L1 Loop Research Articles

Structures of the catalytic EAL domain of theEscherichia colidirect oxygen sensor

The direct oxygen sensor DosP is a multidomain protein that contains a gas-sensing haem domain and an EAL effector domain. EAL domains are omnipresent signal transduction domains in bacteria. Many EAL domains are active phosphodiesterases and are involved in breakdown of the ubiquitous bacterial second messenger cyclic di-GMP. Despite a great deal of information on the functional and structural aspects of active and inactive EAL domains, little is known about the structural basis of their regulation by their associated sensory domains. Here, two crystal structures of the Escherichia coli DosP EAL domain derived from cubic and monoclinic crystal forms that were obtained under tartrate and PEG conditions, respectively, are described. Both of the structures display the typical TIM (triosephosphate isomerase) barrel fold with one antiparallel β-strand. However, unlike other EAL structures, access to the active site in DosP EAL is sterically restricted by the presence of a short helical stretch (Ser637-Ala-Leu-His640) in loop L3 between strand β3 and helix α3. This element, together with an unordered fragment, replaces the short α-helix (named α5 in Tbd1265 EAL) that is found in other EAL-domain structures. Since DosP EAL is an active c-di-GMP phosphodiesterase, the observed inactive conformation is suggested to be of functional relevance for the regulation mechanism of DosP.

Mutations in human papillomavirus type 16 L1 hypervariable surface-exposed loops affect L2 binding and DNA encapsidation

Prophylactic vaccines against human papillomavirus (HPV) based on virus-like particles (VLP) induce type-specific neutralizing antibodies against a small number of hypervariable residues positioned in surface-exposed loops of the major capsid protein L1. To investigate the importance of these residues for neutralization, cross-neutralization, L2 incorporation and genome encapsidation, ten surface-exposed amino acid residues in four hypervariable loops of L1 were mutated. VLPs containing mutated or WT L1, with or without WT L2, were produced in 293TT cells using pseudovirion expression vectors. The mutations reduced the ability to induce neutralizing antibodies and to incorporate the L2 protein in the capsid. Ability to induce cross-neutralizing antibodies and to encapsidate pseudogenomes were completely abrogated. In summary, the surface-exposed L1 loops are important for the function of the HPV particle.

Complex Structure of OspI and Ubc13: The Molecular Basis of Ubc13 Deamidation and Convergence of Bacterial and Host E2 Recognition

Ubc13 is an important ubiquitin-conjugating (E2) enzyme in the NF-κB signaling pathway. The Shigella effector OspI targets Ubc13 and deamidates Gln100 of Ubc13 to a glutamic acid residue, leading to the inhibition of host inflammatory responses. Here we report the crystal structure of the OspI-Ubc13 complex at 2.3 Å resolution. The structure reveals that OspI uses two differently charged regions to extensively interact with the α1 helix, L1 loop and L2 loop of Ubc13. The Gln100 residue is bound within the hydrophilic catalytic pocket of OspI. A comparison between Ubc13-bound and wild-type free OspI structures revealed that Ubc13 binding induces notable structural reassembly of the catalytic pocket, suggesting that substrate binding might be involved in the catalysis of OspI. The OspI-binding sites in Ubc13 largely overlap with the binding residues for host ubiquitin E3 ligases and a deubiquitinating enzyme, which suggests that the bacterial effector and host proteins exploit the same surface on Ubc13 for specific recognition. Biochemical results indicate that both of the differently charged regions in OspI are important for the interaction with Ubc13, and the specificity determinants in Ubc13 for OspI recognition reside in the distinct residues in the α1 helix and L2 region. Our study reveals the molecular basis of Ubc13 deamidation by OspI, as well as a convergence of E2 recognition by bacterial and host proteins.

