A selection of this week’s interesting papers, brought to you via the Furman Lab
Journal of the American Chemical Society (6 April 2012), doi:10.1021/ja301868p
The reversible photo-control of biomolecules requires chromophores that can efficiently undergo large conformational changes upon exposure to wavelengths of light that are compatible with living systems. We designed a benzylidene pyrroline chromophore that mimics the Schiff base of rhodopsin and can be used to introduce light-switchable intramolecular cross-links in peptides and proteins. This new class of photoswitch undergoes a ~10Å change in end-to-end distance upon isomerization and can be used to efficiently and reversibly control the conformation of a target peptide using, alternately, violet (400 nm) and blue (446 nm) light.
Marina Blanco-Lomas, Subhas Samanta, Pedro José Campos, Andrew Woolley, Diego Sampedro
Psoromic Acid is a Selective and Covalent Rab-Prenylation Inhibitor Targeting Autoinhibited RabGGTase.
Journal of the American Chemical Society (5 April 2012), doi:10.1021/ja211305j
Posttranslational attachment of geranylgeranyl isoprenoids to RabGTPases, the key organizers of intracellular vesicular transport, is essential for their function. Rab geranylgeranyl transferase (RabGGTase) is responsible for prenylation of Rab proteins. Recently, RabGGTase inhibitors have been proposed to be potential therapeutics for treatment of cancer and osteoporosis. However, the development of RabGGTase selective inhibitors is complicated by its structural and functional similarity to other protein prenyltransferases. Herein we report identification of the natural product psoromic acid (PA) that potently and selectively inhibits of RabGGTase with an IC50 of 1.3 µM. Structure-activity relationship analysis suggested a minimal structure involving the depsidone core with a 3-hydroxyl and 4-aldehyde motif for binding to RabGGTase. Analysis of crystal structure of RabGGTase:PA complex revealed that PA forms largely hydrophobic interactions with the isoprenoid binding site of RabGGTase and that it attaches covalently to the N-terminus of the ? subunit. We found that in contrast to other protein prenyltransferases, RabGGTase is autoinhibited through N-terminal ?His2 coordination with the catalytic zinc ion. Mutation of ?His dramatically enhances the reaction rate, indicating that the activity of RabGGTase is likely regulated in vivo. The covalent binding of PA to the N-terminus of the RabGGTase ? subunit seems to potentiate its interaction with the active site and explains the selectivity of PA for RabGGTase. Therefore, psoromic acid provides a new starting point for the development of selective RabGGTase inhibitors.
Céline Deraeve, Zhong Guo, Robin Stefan Bon, Wulf Blankenfeldt, Raffaella Dilucrezia, Alexander Wolf, Sascha Menninger, Anouk Stigter, Stefan Wetzel, Axel Choidas, Kirill Alexandrov, Herbert Waldmann, Roger Goody, Yao-Wen Wu
Journal of the American Chemical Society (11 April 2012), doi:10.1021/ja3017297
Ribosomally synthesized and post-translationally modified peptides are a rapidly expanding class of natural products. They are typically biosynthesized by modification of a C-terminal segment of the precursor peptide (the core peptide). The precursor peptide also contains an N-terminal leader peptide that is required to guide the biosynthetic enzymes. For bioengineering purposes, the leader peptide is beneficial because it allows promiscuous activity of the biosynthetic enzymes with respect to modification of the core peptide sequence. However, the leader peptide also presents drawbacks as it needs to be present on the core peptide and then removed in a later step. We show that fusing the leader peptide for the lantibiotic lacticin 481 to its biosynthetic enzyme LctM allows the protein to act on core peptides without a leader peptide. We illustrate the use of this methodology for preparation of improved lacticin 481 analogues containing non-proteinogenic amino acids.
