24+/-0.15. The variations of free energy in the absence of denaturant [DeltaG(H(2)O)] and its coefficients of urea dependence (m), calculated by the linear extrapolation model, were 36.15+/-2.3 kJ.mol(-1) and 19.87+/-0.71 kJ.mol(-1).M(-1) for the dissociation of the native dimer and 14.77+/-1.63 kJ.mol(-1) and 5.23+/-0.21 kJ.mol(-1).M(-1) for the unfolding of the monomeric intermediate respectively. Thus the global free energy change in the absence of denaturant and the m coefficient were calculated to be 65.69 kJ.mol(-1) and 30.33 kJ.mol(-1).M(-1) respectively. Analysis of the calculated thermodynamical parameters indicate the instability of the dimer in the presence of denaturant, and that the major exposure to the solvent is due to dimer dissociation. Finally, a minimum-folding mechanism for methionine adenosyltransferase III is established. PMID:11772402

A versatile tripodal amide receptor for the encapsulation of anions or hydrated anions via formation of dimeric capsules.

PubMed

Arunachalam, M; Ghosh, Pradyut

2010-02-01

A bowl-shaped tripodal receptor with an appropriately positioned amide functionality on the benzene platform and electron-withdrawing p-nitrophenyl terminals (L(1)) has been designed, synthesized, and studied for the anion binding properties. The single-crystal X-ray crystallographic analysis on crystals of L(1) with tetrabutylammonium salts of nitrate (1), acetate (2), fluoride (3), and chloride (4) obtained in moist dioxane medium showed encapsulation of two NO(3)(-), [(AcO)(2)(H(2)O)(4)](2-), [F(2)(H(2)O)(6)](2-), and [Cl(2)(H(2)O)(4)](2-) respectively as the anionic guests inside the staggered dimeric capsular assembly of L(1). The p-nitro substitution in the aryl terminals assisted the formation of dimeric capsular assembly of L(1) exclusively upon binding/encapsulating above different guests. Though L(1) demonstrates capsule formation upon anion or hydrated anion complexation for all of the anions studied here, its positional isomer with the o-nitro-substituted tripodal triamide receptor L(2) selectively formed the dimeric capsular assembly upon encapsulation of [F(2)(H(2)O)(6)](2-) and noncapsular aggregates in the cases of other anions such as Cl(-), NO(3)(-), and AcO(-). Interestingly, structural investigations upon anion exchange of the complexes revealed that both isomers have selectivity toward the formation of a [F(2)(H(2)O)(6)](2-) encapsulated dimeric capsule. In contrast, solution-state (1)H NMR titration studies of L(1) and L(2) in DMSO-d(6) with AcO(-) indicated 1:3 (host:guest) binding.

Monoatomic and dimer Mn adsorption on the Au(111) surface from first principles

NASA Astrophysics Data System (ADS)

Muñoz, Francisco; Romero, Aldo H.; Mejía-López, Jose; Morán-López, J. L.

2011-05-01

A theoretical study based on the density functional theory of the adsorption of Mn monomers and dimers on a Au-(111) surface is presented. As necessary preliminary steps, the bulk and clean surface electronic structure are calculated, which agree well with previous reports. Then, the electronic structure of the Mn adatom, chemisorbed on four different surface geometries, is analyzed. It is found that the most stable geometry is when the Mn atom is chemisorbed on threefold coordinated sites. Using this geometry for a single adatom a second Mn atom is chemisorbed and the most stable dimer geometrical structure is calculated. The lowest-energy configuration corresponds to the molecule lying parallel to the surface, adsorbed on two topological equivalent threefold coordinated sites. It is also found that the lowest-energy magnetic configuration corresponds to the antiferromagnetic arrangement with individual magnetic moments of 4.64μB. Finally, it is concluded that the dimer is not stable and should fragment at the surface.

Extra-domain B in oncofetal fibronectin structurally promotes fibrillar head-to-tail dimerization of extracellular matrix protein.

PubMed

Schiefner, André; Gebauer, Michaela; Skerra, Arne

2012-05-18

The type III extra-domain B (ED-B) is specifically spliced into fibronectin (Fn) during embryogenesis and neoangiogenesis, including many cancers. The x-ray structure of the recombinant four-domain fragment Fn(III)7B89 reveals a tightly associated, extended head-to-tail dimer, which is stabilized via pair-wise shape and charge complementarity. A tendency toward ED-B-dependent dimer formation in solution was supported by size exclusion chromatography and analytical ultracentrifugation. When amending the model with the known three-dimensional structure of the Fn(III)10 domain, its RGD loop as well as the adhesion synergy region in Fn(III)9-10 become displayed on the same face of the dimer; this should allow simultaneous binding of at least two integrins and, thus, receptor clustering on the cell surface and intracellular signaling. Insertion of ED-B appears to stabilize overall head-to-tail dimerization of two separate Fn chains, which, together with alternating homodimer formation via disulfide bridges at the C-terminal Fn tail, should lead to the known macromolecular fibril formation.

Active and regulatory sites of cytosolic 5'-nucleotidase.

PubMed

Pesi, Rossana; Allegrini, Simone; Careddu, Maria Giovanna; Filoni, Daniela Nicole; Camici, Marcella; Tozzi, Maria Grazia

2010-12-01

Cytosolic 5'-nucleotidase (cN-II), which acts preferentially on 6-hydroxypurine nucleotides, is essential for the survival of several cell types. cN-II catalyses both the hydrolysis of nucleotides and transfer of their phosphate moiety to a nucleoside acceptor through formation of a covalent phospho-intermediate. Both activities are regulated by a number of phosphorylated compounds, such as diadenosine tetraphosphate (Ap₄A), ADP, ATP, 2,3-bisphosphoglycerate (BPG) and phosphate. On the basis of a partial crystal structure of cN-II, we mutated two residues located in the active site, Y55 and T56. We ascertained that the ability to catalyse the transfer of phosphate depends on the presence of a bulky residue in the active site very close to the aspartate residue that forms the covalent phospho-intermediate. The molecular model indicates two possible sites at which adenylic compounds may interact. We mutated three residues that mediate interaction in the first activation site (R144, N154, I152) and three in the second (F127, M436 and H428), and found that Ap₄A and ADP interact with the same site, but the sites for ATP and BPG remain uncertain. The structural model indicates that cN-II is a homotetrameric protein that results from interaction through a specific interface B of two identical dimers that have arisen from interaction of two identical subunits through interface A. Point mutations in the two interfaces and gel-filtration experiments indicated that the dimer is the smallest active oligomerization state. Finally, gel-filtration and light-scattering experiments demonstrated that the native enzyme exists as a tetramer, and no further oligomerization is required for enzyme activation. © 2010 The Authors Journal compilation © 2010 FEBS.