Phosphorylation of the Retinoic Acid Receptor Alpha Induces a Mechanical Allosteric Regulation and Changes in Internal Dynamics

Nuclear receptor proteins constitute a superfamily of proteins that function as ligand dependent transcription factors. They are implicated in the transcriptional cascades underlying many physiological phenomena, such as embryogenesis, cell growth and differentiation, and apoptosis, making them one of the major signal transduction paradigms in metazoans. Regulation of these receptors occurs through the binding of hormones, and in the case of the retinoic acid receptor (RAR), through the binding of retinoic acid (RA). In addition to this canonical scenario of RAR activity, recent discoveries have shown that RAR regulation also occurs as a result of phosphorylation. In fact, RA induces non-genomic effects, such as the activation of kinase signaling pathways, resulting in the phosphorylation of several targets including RARs themselves. In the case of RARα, phosphorylation of Ser369 located in loop L9–10 of the ligand-binding domain leads to an increase in the affinity for the protein cyclin H, which is part of the Cdk-activating kinase complex of the general transcription factor TFIIH. The cyclin H binding site in RARα is situated more than 40 Å from the phosphorylated serine. Using molecular dynamics simulations of the unphosphorylated and phosphorylated forms of the receptor RARα, we analyzed the structural implications of receptor phosphorylation, which led to the identification of a structural mechanism for the allosteric coupling between the two remote sites of interest. The results show that phosphorylation leads to a reorganization of a local salt bridge network, which induces changes in helix extension and orientation that affects the cyclin H binding site. This results in changes in conformation and flexibility of the latter. The high conservation of the residues implicated in this signal transduction suggests a mechanism that could be applied to other nuclear receptor proteins.

Abstract 2316: Mutations in the p53 gene family reveal conservation of structure-function in the L1 and L3 loops and a response element code for transcriptional specificity.

Abstract The p53, p63 and p73 family of sequence-specific transcription factors share similar biochemical properties and structural features resulting in the recognition of DNA with identical amino acids and similar binding affinity. However, loss of individual members results in very different phenotypes in mice as well as humans. We have addressed functional relationships between p53 family proteins using a combination of mutants, target response elements (REs) and sequence-specific transactivation assays based in yeast and human cells. p53 alleles exhibiting enhanced transcriptional activation and altered promoter selectivity were previously identified in the L1 and L3 loop of the DNA binding domain of the protein. Included was S121F that results in defective p21 induction and confers a strong apoptotic phenotype in mammalian cells. Guided by result with p53-S121F, -T123A and -S240N mutants, the corresponding p63 and p73 alleles were investigated for ability to drive transactivation at over fifty p53 REs in isogenic yeast reporter strains. The phenotypes observed with p53 alleles were reproduced with the p63- and p73- alleles. Focusing on p73-S139F (corresponding to p53-S121F) transactivation at many RE sequences, we identify and experimentally confirm an RE target sequence code that predicts enhanced or reduced transactivation potentials. The correlation is particularly strong for induction at p53 targets within promoters of genes associated with apoptosis. Furthermore, p73 S139F showed an enhanced DNA-binding cooperativity in vitro, and loop L1 shows conformational changes in the crystal structure of the protein-DNA complex. The altered function phenotype was confirmed not only in gene reporter assays but also for endogenous gene expression in the p53 null HCT116 human cell line, suggesting that target recognition can predict relative changes in transcription rates at different loci. Based on the correlation between RE code and associated gene functions, we propose the selection during evolution of an RE sequence code within target promoters that can affect intrinsic conformational flexibility / DNA binding affinity of p53 proteins thereby modulating in vivo selectivity, specifically favoring the activation of apoptotic target genes Citation Format: Yari Ciribilli, Paola Monti, Alessandra Bisio, H. T. Nguyen, A S. Ethayathulla, Micheal A. Resnick, Hector Viadiu, Gilberto Fronza, Alberto Inga. Mutations in the p53 gene family reveal conservation of structure-function in the L1 and L3 loops and a response element code for transcriptional specificity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2316. doi:10.1158/1538-7445.AM2013-2316

Adenoviral Gizzard Erosion in Broiler Chickens in Germany

Avian adenovirus infections cause important disease complexes in chickens, but many of the viruses also infect chickens without resulting in overt disease. Previously several outbreaks of gizzard erosions caused by a fowl adenovirus A serotype-1 (FAdV-1) were reported from Japan. Here we report an outbreak of gizzard erosions in 12 broiler flocks in Germany in 2011. Chickens had a reduced daily weight gain and a higher total mortality rate of up to 8%. The birds showed a severe detachment of the koilin layer and ulcerative to necrotizing lesions of the underlying mucosa. Histopathologically, necrotizing ventriculitis with basophilic, intranuclear inclusion bodies in epithelial cells was diagnosed. Immunohistochemistry, egg culture, and electron microscopic examination revealed adenovirus-like particles in the samples. No concurrent infectious agent could be identified. The virus was genotyped as FAdV-1 by PCR and subsequent sequencing. Phylogenetic analysis of the hexon loop L1 gene yielded 100% sequence identity to the chicken embryo lethal orphan strain. These findings suggest that outbreaks of adenoviral gizzard erosion can lead to significant economic losses in Germany and may be caused by an unusual virulent FAdV-1 strain.