Trent Oman, Patrick Knerr, Noah Bindman, Juan Velásquez, Wilfred van der Donk
Proceedings of the National Academy of Sciences of the United States of America (12 April 2012), doi:10.1073/pnas.1203767109
Genome-wide protein-DNA binding dynamics suggest a molecular clutch for transcription factor function
Nature, Vol. 484, No. 7393. (12 April 2012), pp. 251-255, doi:10.1038/nature10985
Dynamic access to genetic information is central to organismal development and environmental response. Consequently, genomic processes must be regulated by mechanisms that alter genome function relatively rapidly. Conventional chromatin immunoprecipitation (ChIP) experiments measure transcription factor occupancy, but give no indication of kinetics and are poor predictors of transcription factor function at a given locus. To measure transcription-factor-binding dynamics across the genome, we performed competition ChIP (refs 6, 7) with a sequence-specific Saccharomyces cerevisiae transcription factor, Rap1 (ref. 8). Rap1-binding dynamics and Rap1 occupancy were only weakly correlated (R(2) = 0.14), but binding dynamics were more strongly linked to function than occupancy. Long Rap1 residence was coupled to transcriptional activation, whereas fast binding turnover, which we refer to as ‘treadmilling’, was linked to low transcriptional output. Thus, DNA-binding events that seem identical by conventional ChIP may have different underlying modes of interaction that lead to opposing functional outcomes. We propose that transcription factor binding turnover is a major point of regulation in determining the functional consequences of transcription factor binding, and is mediated mainly by control of competition between transcription factors and nucleosomes. Our model predicts a clutch-like mechanism that rapidly engages a treadmilling transcription factor into a stable binding state, or vice versa, to modulate transcription factor function.
Colin Lickwar, Florian Mueller, Sean Hanlon, James McNally, Jason Lieb
PLoS computational biology, Vol. 8, No. 4. (April 2012), doi:10.1371/journal.pcbi.1002463
Models of early protein evolution posit the existence of short peptides that bound metals and ions and served as transporters, membranes or catalysts. The Cys-X-X-Cys-X-X-Cys heptapeptide located within bacterial ferredoxins, enclosing an Fe(4)S(4) metal center, is an attractive candidate for such an early peptide. Ferredoxins are ancient proteins and the simple ?+? fold is found alone or as a domain in larger proteins throughout all three kingdoms of life. Previous analyses of the heptapeptide conformation in experimentally determined ferredoxin structures revealed a pervasive right-handed topology, despite the fact that the Fe(4)S(4) cluster is achiral. Conformational enumeration of a model CGGCGGC heptapeptide bound to a cubane iron-sulfur cluster indicates both left-handed and right-handed folds could exist and have comparable stabilities. However, only the natural ferredoxin topology provides a significant network of backbone-to-cluster hydrogen bonds that would stabilize the metal-peptide complex. The optimal peptide configuration (alternating ?(L),?(R)) is that of an ?-sheet, providing an additional mechanism where oligomerization could stabilize the peptide and facilitate iron-sulfur cluster binding.
Dongun Kim, Agustina Rodriguez-Granillo, David Case, Vikas Nanda, Paul Falkowski
Science, Vol. 336, No. 6078. (13 April 2012), pp. 229-233, doi:10.1126/science.1216533
The mechanism of ion channel voltage gating-how channels open and close in response to voltage changes-has been debated since Hodgkin and Huxley’s seminal discovery that the crux of nerve conduction is ion flow across cellular membranes. Using all-atom molecular dynamics simulations, we show how a voltage-gated potassium channel (KV) switches between activated and deactivated states. On deactivation, pore hydrophobic collapse rapidly halts ion flow. Subsequent voltage-sensing domain (VSD) relaxation, including inward, 15-angstrom S4-helix motion, completes the transition. On activation, outward S4 motion tightens the VSD-pore linker, perturbing linker-S6-helix packing. Fluctuations allow water, then potassium ions, to reenter the pore; linker-S6 repacking stabilizes the open pore. We propose a mechanistic model for the sodium/potassium/calcium voltage-gated ion channel superfamily that reconciles apparently conflicting experimental data.
Morten, Vishwanath Jogini, David Borhani, Abba Leffler, Ron Dror, David Shaw
J. Chem. Inf. Model. In Journal of Chemical Information and Modeling (29 March 2012), doi:10.1021/ci200608b
The ligand binding pockets of proteins have preponderance of hydrophobic amino acids and are typically within the apolar interior of the protein; nevertheless, they are able to bind low complexity, polar, water-soluble fragments. In order to understand this phenomenon, we analyzed high resolution X-ray data of protein?ligand complexes from the Protein Data Bank and found that fragments bind to proteins with two near optimal geometry H-bonds on average. The linear extent of the fragment binding site was found not to be larger than 10 Å, and the H-bonding region was found to be restricted to about 5 Å on average. The number of conserved H-bonds in proteins cocrystallized with multiple different fragments is also near to 2. These fragment binding sites that are able to form limited number of strong H-bonds in a hydrophobic environment are identified as hot spots. An estimate of the free-energy gain of H-bond formation versus apolar desolvation supports that fragment sized compounds need H-bonds to achieve detectable binding. This suggests that fragment binding is mostly enthalpic that is in line with their observed binding thermodynamics documented in Isothermal Titration Calorimetry (ITC) data sets and gives a thermodynamic rationale for fragment based approaches. The binding of larger compounds tends to more rely on apolar desolvation with a corresponding increase of the entropy content of their binding free-energy. These findings explain the reported size-dependence of maximal available affinity and ligand efficiency both behaving differently in the small molecule region featured by strong H-bond formation and in the larger molecule region featured by apolar desolvation.
