Journal of Molecular Biology

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

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

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

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

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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Omics approaches deciphering molecular function in large biological systems

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

Author(s): Marina Ostankovitch, Igor Stagliar







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Nuclear reformation at the end of mitosis

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

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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The HADDOCK2.2 web server: User-friendly integrative modeling of biomolecular complexes

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

Author(s): G.C.P. van Zundert, J.P.G.L.M. Rodrigues, M. Trellet, C. Schmitz, P.L. Kastritis, E. Karaca, A.S.J. Melquiond, M. van Dijk, S.J. de Vries, A.M.J.J. Bonvin

The prediction of the quaternary structure of biomolecular macromolecules is of paramount importance for fundamental understanding of cellular processes and drug design. In the era of integrative structural biology, one way of increasing the accuracy of modelling methods used to predict the structure of biomolecular complexes is to include as much experimental or predictive information as possible in the process. This has been at the core of our information-driven docking approach HADDOCK. We present here the updated version 2.2 of the HADDOCK portal, which offers new features such as support for mixed molecule types, additional experimental restraints and improved protocols, all of this in a user-friendly interface. With well over 6000 registered users and 108000 jobs served, an increasing fraction of which on grid resources, we hope this timely upgrade will help the community to solve important biological questions and further advance the field. The HADDOCK2.2 web server is freely accessible to non-profit users at http://haddock.science.uu.nl/services/HADDOCK2.2.
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Editorial Board/Cover Legend

Wed, 09/30/2015 - 00:34
Publication date: 25 September 2015
Source:Journal of Molecular Biology, Volume 427, Issue 19









Categories: Journal Articles

Contents List

Wed, 09/30/2015 - 00:34
Publication date: 25 September 2015
Source:Journal of Molecular Biology, Volume 427, Issue 19









Categories: Journal Articles

Dividing and Conquering the Family of RNA Recognition Motifs: A Representative Case Based on hnRNP L

Wed, 09/30/2015 - 00:34
Publication date: 25 September 2015
Source:Journal of Molecular Biology, Volume 427, Issue 19

Author(s): Sarah Loerch, Clara L. Kielkopf







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The Signature of the Five-Stranded vRRM Fold Defined by Functional, Structural and Computational Analysis of the hnRNP L Protein

Wed, 09/30/2015 - 00:34
Publication date: 25 September 2015
Source:Journal of Molecular Biology, Volume 427, Issue 19

Author(s): Markus Blatter, Stanislaw Dunin-Horkawicz, Inna Grishina, Christophe Maris, Stephane Thore, Timm Maier, Albrecht Bindereif, Janusz M. Bujnicki, Frédéric H.-T. Allain

The RNA recognition motif (RRM) is the far most abundant RNA binding domain. In addition to the typical β1α1β2β3α2β4 fold, various sub-structural elements have been described and reportedly contribute to the high functional versatility of RRMs. The heterogeneous nuclear ribonucleoprotein L (hnRNP L) is a highly abundant protein of 64kDa comprising four RRM domains. Involved in many aspects of RNA metabolism, hnRNP L specifically binds to RNAs containing CA repeats or CA-rich clusters. However, a comprehensive structural description of hnRNP L including its sub-structural elements is missing. Here, we present the structural characterization of the RRM domains of hnRNP L and demonstrate their function in repressing exon 4 of SLC2A2. By comparison of the sub-structural elements between the two highly similar paralog families of hnRNP L and PTB, we defined signatures underlying interacting C-terminal coils (ICCs), the RRM34 domain interaction and RRMs with a C-terminal fifth β-strand, a variation we denoted vRRMs. Furthermore, computational analysis revealed new putative ICC-containing RRM families and allowed us to propose an evolutionary scenario explaining the origins of the ICC and fifth β-strand sub-structural extensions. Our studies provide insights of domain requirements in alternative splicing mediated by hnRNP L and molecular descriptions for the sub-structural elements. In addition, the analysis presented may help to classify other abundant RRM extensions and to predict structure–function relationships.
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The Telomere Binding Protein Cdc13 and the Single-Stranded DNA Binding Protein RPA Protect Telomeric DNA from Resection by Exonucleases