Allosteric inhibition of VIM metallo-β-lactamases by a camelid nanobody

MβL (metallo-β-lactamase) enzymes are usually produced by multi-resistant Gram-negative bacterial strains and have spread worldwide. An approach on the basis of phage display was used to select single-domain antibody fragments (VHHs, also called nanobodies) that would inhibit the clinically relevant VIM (Verona integron-encoded MβL)-4MβL. Out of more than 50 selected nanobodies, only the NbVIM_38 nanobody inhibited VIM-4. The paratope, inhibition mechanism and epitope of the NbVIM_38 nanobody were then characterized. An alanine scan of the NbVIM_38 paratope showed that its binding was driven by hydrophobic amino acids. The inhibitory concentration was in the micromolar range for all β-lactams tested. In addition, the inhibition was found to follow a mixed hyperbolic profile with a predominantly uncompetitive component. Moreover, substrate inhibition was recorded only after nanobody binding. These kinetic data are indicative of a binding site that is distant from the active site. This finding was confirmed by epitope mapping analysis that was performed using peptides, and which identified two stretches of amino acids in the L6 loop and at the end of the α2 helix. Because this binding site is distant from the active site and alters both the substrate binding and catalytic properties of VIM-4, this nanobody can be considered as an allosteric inhibitor.

Structural Insights into a Unique Inhibitor Binding Pocket in Kinesin Spindle Protein

Human kinesin Eg5 is a target for drug development in cancer chemotherapy with compounds in phase II clinical trials. These agents bind to a well-characterized allosteric pocket involving the loop L5 region, a structural element in kinesin-5 family members thought to provide inhibitor specificity. Using X-ray crystallography, kinetic, and biophysical methods, we have identified and characterized a distinct allosteric pocket in Eg5 able to bind inhibitors with nanomolar K(d). This pocket is formed by key structural elements thought to be pivotal for force generation in kinesins and may represent a novel site for therapeutic intervention in this increasingly well-validated drug target.

Crystal Structures of the DNA-binding Domain Tetramer of the p53 Tumor Suppressor Family Member p73 Bound to Different Full-site Response Elements

How cells choose between developmental pathways remains a fundamental biological question. In the case of the p53 protein family, its three transcription factors (p73, p63, and p53) each trigger a gene expression pattern that leads to specific cellular pathways. At the same time, these transcription factors recognize the same response element (RE) consensus sequences, and their transactivation of target genes overlaps. We aimed to understand target gene selectivity at the molecular level by determining the crystal structures of the p73 DNA-binding domain (DBD) in complex with full-site REs that vary in sequence. We report two structures of the p73 DBD bound as a tetramer to 20-bp full-site REs based on two distinct quarter-sites: GAACA and GAACC. Our study confirms that the DNA-binding residues are conserved within the p53 family, whereas the dimerization and tetramerization interfaces diverge. Moreover, a conserved lysine residue in loop L1 of the DBD senses the presence of guanines in positions 2 and 3 of the quarter-site RE, whereas a conserved arginine in loop 3 adapts to changes in position 5. Sequence variations in the RE elicit a p73 conformational response that might explain target gene specificity.

Computational identification of a transiently open L1/S3 pocket for reactivation of mutant p53.

The tumour suppressor p53 is the most frequently mutated gene in human cancer. Reactivation of mutant p53 by small molecules is an exciting potential cancer therapy. Although several compounds restore wild-type function to mutant p53, their binding sites and mechanisms of action are elusive. Here computational methods identify a transiently open binding pocket between loop L1 and sheet S3 of the p53 core domain. Mutation of residue Cys124, located at the centre of the pocket, abolishes p53 reactivation of mutant R175H by PRIMA-1, a known reactivation compound. Ensemble-based virtual screening against this newly revealed pocket selects stictic acid as a potential p53 reactivation compound. In human osteosarcoma cells, stictic acid exhibits dose-dependent reactivation of p21 expression for mutant R175H more strongly than does PRIMA-1. These results indicate the L1/S3 pocket as a target for pharmaceutical reactivation of p53 mutants.