György Ferenczy, György Keser?
Current opinion in structural biology (2 April 2012), doi:10.1016/j.sbi.2012.03.008
Knowledge of the transition state is key to understanding a reaction mechanism. This vital information has been lacking for integral membrane protein folding, but now recent advances have given insight into the structure of their folding transition state. This progress has arisen through the successful translation of a classical protein engineering method, ? value analysis, from water-soluble proteins to the hydrophobic, membrane-embedded protein class. This review covers the transition state for the folding of ? helical membrane proteins. Helix formation in the transition state correlates with sequence position and the order of transmembrane insertion into the cell membrane, showing that in vitro measurements, in entirely different conditions to natural membranes, may reflect the cellular situation.
Current opinion in structural biology (3 April 2012), doi:10.1016/j.sbi.2012.03.004
Tight regulation of gene products from transcription to protein degradation is required for reliable and robust control of eukaryotic cell physiology. Many of the mechanisms directing cell regulation rely on proteins detecting the state of the cell through context-dependent, tuneable interactions. These interactions underlie the ability of proteins to make decisions by combining regulatory information encoded in a protein’s expression level, localisation and modification state. This raises the question, how do proteins integrate available information to correctly make decisions? Over the past decade pioneering work on the nature and function of intrinsically disordered protein regions has revealed many elegant switching mechanisms that underlie cell signalling and regulation, prompting a reevaluation of their role in cooperative decision-making.
Kim Van Roey, Toby Gibson, Norman Davey
How conformational dynamics of DNA polymerase select correct substrates: experiments and simulations.
Structure (London, England : 1993), Vol. 20, No. 4. (4 April 2012), pp. 618-627, doi:10.1016/j.str.2012.02.018
Nearly every enzyme undergoes a significant change in structure after binding it’s substrate. Experimental and theoretical analyses of the role of changes in HIV reverse transcriptase structure in selecting a correct substrate are presented. Atomically detailed simulations using the Milestoning method predict a rate and free energy profile of the conformational change commensurate with experimental data. A large conformational change occurring on a millisecond timescale locks the correct nucleotide at the active site but promotes release of a mismatched nucleotide. The positions along the reaction coordinate that decide the yield of the reaction are not determined by the chemical step. Rather, the initial steps of weak substrate binding and protein conformational transition significantly enrich the yield of a reaction with a correct substrate, whereas the same steps diminish the reaction probability of an incorrect substrate.
Serdal Kirmizialtin, Virginia Nguyen, Kenneth Johnson, Ron Elber
Structure (London, England : 1993), Vol. 20, No. 4. (4 April 2012), pp. 676-687, doi:10.1016/j.str.2012.02.010
Lacking any discernible sequence similarity, interleukin-34 (IL-34) and colony stimulating factor 1 (CSF-1) signal through a common receptor CSF-1R on cells of mononuclear phagocyte lineage. Here, the crystal structure of dimeric IL-34 reveals a helical cytokine fold homologous to CSF-1, and we further show that the complex architecture of IL-34 bound to the N-terminal immunoglobulin domains of CSF-1R is similar to the CSF-1/CSF-1R assembly. However, unique conformational adaptations in the receptor domain geometry and intermolecular interface explain the cross-reactivity of CSF-1R for two such distantly related ligands. The docking adaptations of the IL-34 and CSF-1 quaternary complexes, when compared to the stem cell factor assembly, draw a common evolutionary theme for transmembrane signaling. In addition, the structure of IL-34 engaged by a Fab fragment reveals the mechanism of a neutralizing antibody that can help deconvolute IL-34 from CSF-1 biology, with implications for therapeutic intervention in diseases with myeloid pathogenic mechanisms.