Wed, 09/30/2015 - 00:34
Publication date: 25 September 2015
Source:Journal of Molecular Biology, Volume 427, Issue 19

Author(s): Matthew Greetham, Emmanuel Skordalakes, David Lydall, Bernard A. Connolly

The telomere is present at the ends of all eukaryotic chromosomes and usually consists of repetitive TG-rich DNA that terminates in a single-stranded 3′ TG extension and a 5′ CA-rich recessed strand. A biochemical assay that allows the in vitro observation of exonuclease-catalyzed degradation (resection) of telomeres has been developed. The approach uses an oligodeoxynucleotide that folds to a stem–loop with a TG-rich double-stranded region and a 3′ single-stranded extension, typical of telomeres. Cdc13, the major component of the telomere-specific CST complex, strongly protects the recessed strand from the 5′→3′ exonuclease activity of the model exonuclease from bacteriophage λ. The isolated DNA binding domain of Cdc13 is less effective at shielding telomeres. Protection is specific, not being observed in control DNA lacking the specific TG-rich telomere sequence. RPA, the eukaryotic single-stranded DNA binding protein, also inhibits telomere resection. However, this protein is non-specific, equally hindering the degradation of non-telomere controls.
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Updates to the Integrated Protein–Protein Interaction Benchmarks: Docking Benchmark Version 5 and Affinity Benchmark Version 2

Wed, 09/30/2015 - 00:34
Publication date: 25 September 2015
Source:Journal of Molecular Biology, Volume 427, Issue 19

Author(s): Thom Vreven, Iain H. Moal, Anna Vangone, Brian G. Pierce, Panagiotis L. Kastritis, Mieczyslaw Torchala, Raphael Chaleil, Brian Jiménez-García, Paul A. Bates, Juan Fernandez-Recio, Alexandre M.J.J. Bonvin, Zhiping Weng

We present an updated and integrated version of our widely used protein–protein docking and binding affinity benchmarks. The benchmarks consist of non-redundant, high-quality structures of protein–protein complexes along with the unbound structures of their components. Fifty-five new complexes were added to the docking benchmark, 35 of which have experimentally measured binding affinities. These updated docking and affinity benchmarks now contain 230 and 179 entries, respectively. In particular, the number of antibody–antigen complexes has increased significantly, by 67% and 74% in the docking and affinity benchmarks, respectively. We tested previously developed docking and affinity prediction algorithms on the new cases. Considering only the top 10 docking predictions per benchmark case, a prediction accuracy of 38% is achieved on all 55 cases and up to 50% for the 32 rigid-body cases only. Predicted affinity scores are found to correlate with experimental binding energies up to r =0.52 overall and r =0.72 for the rigid complexes.
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Characterization of the Catalytic Domain of Human APOBEC3B and the Critical Structural Role for a Conserved Methionine

Wed, 09/30/2015 - 00:34
Publication date: 25 September 2015
Source:Journal of Molecular Biology, Volume 427, Issue 19

Author(s): Sachini U. Siriwardena, Thisari A. Guruge, Ashok S. Bhagwat

Human APOBEC3B deaminates cytosines in DNA and belongs to the AID/APOBEC family of enzymes. These proteins are involved in innate and adaptive immunity and may cause mutations in a variety of cancers. To characterize its ability to convert cytosines into uracils, we tested several derivatives of APOBEC3B gene for their ability to cause mutations in Escherichia coli. Through this analysis, a methionine residue at the junction of the amino-terminal domain (NTD) and the carboxy-terminal domain (CTD) was found to be essential for high mutagenicity. Properties of mutants with substitutions at this position, examination of existing molecular structures of APOBEC3 family members and molecular modeling suggest that this residue is essential for the structural stability of this family of proteins. The APOBEC3B CTD with the highest mutational activity was purified to homogeneity and its kinetic parameters were determined. Size-exclusion chromatography of the CTD monomer showed that it is in equilibrium with its dimeric form and matrix-assisted laser desorption ionization time-of-flight analysis of the protein suggested that the dimer may be quite stable. The partially purified NTD did not show intrinsic deamination activity and did not enhance the activity of the CTD in biochemical assays. Finally, APOBEC3B was at least 10-fold less efficient at mutating 5-methylcytosine (5mC) to thymine than APOBEC3A in a genetic assay and was at least 10-fold less efficient at deaminating 5mC compared to C in biochemical assays. These results shed light on the structural organization of APOBEC3B catalytic domain, its substrate specificity and its possible role in causing genome-wide mutations.
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Analysis of a FANCE Splice Isoform in Regard to DNA Repair