The Pekin duck IL‐10R2 common chain: cDNA cloning, genomic structure, molecular characterization and mRNA expression analysis

The interleukin-10 receptor 2 (IL-10R2, IL-10Rβ) is required for the signalling of the class 2 cytokines IL-10, IL-22, IL-26 and IFN-λ1-3 . Here, we describe the identification of the Pekin duck IL-10R2 (duIL-10R2) common chain and its gene structure. The duIL-10R2 cDNA encodes a 343 amino acid protein that has an amino acid identity of 76% and 42% with chicken and human IL-10R2, respectively. Binding residues of human IL-10R2 for IL-10 and IL-22 were mostly conserved in the avian IL-10R2 proteins within loops L3 and L5, but not within loops L2 and L6. Homology modelling of the duIL-10R2 extracellular domain structure using soluble human IL-10R2 (shIL-10R2, PDB ID: 3LQM) as a template revealed a protruding loop L5 and two distinct clefts between loops L2/L3 and L3/L5, similar to shIL-10R2. However, in contrast to the three amino acid β-hairpin loop L2 of shIL-10R2, loop L2 of duIL-10R2 is five residues longer. Residues within a putative Tyk2 binding site were highly conserved across all vertebrate IL-10R2 proteins examined. The duIL-10R2 gene shares a seven exon-six intron structure with chicken and human IL-10R2 genes, but avian genes are more compact. DuIL-10R2 mRNA was constitutively expressed in all tissues. Mitogen stimulation of duck peripheral blood mononuclear cells (PBMC) did not alter transcript levels. Our observations suggest that genomic organization and structural features implicated in multiple cytokine-binding properties of human IL-10R2 are conserved in duck IL-10R2, but the evolutionary changes that appear to have lead to low-affinity cytokine interaction within loop L2 are distinct to mammalian species.

Structure and function of the PorB porin from disseminating Neisseria gonorrhoeae

The outer membrane of Gram-negative bacteria contains a large number of channel-forming proteins, porins, for the uptake of small nutrient molecules. Neisseria gonorrhoeae PorBIA (PorB of serotype A) are associated with disseminating diseases and mediate a rapid bacterial invasion into host cells in a phosphate-sensitive manner. To gain insights into this structure-function relationship we analysed PorBIA by X-ray crystallography in the presence of phosphate and ATP. The structure of PorBIA in the complex solved at a resolution of 3.3 Å (1 Å=0.1 nm) displays a surplus of positive charges inside the channel. ATP ligand-binding in the channel is co-ordinated by the positively charged residues of the channel interior. These residues ligate the aromatic, sugar and pyrophosphate moieties of the ligand. Two phosphate ions were observed in the structure, one of which clamped by two arginine residues (Arg92 and Arg124) localized at the extraplasmic channel exit. A short β-bulge in β2-strand together with the long L3 loop narrow the barrel diameter significantly and further support substrate specificity through hydrogen bond interactions. Interestingly the structure also comprised a small peptide as a remnant of a periplasmic protein which physically links porin molecules to the peptidoglycan network. To test the importance of Arg92 on bacterial invasion the residue was mutated. In vivo assays of bacteria carrying a R92S mutation confirmed the importance of this residue for host-cell invasion. Furthermore systematic sequence and structure comparisons of PorBIA from Neisseriaceae indicated Arg92 to be unique in disseminating N. gonorrhoeae thereby possibly distinguishing invasion-promoting porins from other neisserial porins.