Xiaolei Ma, Wei Yu Lin, Yongmei Chen, Scott Stawicki, Kiran Mukhyala, Yan Wu, Flavius Martin, Fernando Bazan, Melissa Starovasnik
Structure (London, England : 1993), Vol. 20, No. 4. (4 April 2012), pp. 718-728, doi:10.1016/j.str.2012.01.024
Protein structures are frequently related by spectacular and often surprising similarities. Structural correlations among protein chains are routinely detected by various structure-matching techniques, but the comparison of oligomers and molecular complexes is largely uncharted territory. Here we solve the structure-matching problem for oligomers and large molecular aggregates, including the largest molecular complexes known today. We provide several challenging examples that cannot be handled by conventional structure-matching techniques and we report on a number of remarkable correlations. The examples cover the cell-puncturing device of bacteriophage T4, the secretion system of P. aeruginosa, members of the dehydrogenase family, DNA clamps, ferredoxin iron-storage cages, and virus capsids.
Manfred Sippl, Markus Wiederstein
Two Distinct Binding Modes Define the Interaction of Brox with the C-Terminal Tails of CHMP5 and CHMP4B.
Structure (London, England : 1993) (5 April 2012), doi:10.1016/j.str.2012.03.001
Interactions of the CHMP protein carboxyl terminal tails with effector proteins play important roles in retroviral budding, cytokinesis, and multivesicular body biogenesis. Here we demonstrate that hydrophobic residues at the CHMP4B C-terminal amphipathic ? helix bind a concave surface of Brox, a mammalian paralog of Alix. Unexpectedly, CHMP5 was also found to bind Brox and specifically recruit endogenous Brox to detergent-resistant membrane fractions through its C-terminal 20 residues. Instead of an ? helix, the CHMP5 C-terminal tail adopts a tandem ?-hairpin structure that binds Brox at the same site as CHMP4B. Additional Brox:CHMP5 interface is furnished by a unique CHMP5 hydrophobic pocket engaging the Brox residue Y348 that is not conserved among the Bro1 domains. Our studies thus unveil a ?-hairpin conformation of the CHMP5 protein C-terminal tail, and provide insights into the overlapping but distinct binding profiles of ESCRT-III and the Bro1 domain proteins.
Ruiling Mu, Vincent Dussupt, Jiansheng Jiang, Paola Sette, Victoria Rudd, Watchalee Chuenchor, Nana Bello, Fadila Bouamr, Tsan Sam Xiao
The Journal of biological chemistry (12 April 2012), doi:10.1074/jbc.M111.335752
The medium-chain dehydrogenase/reductase (MDR) superfamily consists of a large group of enzymes with broad range of activities. Members of this superfamily are currently the subject of intensive investigation, but many aspects, including the Zn dependence of MDR superfamily proteins, have not yet been adequately investigated. Using a density functional theory (DFT)-based screening strategy, we have identified a strictly conserved glycine residue (GLY) in the Zn-dependent MDR superfamily. To elucidate the role of this conserved GLY in MDR, we carried out a comprehensive structural, functional, and computational analysis of four MDR enzymes through a series of studies including site-directed mutagenesis, isothermal titration calorimetry (ITC), electron paramagnetic resonance (EPR), quantum mechanics (QM), and molecular mechanics (MM) analysis. GLY substitution by other amino acids posed a significant threat to the metal binding affinity and activity of MDR superfamily enzymes. Mutagenesis at the conserved GLY resulted in alterations in the coordination of the catalytic Zn ion, with concomitant changes in metal-ligand bond length, bond angle, and the affinity (Kd) towards the Zn ion. The GLY mutants also showed different spectroscopic properties in EPR compared to those of the wild-type, indicating that the binding geometries of the Zn to the Zn-binding ligands were changed by the mutation. The present results demonstrate that the conserved Gly in the GHE motif plays a role in maintaining the metal binding affinity and the electronic state of the catalytic Zn ion during catalysis of the MDR superfamily enzymes.
