Journal Articles

Computer-aided design of aptamers for cytochrome p450

Journal of Structural Biology - Wed, 09/30/2015 - 03:56
Publication date: August 2015
Source:Journal of Structural Biology, Volume 191, Issue 2

Author(s): Dmitrii S. Shcherbinin, Oksana V. Gnedenko, Svetlana A. Khmeleva, Sergey A. Usanov, Andrei A. Gilep, Aliaksei V. Yantsevich, Tatsiana V. Shkel, Ivan V. Yushkevich, Sergey P. Radko, Alexis S. Ivanov, Alexander V. Veselovsky, Alexander I. Archakov

Aptamers are short single-stranded DNA or RNA oligonucleotides that can bind to their targets with high affinity and specificity. Usually, they are experimentally selected using the SELEX method. Here, we describe an approach toward the in silico selection of aptamers for proteins. This approach involves three steps: finding a potential binding site, designing the recognition and structural parts of the aptamers and evaluating the experimental affinity. Using this approach, a set of 15-mer aptamers for cytochrome P450 51A1 was designed using docking and molecular dynamics simulation. An experimental evaluation of the synthesized aptamers using SPR biosensor showed that these aptamers interact with cytochrome P450 51A1 with K d values in the range of 10−6–10−7 M.





Categories: Journal Articles

Molecular dynamics simulation study reveals potential substrate entry path into γ-secretase/presenilin-1

Journal of Structural Biology - Wed, 09/30/2015 - 03:56
Publication date: August 2015
Source:Journal of Structural Biology, Volume 191, Issue 2

Author(s): Ren Kong, Shan Chang, Weiming Xia, Stephen T.C. Wong

Presenilin 1 (PS1) is the catalytic unit of γ-secretase which cleaves more than one hundred substrates. Among them, amyloid precursor protein (APP) and Notch are notable for their pivotal role in the pathogenesis of Alzheimer’s disease (AD) and certain types of cancer. The hydrolysis process occurring inside the hydrophobic lipid bilayer remains unclear. With the aim to understand the mechanism of intramembrane proteolysis by γ-secretase, we constructed a homology model of human PS1 and performed molecular dynamics simulation in explicit membrane phospholipids with different components. During the simulation, TM9 was found to exhibit a high level of flexibility that involved in “gate-open” movement of TM2 and TM6, and thus partially exposed the catalytic residues. The highly conserved PALP motif acts as an anchor to mediate the conformation changes of TM6 induced by TM9. Moreover, direct interactions were observed between 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and the active site of γ-secretase, indicating that the lipid molecules have the potential to modulate γ-secretase by contacting with the catalytic residues, i.e., ASP 257 and ASP 385 of PS1. The intermediate states indicate a potential substrate penetration pathway through the interface of TM2 and TM6, which may be induced by changes of TM9. To our knowledge, this is the first molecular simulation study that reveals dynamic behavior of the human PS1 structure in the lipid bilayer and provides insight into the substrate entry path for subsequent intramembrane hydrolysis, which is critical information required for new strategy development of γ-secretase modulators to alleviate devastating AD.





Categories: Journal Articles

Ion and seed dependent fibril assembly of a spidroin core domain

Journal of Structural Biology - Wed, 09/30/2015 - 03:56
Publication date: August 2015
Source:Journal of Structural Biology, Volume 191, Issue 2

Author(s): Martin Humenik, Andrew M. Smith, Sina Arndt, Thomas Scheibel

Recombinant eADF4(C16) represents an engineered spider silk variant based on the sequence of the core domain of the natural dragline silk protein ADF4 of Araneus diadematus. Previously eADF4(C16) has been shown to self-assemble into cross-β fibrils in a two-step process of nucleus formation and fibril growth. Here, it is shown that structurally converted low molecular weight oligomers can act as nuclei. Further, it could be determined that specifically potassium and phosphate ions strongly influence both nucleus formation as well as fibril growth. Nucleation of fibril assembly could be surpassed by seeding soluble protein with pre-assembled fibrils but also, unexpectedly, with eADF4(C16) sub-micrometer particles. The latter finding reveals that spider silk fibril assembly seems to be rather dependent on the protein sequence than on the structural features, since cross-seeding with other proteins was not possible.