Structure-based computational study of the hydrolysis of New Delhi metallo-β-lactmase-1

New Delhi metallo-β-lactmase-1 (NDM-1) is an enzyme that confers antibiotic resistance to bacteria and is thus a serious threat to human health. Almost all clinically available β-lactam antibiotics can be hydrolyzed by NDM-1. To determine the mechanism behind the wide substrate diversity and strong catalytic ability of NDM-1, we explored the molecular interactions between NDM-1 and different β-lactam antibiotics using computational methods. Molecular dynamics simulations and binding free energy calculations were performed on enzyme-substrate (ES) complex models of NDM-1-Meropenem, NDM-1-Nitrocefin, and NDM-1-Ampicillin constructed by molecular docking. Our computational results suggest that mutant residues Ile35 and Lys216, and active site loop L1 residues 65–73 in NDM-1 play crucial roles in substrate recognition and binding. The results of our study provide new insights into the mechanism behind the enhanced substrate binding and wider substrate spectrum of NDM-1 compared with its homologous enzymes CcrA and IMP-1. These insights may be useful in the discovery and design of specific and potent inhibitors against NDM-1.

Proteolytic activation of Chlamydia trachomatis HTRA is mediated by PDZ1 domain interactions with protease domain loops L3 and LC and beta strand β5

Chlamydia trachomatis is a bacterial pathogen responsible for one of the most prevalent sexually transmitted infections worldwide. Its unique development cycle has limited our understanding of its pathogenic mechanisms. However, CtHtrA has recently been identified as a potential C. trachomatis virulence factor. CtHtrA is a tightly regulated quality control protein with a monomeric structural unit comprised of a chymotrypsin-like protease domain and two PDZ domains. Activation of proteolytic activity relies on the C-terminus of the substrate allosterically binding to the PDZ1 domain, which triggers subsequent conformational change and oligomerization of the protein into 24-mers enabling proteolysis. This activation is mediated by a cascade of precise structural arrangements, but the specific CtHtrA residues and structural elements required to facilitate activation are unknown. Using in vitro analysis guided by homology modeling, we show that the mutation of residues Arg362 and Arg224, predicted to disrupt the interaction between the CtHtrA PDZ1 domain and loop L3, and between loop L3 and loop LD, respectively, are critical for the activation of proteolytic activity. We also demonstrate that mutation to residues Arg299 and Lys160, predicted to disrupt PDZ1 domain interactions with protease loop LC and strand β5, are also able to influence proteolysis, implying their involvement in the CtHtrA mechanism of activation. This is the first investigation of protease loop LC and strand β5 with respect to their potential interactions with the PDZ1 domain. Given their high level of conservation in bacterial HtrA, these structural elements may be equally significant in the activation mechanism of DegP and other HtrA family members.

61 High-resolution crystal structures of p53 mutants and their interaction with DNA

In response to cellular stress, the tumor suppressor protein p53 acts as a transcription factor by binding to its DNA targets, leading to the expression of several genes that participate in a variety of biological processes including DNA repair, cell cycle arrest or apoptosis. Thus, p53 “protects” the integrity of the genome. p53 binds as a tetramer to DNA response elements made of two decameric half-sites of the consensus sequence RRRCWWGYYY (R = A/G, Y = T/C, W = A/T) separated by a variable number of base pairs (Kitayner et al., 2006; 2010). About 50% of all invasive cancer cases show mutations in p53 and 97% of these mutations occur within the DNA-binding domain (DBD), among them, mutations in six “hot spot” codons account for more than 30% of cancer cases. These mutations lead to p53 loss of function. It was shown that several oncogenic mutants can be rescued by second-site suppressor mutations, resulting in wild-type-like activity in terms of DNA binding and transcriptional activation. To understand the structural effects caused by hot-spot mutations and the mechanisms of their rescue by suppressor mutations, we determined several crystal structures of human p53DBD incorporating these mutations as well as the rescued proteins and their complexes with DNA. These included DNA contact mutants, R273H and R273C, a structural mutant G245S, and the rescued proteins incorporating both the oncogenic mutation and the corresponding suppressor mutation. The crystal structures elucidate the structural basis of loss of function caused by the hot-spot mutations. The crystal structures of the rescued proteins bound to DNA reveal different rescue mechanisms: formation of alternative p53-DNA interactions for the DNA-contact mutants, and intramolecular or intermolecular stabilizing interactions for the structural mutant which compensate for the loss of p53 stability caused by the oncogenic mutation. In contrast to cancer-related mutations, specific mutations in other regions of p53DBD such as its DNA-binding loop (L1), which is part of the loop–sheet–helix motif at the p53-DNA interface, were shown to modulate the protein’s DNA-binding affinity and transactivation (Zupnick & Prives, 2006; Resnick & Inga, 2003). In particular, the replacement of T123 in the L1 loop by T123A or T123P largely increases p53 transactivation capacity relative to wild-type p53 (Resnick & Inga, 2003). The crystal structures of these mutants and their complexes with DNA provide a structural basis for understanding their biological activity.