Wed, 09/30/2015 - 00:34
Publication date: 25 September 2015
Source:Journal of Molecular Biology, Volume 427, Issue 19

Author(s): Frédérick Bouffard, Karine Plourde, Simon Bélanger, Geneviève Ouellette, Yvan Labrie, Francine Durocher

The FANC-BRCA DNA repair pathway is activated in response to interstrand crosslinks formed in DNA. A homozygous mutation in 1 of the 17 Fanconi anemia (FA) genes results in malfunctions of this pathway and development of FA syndrome. The integrity of this protein network is essential for good maintenance of DNA repair process and genome stability. Following the identification of an alternatively splice isoform of FANCE (Fanconi anemia complementation group E) significantly expressed in breast cancer individuals from high-risk non-BRCA1/2 families, we studied the impact of this FANCE splice isoform (FANCEΔ4) on DNA repair processes. We have demonstrated that FANCEΔ4 mRNA was efficiently translated into a functional protein and expressed in normal and breast cancer cell lines. Following treatment with the crosslinking agent mitomycin C, EUFA130 (FANCE-deficient) cells infected with FANCEΔ4 were blocked into G2/M phase, while cell survival was significantly reduced compared with FANCE-infected EUFA130 cells. In addition, FANCEΔ4 did not allow FANCD2 and FANCI monoubiquitination, which represents a crucial step of the FANC-BRCA functional pathway. As observed for FANCE wild-type protein, localization of FANCEΔ4 protein was confined to the nucleus following mitomycin C treatment. Although FANCEΔ4 protein showed interaction with FANCE, FANCEΔ4 did not support normal function of FANCE protein in this pathway and could have deleterious effects on FANCE protein activity. We have demonstrated that FANCEΔ4 seems to act as a regulator of FANCD2 protein expression level by promoting its degradation. This study highlights the importance of an efficient regulation of alternative splicing expression of FA genes for proper DNA repair.
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The Structure of Escherichia coli TcdA (Also Known As CsdL) Reveals a Novel Topology and Provides Insight into the tRNA Binding Surface Required for N6-Threonylcarbamoyladenosine Dehydratase Activity

Wed, 09/30/2015 - 00:34
Publication date: 25 September 2015
Source:Journal of Molecular Biology, Volume 427, Issue 19

Author(s): Sunmin Kim, Hyuk Lee, SangYoun Park

Escherichia coli TcdA (also known as CsdL) was previously shown to catalyze the ATP-dependent dehydration/cyclization of hypermodified tRNA N 6-threonylcarbamoyladenosine into further cyclic N 6-threonylcarbamoyladenosine. In this study, we report the X-ray crystal structures of E. coli TcdA with either AMP or ATP bound. The AMP/ATP-bound N-terminal sub-domain of TcdA resembles the ATP-binding Rossmann fold of E. coli ThiF and MoeB that are enzymes respectively taking part in the biosynthesis of thiamine and molybdopterin; however, the remaining C-terminal sub-domain of TcdA adopts a structure unrelated to any other known folds. In TcdA, the ATP-utilizing adenylation of tRNA N 6-threonylcarbamoyladenosine and a subsequent thioester formation via an active cysteine, similar to the mechanisms in ThiF and MoeB, could take place for the dehydratase function. Analysis of the structure with sequence alignment suggests the disordered Cys234 of TcdA as the most likely catalytic residue. The structure further indicates that the C-terminal sub-domain can provide a binding interface for the tRNA substrate. Binding study using the surface mutants of TcdA and tRNA reveals that the positively charged regions of mainly the C-terminal sub-domain are important for the tRNA recognition.
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Binding of the 5′-Triphosphate End of mRNA to the γ-Subunit of Translation Initiation Factor 2 of the Crenarchaeon Sulfolobus solfataricus