Categories: Journal Articles

Editorial Board

Journal of Molecular Biology - Wed, 09/30/2015 - 00:34
Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20









Categories: Journal Articles

Contents List

Journal of Molecular Biology - Wed, 09/30/2015 - 00:34
Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20









Categories: Journal Articles

Influence of Internal DNA Pressure on Stability and Infectivity of Phage λ

Journal of Molecular Biology - Wed, 09/30/2015 - 00:34
Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20

Author(s): D.W. Bauer, A. Evilevitch

Viruses must remain infectious while in harsh extracellular environments. An important aspect of viral particle stability for double-stranded DNA viruses is the energetically unfavorable state of the tightly confined DNA chain within the virus capsid creating pressures of tens of atmospheres. Here, we study the influence of internal genome pressure on the thermal stability of viral particles. Using differential scanning calorimetry to monitor genome loss upon heating, we find that internal pressure destabilizes the virion, resulting in a smaller activation energy barrier to trigger DNA release. These experiments are complemented by plaque assay and electron microscopy measurements to determine the influence of intra-capsid DNA pressure on the rates of viral infectivity loss. At higher temperatures (65–75°C), failure to retain the packaged genome is the dominant mechanism of viral inactivation. Conversely, at lower temperatures (40–55°C), a separate inactivation mechanism dominates, which results in non-infectious particles that still retain their packaged DNA. Most significantly, both mechanisms of infectivity loss are directly influenced by internal DNA pressure, with higher pressure resulting in a more rapid rate of inactivation at all temperatures.
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Categories: Journal Articles

Epigenetic Modulation of Human Podocyte Vitamin D Receptor in HIV Milieu

Journal of Molecular Biology - Wed, 09/30/2015 - 00:34
Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20

Author(s): Nirupama Chandel, Kameshwar S. Ayasolla, Xiqian Lan, Maria Sultana-Syed, Amrita Chawla, Rivka Lederman, Vasupradha Vethantham, Moin A. Saleem, Praveen N. Chander, Ashwani Malhotra, Pravin C. Singhal

HIV (human immunodeficiency virus) has been reported to induce podocyte injury through down regulation of vitamin D receptor (VDR) and activation of renin angiotensin system; however, the involved mechanism is not clear. Since HIV has been reported to modulate gene expression via epigenetic phenomena, we asked whether epigenetic factors contribute to down regulation of VDR. Kidney cells in HIV transgenic mice and HIV-infected podocytes (HIV/HPs) displayed enhanced expression of SNAIL, a repressor of VDR. To elucidate the mechanism, we studied the effect of HIV on expression of molecules involved in SNAIL repressor complex formation and demonstrated that HIV enhances expression of the histone deacetylase HDAC1 and DNA methyl transferases DNMT3b and DNMT1. 293T cells, when stably transfected with SNAIL (SNAIL/293T), displayed suppressed transcription and translation of VDR. In SNAIL/293T cells, co-immunoprecipitation studies revealed the association of HDAC1, DNMT3b, DNMT1, and mSin3A with SNAIL. Chromatin immunoprecipitation experiments confirmed the presence of the SNAIL repressor complex at the VDR promoter. Consistent with the enhanced DNA methyl transferase expression in HIV/HPs, there was an increased CpG methylation at the VDR promoter. Chromatin immunoprecipitation assay confirmed occurrence of H3K4 trimethylation on SNAIL promoter. Neither a VDR agonist (VDA) nor an HDAC inhibitor (HDACI) nor a demethylating agent (DAC) individually could optimally up regulate VDR in HIV milieu. However, VDA and HDACI when combined were successful in de-repressing VDR expression. Our findings demonstrate that SNAIL recruits multiple chromatin enzymes to form a repressor complex in HIV milieu that down regulates VDR expression.
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Categories: Journal Articles

Structural Insights into Nonspecific Binding of DNA by TrmBL2, an Archaeal Chromatin Protein

Journal of Molecular Biology - Wed, 09/30/2015 - 00:34
Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20

Author(s): Misbha Ud Din Ahmad, Ingrid Waege, Winfried Hausner, Michael Thomm, Winfried Boos, Kay Diederichs, Wolfram Welte

The crystal structure of TrmBL2 from the archaeon Pyrococcus furiosus shows an association of two pseudosymmetric dimers. The dimers follow the prototypical design of known bacterial repressors with two helix–turn–helix (HTH) domains binding to successive major grooves of the DNA. However, in TrmBL2, the two dimers are arranged at a mutual displacement of approximately 2bp so that they associate with the DNA along the double-helical axis at an angle of approximately 80°. While the deoxyribose phosphate groups of the double-stranded DNA (dsDNA) used for co-crystallization are clearly seen in the electron density map, most of the nucleobases are averaged out. Refinement required to assume a superposition of at least three mutually displaced dsDNAs. The HTH domains interact primarily with the deoxyribose phosphate groups and polar interactions with the nucleobases are almost absent. This hitherto unseen mode of DNA binding by TrmBL2 seems to arise from nonoptimal protein–DNA contacts made by its four HTH domains resulting in a low-affinity, nonspecific binding to DNA.
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Categories: Journal Articles