Role of the external loops in the formation of antigenic structure of OmpF porin from Yersinia pseudotuberculosis

Event Abstract Back to Event Role of the external loops in the formation of antigenic structure of OmpF porin from Yersinia pseudotuberculosis Olga Portnyagina1*, Olga Novikova2, Olga Sidorova3 and Valentina Khomenko4 1 G.B. Elyakov Pacific Institute of Bioorganic Chemistry Far Eastern Branch, Russian Academy of Sciences, Molecular Immunology, Russia 2 G.B. Elyakov Pacific Institute of Bioorganic Chemistry Far Eastern Branch, Russian Academy of Sciences, Russia 3 G.B. Elyakov Pacific Institute of Bioorganic Chemistry Far Eastern Branch, Russian Academy of Sciences, Molecular Immunology, Russia 4 G.B. Elyakov Pacific Institute of Bioorganic Chemistry Far Eastern Branch, Russian Academy of Sciences, Russia Porins are the components of Gram-negative bacteria outer membrane (OM) playing a crucial role at the pathogen - host interrelations. Porins are the factors of microbial pathogenicity, participating in the forming of protective immunity at the same time. We have investigated the antigenic structure of the recombinant porin from OM of Yersinia pseudotuberculosis, the causative agents of pseudotuberculosis, using polyclonal antibodies against the full -sized OmpF porin (RP) and against the mutant porins with deletions of the external loops of the protein (del1, del4, del6, del8). As result of refolding conducted by dialysis and ion-exchange chromatography, the recombinant proteins were obtained as trimers and were used for immunization. It is interesting to note the most immunogenicity of del4 and del6 samples for mice, compared with RP. Perhaps, the absence of the loop sites of the protein sequence makes some of T-epitopes more "accessible" to form a ternary complex of antigen / T-receptor / MHC receptor. Deletion loops L1, by contrast, led to 50% loss of antigenic epitopes involved in the formation of the humoral immune response. Analysis of the interaction of specific antibodies against mutant proteins with RP as antigen showed no effect of the lack of loops L6 and L8 on the structure of B-epitopes formation as soon as the efficiency of antibody - antigen binding is similar for the RP and for the mutants. At the same time, deletion of loops L1 and L4 cause loss of 10% and 20% of antigenic epitopes, respectively. Keywords: Yerinia pseudotuberculosis, OmpF porin, antigenic structure, external loops, recombinant protein Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Host-pathogen interactions Citation: Portnyagina O, Novikova O, Sidorova O and Khomenko V (2013). Role of the external loops in the formation of antigenic structure of OmpF porin from Yersinia pseudotuberculosis. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00285 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 13 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Olga Portnyagina, G.B. Elyakov Pacific Institute of Bioorganic Chemistry Far Eastern Branch, Russian Academy of Sciences, Molecular Immunology, Vladivostok, 690022, Russia, odd64@mail.ru Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Olga Portnyagina Olga Novikova Olga Sidorova Valentina Khomenko Google Olga Portnyagina Olga Novikova Olga Sidorova Valentina Khomenko Google Scholar Olga Portnyagina Olga Novikova Olga Sidorova Valentina Khomenko PubMed Olga Portnyagina Olga Novikova Olga Sidorova Valentina Khomenko Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The Structural Basis of Force Generation by the Mitotic Motor Kinesin-5