Manish Tiwari, Raushan Singh, Ranjitha Singh, Marimuthu Jeya, Huimin Zhao, Jung-Kul Lee
Intrinsic protein-protein interaction mediated and chaperonin assisted sequential assembly of a stable Bardet Biedl syndome protein complex, the BBSome
Journal of Biological Chemistry (12 April 2012), doi:10.1074/jbc.M112.341487
The pleiotropic features of obesity, retinal degeneration, polydactyly, kidney abnormalities, cognitive impairment, hypertension and diabetes found in Bardet Biedl Syndrome (BBS) make this disorder an important model disorder for identifying molecular mechanisms involved in common human diseases. To date, sixteen BBS genes have been reported, seven of which (BBS1, 2, 4, 5, 7, 8, and 9) code for proteins that form a complex known as the BBSome. The function of the BBSome involves ciliary membrane biogenesis. Three additional BBS genes (BBS6, BBS10 and BBS12) have homology to type II chaperonins, and interact with CCT/TRiC proteins and BBS7 to form a complex termed the BBS-chaperonin complex. This complex is required for BBSome assembly. Little is known about the process and the regulation of BBSome formation. We utilized point mutations and null alleles of BBS proteins to disrupt assembly of the BBSome leading to the accumulation of BBSome assembly intermediates. By characterizing BBSome assembly intermediates, we show that the BBS-chaperonin complex plays a role in BBS7 stability. BBS7 interacts with BBS2 and becomes part of a BBS7/BBS2/BBS9 assembly intermediate referred to as the BBSome-core complex because it forms the core of the BBSome. BBS1, BBS5, BBS8, and finally BBS4 are added to the BBSome-core to form the complete BBSome.
Qihong Zhang, Dahai Yu, Seongjing Seo, Edwin Stone, Val Sheffield
Proceedings of the National Academy of Sciences of the United States of America (9 April 2012), doi:10.1073/pnas.1119456109
Staphylococcus aureus and Staphylococcus epidermidis form communities (called biofilms) on inserted medical devices, leading to infections that affect many millions of patients worldwide and cause substantial morbidity and mortality. As biofilms are resistant to antibiotics, device removal is often required to resolve the infection. Thus, there is a need for new therapeutic strategies and molecular data that might assist their development. Surface proteins S. aureus surface protein G (SasG) and accumulation-associated protein (S. epidermidis) promote biofilm formation through their “B” regions. B regions contain tandemly arrayed G5 domains interspersed with approximately 50 residue sequences (herein called E) and have been proposed to mediate intercellular accumulation through Zn(2+)-mediated homodimerization. Although E regions are predicted to be unstructured, SasG and accumulation-associated protein form extended fibrils on the bacterial surface. Here we report structures of E-G5 and G5-E-G5 from SasG and biophysical characteristics of single and multidomain fragments. E sequences fold cooperatively and form interlocking interfaces with G5 domains in a head-to-tail fashion, resulting in a contiguous, elongated, monomeric structure. E and G5 domains lack a compact hydrophobic core, and yet G5 domain and multidomain constructs have thermodynamic stabilities only slightly lower than globular proteins of similar size. Zn(2+) does not cause SasG domains to form dimers. The work reveals a paradigm for formation of fibrils on the 100-nm scale and suggests that biofilm accumulation occurs through a mechanism distinct from the “zinc zipper.” Finally, formation of two domains by each repeat (as in SasG) might reduce misfolding in proteins when the tandem arrangement of highly similar sequences is advantageous.
Dominika Gruszka, Justyna Wojdyla, Richard Bingham, Johan Turkenburg, Iain Manfield, Annette Steward, Andrew Leech, Joan Geoghegan, Timothy Foster, Jane Clarke, Jennifer Potts
The E. coli CsgB nucleator of curli assembles to ?-sheet oligomers that alter the CsgA fibrillization mechanism.
Proceedings of the National Academy of Sciences of the United States of America (9 April 2012), doi:10.1073/pnas.1204161109
Curli are extracellular proteinaceous functional amyloid aggregates produced by Escherichia coli, Salmonella spp., and other enteric bacteria. Curli mediate host cell adhesion and invasion and play a critical role in biofilm formation. Curli filaments consist of CsgA, the major subunit, and CsgB, the minor subunit. In vitro, purified CsgA and CsgB exhibit intrinsically disordered properties, and both are capable of forming amyloid fibers similar in morphology to those formed in vivo. However, in vivo, CsgA alone cannot form curli fibers, and CsgB is required for filament growth. Thus, we studied the aggregation of CsgA and CsgB both alone and together in vitro to investigate the different roles of CsgA and CsgB in curli formation. We found that though CsgA and CsgB individually are able to self-associate to form aggregates/fibrils, they do so using different mechanisms and with different kinetic behavior. CsgB rapidly forms structured oligomers, whereas CsgA aggregation is slower and appears to proceed through large amorphous aggregates before forming filaments. Substoichiometric concentrations of CsgB induce a change in the mechanism of CsgA aggregation from that of forming amorphous aggregates to that of structured intermediates similar to those of CsgB alone. Oligomeric CsgB accelerated the aggregation of CsgA, in contrast to monomeric CsgB, which had no effect. The structured ?-strand oligomers formed by CsgB serve as nucleators for CsgA aggregation. These results provide insights into the formation of curli in vivo, especially the nucleator function of CsgB.