Mis16 Independently Recognizes Histone H4 and the CENP-ACnp1-Specific Chaperone Scm3sp

Journal of Molecular Biology - Wed, 09/30/2015 - 00:34
Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20

Author(s): Sojin An, Hanseong Kim, Uhn-Soo Cho

CENP-A is a centromere-specific histone H3 variant that is required for kinetochore assembly and accurate chromosome segregation. For it to function properly, CENP-A must be specifically localized to centromeres. In fission yeast, Scm3sp and the Mis18 complex, composed of Mis16, Eic1, and Mis18, function as a CENP-ACnp1-specific chaperone and a recruiting factor, respectively, and together ensure accurate delivery of CENP-ACnp1 to centromeres. Although how Scm3sp specifically recognizes CENP-ACnp1 has been revealed recently, the recruiting mechanism of CENP-ACnp1 via the Mis18 complex remains unknown. In this study, we have determined crystal structures of Schizosaccharomyces japonicus Mis16 alone and in complex with the helix 1 of histone H4 (H4α1). Crystal structures followed by mutant analysis and affinity pull-downs have revealed that Mis16 recognizes both H4α1 and Scm3sp independently within the CENP-ACnp1/H4:Scm3sp complex. This observation suggests that Mis16 gains CENP-ACnp1 specificity by recognizing both Scm3sp and histone H4. Our studies provide insights into the molecular mechanisms underlying specific recruitment of CENP-ACnp1/H4:Scm3sp into centromeres.
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Categories: Journal Articles

The First Crystal Structure of the UP1 Domain of hnRNP A1 Bound to RNA Reveals a New Look for an Old RNA Binding Protein

Journal of Molecular Biology - Wed, 09/30/2015 - 00:34
Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20

Author(s): Christopher E. Morgan, Jennifer L. Meagher, Jeffrey D. Levengood, James Delproposto, Carrie Rollins, Jeanne A. Stuckey, Blanton S. Tolbert

The heterogeneous nuclear ribonucleoprotein (hnRNP) A1 protein is a multifunctional RNA binding protein implicated in a wide range of biological functions. Mechanisms and putative hnRNP A1–RNA interactions have been inferred primarily from the crystal structure of its UP1 domain bound to ssDNA. RNA stem loops represent an important class of known hnRNP A1 targets, yet little is known about the structural basis of hnRNP A1–RNA recognition. Here, we report the first high-resolution structure (1.92Å) of UP1 bound to a 5′-AGU-3′ trinucleotide that resembles sequence elements of several native hnRNP A1–RNA stem loop targets. UP1 interacts specifically with the AG dinucleotide sequence via a “nucleobase pocket” formed by the β-sheet surface of RRM1 and the inter-RRM linker; RRM2 does not contact the RNA. The inter-RRM linker forms the lid of the nucleobase pocket and we show using structure-guided mutagenesis that the conserved salt-bridge interactions (R75:D155 and R88:D157) on the α-helical side of the RNA binding surface stabilize the linker in a geometry poised to bind RNA. We further investigated the structural basis of UP1 binding HIViSL3ESS3 by determining a structural model of the complex scored by small-angle X-ray scattering. UP1 docks on the apical loop of SL3ESS3 using its RRM1 domain and inter-RRM linker only. The biophysical implications of the structural model were tested by measuring kinetic binding parameters, where mutations introduced within the apical loop reduce binding affinities by slowing down the rate of complex formation. Collectively, the data presented here provide the first insights into hnRNP A1–RNA interactions.
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Categories: Journal Articles

Snapshots of Conformational Changes Shed Light into the NtrX Receiver Domain Signal Transduction Mechanism

Journal of Molecular Biology - Wed, 09/30/2015 - 00:34
Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20

Author(s): Ignacio Fernández, Lisandro H. Otero, Sebastián Klinke, Mariela del Carmen Carrica, Fernando A. Goldbaum