Kinesin-5s are essential for forming the bipolar spindle during mitosis in most eukaryotes. The kinesin-5 motor domain contains conserved nucleotide and microtubule binding sites and mechanical elements to generate force. However, biochemical and biophysical studies have suggested that the mechano-chemistry of the kinesin-5 motor is very different from other kinesins. using cryo-electron microscopy and image reconstruction, we have calculated sub-nanometer resolution structures of microtubule-bound human kinesin-5 before and after nucleotide binding. Our structures reveal that, despite its mechanistic differences with conventional kinesin, kinesin-5 has the same coupled, nucleotide-dependent conformational changes as seen in conventional kinesins, including a ratchet-like docking of the neck linker, and simultaneous, parallel docking of the amino-terminal cover strand. These observations are supported by kinetic experiments that indicate a cooperative rearrangement of the kinesin-5-specific neck linker with the amino-terminal cover strand during the motor's ATPase cycle. In contrast to conventional kinesin however, our structures reveal a dramatic reorientation of Loop L5 - the binding site for allosteric inhibitors of kinesin-5-following ATP binding. This reorientation suggests that L5 is directly involved in controlling nucleotide binding by acting as an intra-molecular competitive inhibitor. Our structures indicate that allosteric inhibitors of human kinesin-5 bind to a motor conformation that occurs in the course of normal function. However, due to evolutionarily defined sequence variations in L5, this conformation is not adopted by invertebrate kinesin-5s, explaining their resistance to drug inhibition. Our data reveal the structural basis for kinesin-5 force generation that has evolved in the physiological context of the mitotic spindle.

2P175 有糸分裂キネシンEg5の機能性ループL5へのフォトクロミック分子導入と光制御(11.分子モーター,ポスター,日本生物物理学会年会第51回(2013年度))

2P175 有糸分裂キネシンEg5の機能性ループL5へのフォトクロミック分子導入と光制御(11.分子モーター,ポスター,日本生物物理学会年会第51回(2013年度))

Src Kinase regulates the Human Kinesin-5, Eg5, by Phosphorylating Tyrosines in the Eg5 Motor Domain

The human kinesin-5, Eg5, is required to establish and maintain the mitotic spindle. using in silico, in vitro and cell culture methods, we show that Src kinase phosphorylates specific tyrosine residues in Eg5. These residues are located near the nucleotide pocket and the functionally critical L5 loop. Phosphomimetic and non-phosphorylatable Eg5 mutant proteins have diminished ATPase activity and microtubule sliding relative to wild-type Eg5. We also report that phosphomimetic proteins have greatly reduced affinity for the Eg5 inhibitor S-trityl-L-cysteine. This finding suggests that Src phosphorylation of Eg5 may provide cells a non-mutagenesis-dependent strategy to evolve resistance to anti-mitotic Eg5 inhibitors. In this case, combination treatment targeting both SFKs and Eg5 may inhibit mitosis more effectively than anti-Eg5 treatment alone. Ultimately, Src phosphorylation of Eg5 represents a novel regulatory mechanism for a human kinesin, and links the chemical and physical processes that cause mitosis.

Analysis of 15N-1H NMR relaxation in proteins by a combined experimental and molecular dynamics simulation approach: picosecond-nanosecond dynamics of the Rho GTPase binding domain of plexin-B1 in the dimeric state indicates allosteric pathways.

We investigate picosecond–nanosecond dynamics of the Rho-GTPase Binding Domain (RBD) of plexin-B1, which plays a key role in plexin-mediated cell signaling. Backbone 15N relaxation data of the dimeric RBD are analyzed with the model-free (MF) method, and with the slowly relaxing local structure/molecular dynamics (SRLS-MD) approach. Independent analysis of the MD trajectories, based on the MF paradigm, is also carried out. MF is a widely popular and simple method, SRLS is a general approach, and SRLS-MD is an integrated approach we developed recently. Corresponding parameters from the RBD dimer, a previously studied RBD monomer mutant, and the previously studied complex of the latter with the GTPase Rac1, are compared. The L2, L3, and L4 loops of the plexin-B1 RBD are involved in interactions with other plexin domains, GTPase binding, and RBD dimerization, respectively. Peptide groups in the loops of both the monomeric and dimeric RBD are found to experience weak and moderately asymmetric local ordering centered approximately at the C(i–1)(α)–C(i)(α) axes, and nanosecond backbone motion. Peptide groups in the α-helices and the β-strands of the dimer (the β-strands of the monomer) experience strong and highly asymmetric local ordering centered approximately at the C(i–1)(α)–C(i)(α) axes (N–H bonds). N–H fluctuations occur on the picosecond time scale. An allosteric pathway for GTPase binding, providing new insights into plexin function, is delineated.