Wed, 09/30/2015 - 00:34
Publication date: 25 September 2015
Source:Journal of Molecular Biology, Volume 427, Issue 19

Author(s): Valentina Arkhipova, Elena Stolboushkina, Olesya Kravchenko, Vladislav Kljashtorny, Azat Gabdulkhakov, Maria Garber, Stanislav Nikonov, Birgit Märtens, Udo Bläsi, Oleg Nikonov

The heterotrimeric archaeal IF2 orthologue of eukaryotic translation initiation factor 2 consists of the α-subunit, β-subunit and γ-subunit. Previous studies showed that the γ-subunit of aIF2, besides its central role in Met-tRNAi binding, has an additional function: it binds to the 5′-triphosphorylated end of mRNA and protects its 5′-part from degradation. Competition studies with nucleotides and mRNA, as well as structural and kinetic analyses of aIF2γ mutants, strongly implicate the canonical GTP/GDP-binding pocket in binding to the 5′-triphosphate end of mRNAs. The biological implication of these findings is being discussed.
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The LcrG Tip Chaperone Protein of the Yersinia pestis Type III Secretion System Is Partially Folded

Wed, 09/30/2015 - 00:34
Publication date: 25 September 2015
Source:Journal of Molecular Biology, Volume 427, Issue 19

Author(s): Sukanya Chaudhury, Clarice de Azevedo Souza, Gregory V. Plano, Roberto N. De Guzman

The type III secretion system (T3SS) is essential in the pathogenesis of Yersinia pestis, the causative agent of plague. A small protein, LcrG, functions as a chaperone to the tip protein LcrV, and the LcrG–LcrV interaction is important in regulating protein secretion through the T3SS. The atomic structure of the LcrG family is currently unknown. However, because of its predicted helical propensity, many have suggested that the LcrG family forms a coiled-coil structure. Here, we show by NMR and CD spectroscopy that LcrG lacks a tertiary structure and it consists of three partially folded α-helices spanning residues 7–38, 41–46, and 58–73. NMR titrations of LcrG with LcrV show that the entire length of a truncated LcrG (residues 7–73) is involved in binding to LcrV. However, there is regional variation in how LcrG binds to LcrV. The C-terminal region of a truncated LcrG (residues 52–73) shows tight binding interaction with LcrV while the N-terminal region (residues 7–51) shows weaker interaction with LcrV. This suggests that there are at least two binding events when LcrG binds to LcrV. Biological assays and mutagenesis indicate that the C-terminal region of LcrG (residues 52–73) is important in blocking protein secretion through the T3SS. Our results reveal structural and mechanistic insights into the atomic conformation of LcrG and how it binds to LcrV.
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Selection of High-Affinity Peptidic Serine Protease Inhibitors with Increased Binding Entropy from a Back-Flip Library of Peptide–Protease Fusions

Wed, 09/30/2015 - 00:34
Publication date: 25 September 2015
Source:Journal of Molecular Biology, Volume 427, Issue 19

Author(s): Hans Peter Sørensen, Peng Xu, Longguang Jiang, Tobias Kromann-Hansen, Knud J. Jensen, Mingdong Huang, Peter A. Andreasen

We have developed a new concept for designing peptidic protein modulators, by recombinantly fusing the peptidic modulator, with randomized residues, directly to the target protein via a linker and screening for internal modulation of the activity of the protein. We tested the feasibility of the concept by fusing a 10-residue-long, disulfide-bond-constrained inhibitory peptide, randomized in selected positions, to the catalytic domain of the serine protease murine urokinase-type plasminogen activator. High-affinity inhibitory peptide variants were identified as those that conferred to the fusion protease the lowest activity for substrate hydrolysis. The usefulness of the strategy was demonstrated by the selection of peptidic inhibitors of murine urokinase-type plasminogen activator with a low nanomolar affinity. The high affinity could not have been predicted by rational considerations, as the high affinity was associated with a loss of polar interactions and an increased binding entropy.
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Categories: Journal Articles