Brucella abortus is an important pathogenic bacterium that has to overcome oxygen deficiency in order to achieve a successful infection. Previously, we proved that a two-component system formed by the histidine kinase NtrY and the response regulator NtrX is essential to achieve an adaptive response to low oxygen tension conditions. Even though the relevance of this signaling pathway has already been demonstrated in other microorganisms, its molecular activation mechanism has not yet been described in detail. In this article, we report the first crystal structures from different conformations of the NtrX receiver domain from B. abortus, and we propose a sequence of events to explain the structural rearrangements along the activation process. The analysis of the structures obtained in the presence of the phosphoryl group analog beryllofluoride led us to postulate that changes in the interface formed by the α4 helix and the β5 strand are important for the activation, producing a reorientation of the α5 helix. Also, a biochemical characterization of the NtrX receiver domain enzymatic activities was performed, describing its autophosphorylation and autodephosphorylation kinetics. Finally, the role of H85, an important residue, was addressed by site-directed mutagenesis. Overall, these results provide significant structural basis for understanding the response regulator activation in this bacterial two-component system.
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Categories: Journal Articles

Septin 9 Exhibits Polymorphic Binding to F-Actin and Inhibits Myosin and Cofilin Activity

Journal of Molecular Biology - Wed, 09/30/2015 - 00:34
Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20

Author(s): Clayton Smith, Lee Dolat, Dimitrios Angelis, Eva Forgacs, Elias T. Spiliotis, Vitold E. Galkin

Septins are a highly conserved family of proteins in eukaryotes that is recognized as a novel component of the cytoskeleton. Septin 9 (SEPT9) interacts directly with actin filaments and functions as an actin stress fiber cross-linking protein that promotes the maturation of nascent focal adhesions and cell migration. However, the molecular details of how SEPT9 interacts with F-actin remain unknown. Here, we use electron microscopy and image analysis to show that SEPT9 binds to F-actin in a highly polymorphic fashion. We demonstrate that the basic domain (B-domain) of the N-terminal tail of SEPT9 is responsible for actin cross-linking, while the GTP-binding domain (G-domain) does not bundle F-actin. We show that the B-domain of SEPT9 binds to three sites on F-actin, and the two of these sites overlap with the binding regions of myosin and cofilin. SEPT9 inhibits actin-dependent ATPase activity of myosin and competes with the weakly bound state of myosin for binding to F-actin. At the same time, SEPT9 significantly reduces the extent of F-actin depolymerization by cofilin. Taken together, these data suggest that SEPT9 protects actin filaments from depolymerization by cofilin and myosin and indicate a mechanism by which SEPT9 could maintain the integrity of growing and contracting actin filaments.
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Categories: Journal Articles

Architecture of the Complex Formed by Large and Small Terminase Subunits from Bacteriophage P22

Journal of Molecular Biology - Wed, 09/30/2015 - 00:34
Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20

Author(s): Reginald McNulty, Ravi Kumar Lokareddy, Ankoor Roy, Yang Yang, Gabriel C. Lander, Albert J.R. Heck, John E. Johnson, Gino Cingolani

Packaging of viral genomes inside empty procapsids is driven by a powerful ATP-hydrolyzing motor, formed in many double-stranded DNA viruses by a complex of a small terminase (S-terminase) subunit and a large terminase (L-terminase) subunit, transiently docked at the portal vertex during genome packaging. Despite recent progress in elucidating the structure of individual terminase subunits and their domains, little is known about the architecture of an assembled terminase complex. Here, we describe a bacterial co-expression system that yields milligram quantities of the S-terminase:L-terminase complex of the Salmonella phage P22. In vivo assembled terminase complex was affinity-purified and stabilized by addition of non-hydrolyzable ATP, which binds specifically to the ATPase domain of L-terminase. Mapping studies revealed that the N-terminus of L-terminase ATPase domain (residues 1–58) contains a minimal S-terminase binding domain sufficient for stoichiometric association with residues 140–162 of S-terminase, the L-terminase binding domain. Hydrodynamic analysis by analytical ultracentrifugation sedimentation velocity and native mass spectrometry revealed that the purified terminase complex consists predominantly of one copy of the nonameric S-terminase bound to two equivalents of L-terminase (1S-terminase:2L-terminase). Direct visualization of this molecular assembly in negative-stained micrographs yielded a three-dimensional asymmetric reconstruction that resembles a “nutcracker” with two L-terminase protomers projecting from the C-termini of an S-terminase ring. This is the first direct visualization of a purified viral terminase complex analyzed in the absence of DNA and procapsid.
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Categories: Journal Articles