Qin Shu, Scott Crick, Jerome Pinkner, Bradley Ford, Scott Hultgren, Carl Frieden
Proceedings of the National Academy of Sciences of the United States of America (10 April 2012), doi:10.1073/pnas.1200915109
It has been known for nearly 100 years that pressure unfolds proteins, yet the physical basis of this effect is not understood. Unfolding by pressure implies that the molar volume of the unfolded state of a protein is smaller than that of the folded state. This decrease in volume has been proposed to arise from differences between the density of bulk water and water associated with the protein, from pressure-dependent changes in the structure of bulk water, from the loss of internal cavities in the folded states of proteins, or from some combination of these three factors. Here, using 10 cavity-containing variants of staphylococcal nuclease, we demonstrate that pressure unfolds proteins primarily as a result of cavities that are present in the folded state and absent in the unfolded one. High-pressure NMR spectroscopy and simulations constrained by the NMR data were used to describe structural and energetic details of the folding landscape of staphylococcal nuclease that are usually inaccessible with existing experimental approaches using harsher denaturants. Besides solving a 100-year-old conundrum concerning the detailed structural origins of pressure unfolding of proteins, these studies illustrate the promise of pressure perturbation as a unique tool for examining the roles of packing, conformational fluctuations, and water penetration as determinants of solution properties of proteins, and for detecting folding intermediates and other structural details of protein-folding landscapes that are invisible to standard experimental approaches.
Julien Roche, Jose Caro, Douglas Norberto, Philippe Barthe, Christian Roumestand, Jamie Schlessman, Angel Garcia, Bertrand García-Moreno E, Catherine Royer
An intersubunit disulfide bridge stabilizes the tetrameric nucleoside diphosphate kinase of Aquifex aeolicus.
Proteins (22 February 2012), doi:10.1002/prot.24062
The nucleoside diphosphate kinase (Ndk) catalyzes the reversible transfer of the ?-phosphate from nucleoside triphosphate to nucleoside diphosphate. Ndks form hexamers or two types of tetramers made of the same building block, namely, the common dimer. The secondary interfaces of the Type I tetramer found in Myxococcus xanthus Ndk and of the Type II found in Escherichia coli Ndk involve the opposite sides of subunits. Up to now, the few available structures of Ndk from thermophiles were hexameric. Here, we determined the X-ray structures of four crystal forms of the Ndk from the hyperthermophilic bacterium Aquifex aeolicus (Aa-Ndk). Aa-Ndk displays numerous features of thermostable proteins and is made of the common dimer but it is a tetramer of Type I. Indeed, the insertion of three residues in a surface-exposed spiral loop, named the Kpn-loop, leads to the formation of a two-turn ?-helix that prevents both hexamer and Type II tetramer assembly. Moreover, the side chain of the cysteine at position 133, which is not present in other Ndk sequences, adopts two alternate conformations. Through the secondary interface, each one forms a disulfide bridge with the equivalent Cys133 from the neighboring subunit. This disulfide bridge was progressively broken during X-ray data collection by radiation damage. Such crosslinks counterbalance the weakness of the common-dimer interface. A 40% decrease of the kinase activity at 60°C after reduction and alkylation of the protein corroborates the structural relevance of the disulfide bridge on the tetramer assembly and enzymatic function.
Fanny Boissier, Florian Georgescauld, Lucile Moynié, Jean-William Dupuy, Claude Sarger, Mircea Podar, Ioan Lascu, Marie-France Giraud, Alain Dautant
Proteins (6 April 2012), doi:10.1002/prot.24079
The tertiary structures of protein complexes provide a crucial insight about the molecular mechanisms that regulate their functions and assembly. However, solving protein complex structures by experimental methods is often more difficult than single protein structures. Here, we have developed a novel computational multiple protein docking algorithm, Multi-LZerD, that builds models of multimeric complexes by effectively reusing pairwise docking predictions of component proteins. A genetic algorithm is applied to explore the conformational space followed by a structure refinement procedure. Benchmark on eleven hetero-multimeric complexes resulted in near native conformations for all but one of them (a root mean square deviation smaller than 2.5Å). We also show that our method copes with unbound docking cases well, outperforming the methodology that can be directly compared to our approach. Multi-LZerD was able to predict near native structures for multimeric complexes of various topologies.