Atomic-Resolution Structures of the APC/C Subunits Apc4 and the Apc5 N-Terminal Domain

Journal of Molecular Biology - Wed, 09/30/2015 - 00:34
Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20

Author(s): Nora B. Cronin, Jing Yang, Ziguo Zhang, Kiran Kulkarni, Leifu Chang, Hiroyuki Yamano, David Barford

Many essential biological processes are mediated by complex molecular machines comprising multiple subunits. Knowledge on the architecture of individual subunits and their positions within the overall multimeric complex is key to understanding the molecular mechanisms of macromolecular assemblies. The anaphase-promoting complex/cyclosome (APC/C) is a large multisubunit complex that regulates cell cycle progression by ubiquitinating cell cycle proteins for proteolysis by the proteasome. The holo-complex is composed of 15 different proteins that assemble to generate a complex of 20 subunits. Here, we describe the crystal structures of Apc4 and the N-terminal domain of Apc5 (Apc5N). Apc4 comprises a WD40 domain split by a long α-helical domain, whereas Apc5N has an α-helical fold. In a separate study, we had fitted these atomic models to a 3.6-Å-resolution cryo-electron microscopy map of the APC/C. We describe how, in the context of the APC/C, regions of Apc4 disordered in the crystal assume order through contacts to Apc5, whereas Apc5N shows small conformational changes relative to its crystal structure. We discuss the complementary approaches of high-resolution electron microscopy and protein crystallography to the structure determination of subunits of multimeric complexes.
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Categories: Journal Articles

When the Scaffold Cannot Be Ignored: The Role of the Hydrophobic Core in Ligand Binding and Specificity

Journal of Molecular Biology - Wed, 09/30/2015 - 00:34
Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20

Author(s): Diana A. Koulechova, Katherine W. Tripp, Geoffrey Horner, Susan Marqusee

The traditional view of protein–ligand binding treats a protein as comprising distinct binding epitopes on the surface of a degenerate structural scaffold, largely ignoring the impact of a protein's energy landscape. To determine the robustness of this simplification, we compared two small helix–turn–helix transcription factors with different energy landscapes. λ-Repressor is stable and well folded, while MarA appears to be marginally stable with multiple native conformations (molten). While λ-repressor is known to tolerate any hydrophobic mutation in the core, we find MarA drastically less tolerant to core mutation. Moreover, core mutations in MarA (distant from the DNA-binding interface) change the relative affinities of its binding partners, altering ligand specificity. These results can be explained by taking into account the effects of mutations on the entire energy landscape and not just the native state. Thus, for proteins with multiple conformations that are close in energy, such as many intrinsically disordered proteins, residues distant from the active site can alter both binding affinity and specificity.
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Categories: Journal Articles

STAC—A New Domain Associated with Transmembrane Solute Transport and Two-Component Signal Transduction Systems

Journal of Molecular Biology - Wed, 09/30/2015 - 00:34
Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20

Author(s): Mateusz Korycinski, Reinhard Albrecht, Astrid Ursinus, Marcus D. Hartmann, Murray Coles, Jörg Martin, Stanislaw Dunin-Horkawicz, Andrei N. Lupas

Transmembrane receptors are integral components of sensory pathways in prokaryotes. These receptors share a common dimeric architecture, consisting in its basic form of an N-terminal extracellular sensor, transmembrane helices, and an intracellular effector. As an exception, we have identified an archaeal receptor family—exemplified by Af1503 from Archaeoglobus fulgidus—that is C-terminally shortened, lacking a recognizable effector module. Instead, a HAMP domain forms the sole extension for signal transduction in the cytosol. Here, we examine the gene environment of Af1503-like receptors and find a frequent association with transmembrane transport proteins. Furthermore, we identify and define a closely associated new protein domain family, which we characterize structurally using Af1502 from A. fulgidus. Members of this family are found both as stand-alone proteins and as domains within extant receptors. In general, the latter appear as connectors between the solute carrier 5 (SLC5)–like transmembrane domains and two-component signal transduction (TCST) domains. This is seen, for example, in the histidine kinase CbrA, which is a global regulator of metabolism, virulence, and antibiotic resistance in Pseudomonads. We propose that this newly identified domain family mediates signal transduction in systems regulating transport processes and name it STAC, for SLC and TCST-Associated Component.
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Categories: Journal Articles

Role of the α Clamp in the Protein Translocation Mechanism of Anthrax Toxin

Journal of Molecular Biology - Wed, 09/30/2015 - 00:34
Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20

Author(s): Michael J. Brown, Katie L. Thoren, Bryan A. Krantz

Membrane-embedded molecular machines are utilized to move water-soluble proteins across these barriers. Anthrax toxin forms one such machine through the self-assembly of its three component proteins—protective antigen (PA), lethal factor, and edema factor. Upon endocytosis into host cells, acidification of the endosome induces PA to form a membrane-inserted channel, which unfolds lethal factor and edema factor and translocates them into the host cytosol. Translocation is driven by the proton motive force, composed of the chemical potential, the proton gradient (ΔpH), and the membrane potential (Δψ). A crystal structure of the lethal toxin core complex revealed an “α clamp” structure that binds to substrate helices nonspecifically. Here, we test the hypothesis that, through the recognition of unfolding helical structure, the α clamp can accelerate the rate of translocation. We produced a synthetic PA mutant in which an α helix was crosslinked into the α clamp to block its function. This synthetic construct impairs translocation by raising a yet uncharacterized translocation barrier shown to be much less force dependent than the known unfolding barrier. We also report that the α clamp more stably binds substrates that can form helices than those, such as polyproline, that cannot. Hence, the α clamp recognizes substrates by a general shape-complementarity mechanism. Substrates that are incapable of forming compact secondary structure (due to the introduction of a polyproline track) are severely deficient for translocation. Therefore, the α clamp and its recognition of helical structure in the translocating substrate play key roles in the molecular mechanism of protein translocation.
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Categories: Journal Articles

Omics approaches deciphering molecular function in large biological systems

Journal of Molecular Biology - Wed, 09/30/2015 - 00:34
Publication date: Available online 28 September 2015
Source:Journal of Molecular Biology

Author(s): Marina Ostankovitch, Igor Stagliar







Categories: Journal Articles

Nuclear reformation at the end of mitosis

Journal of Molecular Biology - Wed, 09/30/2015 - 00:34
Publication date: Available online 28 September 2015
Source:Journal of Molecular Biology

Author(s): Anna Katharina Schellhaus, Paola De Magistris, Wolfram Antonin

Cells have developed highly sophisticated ways to accurately pass on their genetic information to the daughter cells. In animal cells, which undergo open mitosis, the nuclear envelope breaks down at the beginning of mitosis and the chromatin massively condenses to be captured and segregated by the mitotic spindle. These events have to be reverted in order to allow the re-formation of a nucleus competent for DNA transcription and replication, as well as all other nuclear processes occurring in interphase. Here, we summarize our current knowledge of how, in animal cells, the highly compacted mitotic chromosomes are decondensed at the end of mitosis and how a nuclear envelope, including functional nuclear pore complexes, reassembles around these decondensing chromosomes.
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Categories: Journal Articles

Substrate induced allosteric change in the quaternary structure of the spermidine N-acetyltransferase SpeG

Journal of Molecular Biology - Wed, 09/30/2015 - 00:34
Publication date: Available online 26 September 2015
Source:Journal of Molecular Biology

Author(s): Ekaterina V. Filippova, Steven Weigand, Jerzy Osipiuk, Olga Kiryukhina, Andrzej Joachimiak, Wayne F. Anderson

The spermidine N-acetyltransferase SpeG is a dodecameric enzyme that catalyzes the transfer of an acetyl group from acetyl-coenzyme A to polyamines such as spermidine and spermine. SpeG has an allosteric polyamine-binding site and acetylating polyamines regulates their intracellular concentrations. The structures of SpeG from Vibrio cholerae in complexes with polyamines and cofactor have been characterized earlier. Here, we present the dodecameric structure of SpeG from V. cholerae in a ligand-free form in three different conformational states: open, intermediate and closed. All structures were crystallized in C2 space group symmetry and contain 6 monomers in the asymmetric unit cell. Two hexamers related by crystallographic twofold symmetry form the SpeG dodecamer. The open and intermediate states have a unique open dodecameric ring. This SpeG dodecamer is asymmetric except for the one twofold axis and is unlike any known dodecameric structure. Using a fluorescence thermal shift assay, size exclusion chromatography with multi-angle light scattering, small angle X-ray scattering analysis, negative stain electron microscopy, and structural analysis we demonstrate that this unique open dodecameric state exists in solution. Our combined results indicate that polyamines trigger conformational changes and induce the symmetric closed dodecameric state of the protein when they bind to their allosteric sites.
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Categories: Journal Articles
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