Juan Esquivel-Rodríguez, Yifeng David Yang, Daisuke Kihara
Proteins (10 April 2012), doi:10.1002/prot.24085
We present an adaptation of the ART-nouveau energy surface sampling method to the problem of loop structure prediction. This method, previously used to study protein folding pathways and peptide aggregation, is well suited to the problem of sampling the conformation space of large loops by targeting probable folding pathways instead of sampling exhaustively that space. The number of sampled conformations needed by ART nouveau to find the global energy minimum for a loop was found to scale linearly with the sequence length of the loop for loops between 8 and about 20 amino acids. Considering the linear scaling dependance of the computation cost on the loop sequence length for sampling new conformations, we estimate the total computational cost of sampling larger loops to scale quadratically compared to the exponential scaling of exhaustive search methods.
Jean-François St-Pierre, Normand Mousseau
Proteins (10 April 2012), doi:10.1002/prot.24086
Contemporary template-based modeling techniques allow applications of modeling methods to vast biological problems. However, they tend to fail to provide accurate structures for less-conserved local regions in sequence even when the overall structure can be modeled reliably. We call these regions unreliable local regions (ULRs). Accurate modeling of ULRs is of enormous value because they are frequently involved in functional specificity. In this article, we introduce a new method for modeling ULRs in template-based models by employing a sophisticated loop modeling technique. Combined with our previous study on protein termini, the method is applicable to refinement of both loop and terminus ULRs. A large-scale test carried out in a blind fashion in CASP9 (the 9th Critical Assessment of techniques for Protein Structure prediction) shows that ULR structures are improved over initial template-based models by refinement in more than 70% of the successfully detected ULRs. It is also notable that successful modeling of several long ULRs over 12 residues is achieved. Overall, the current results show that a careful application of loop and terminus modeling can be a promising tool for model refinement in template-based modeling.
Hahnbeom Park, Chaok Seok
Homology modeling and molecular dynamics simulations of the active state of the nociceptin receptor (NOP) reveals new insights into agonist binding and activation.
Proteins (6 April 2012), doi:10.1002/prot.24077
The opioid receptor-like receptor ORL1, also known as the nociceptin receptor (NOP), is a Class A GPCR in the opioid receptor family. Although NOP shares a significant homology with the other opioid receptors, it does not bind known opioid ligands and has been shown to have a distinct mechanism of activation compared to the closely-related opioid receptors mu, delta and kappa. Previously reported homology models of the NOP receptor, based on the inactive-state GPCR crystal structures, give limited information on the activation and selectivity features of this fourth member of the opioid receptor family. We report here the first active-state homology model of the NOP receptor based on the opsin GPCR crystal structure. An inactive-state homology model of NOP was also built using a multiple template approach. Molecular dynamics simulation of the active-state NOP model and comparison to the inactive-state model suggests that NOP activation involves movements of TM3 and TM6 and several activation microswitches consistent with GPCR activation. Docking of the selective non-peptidic NOP agonist ligand Ro 64-6198 into the active-state model reveals active-site residues in NOP that play a role in the high selectivity of this ligand for NOP over the other opioid receptors. Docking the shortest active fragment of endogenous agonist nociceptin/orphaninFQ (residues 1-13) shows that the NOP EL2 loop interacts with the positively charged residues (8-13) of N/OFQ. Both agonists show extensive polar interactions with residues at the extracellular end of the transmembrane domain and EL2 loop, suggesting agonist-induced re-organization of polar networks, during receptor activation.
Pankaj Daga, Nurulain Zaveri
Proteins (6 April 2012), doi:10.1002/prot.24078
Inaccuracies in computational molecular modelling methods are often counterweighed by brute-force generation of a plethora of putative solutions. These are then typically sieved via structural clustering based on similarity measures such as the root mean square deviation of atomic positions. Albeit widely used, these measures suffer from several theoretical and technical limitations (e.g. choice of regions for fitting) that impair their application in multi-component systems (N>2), large-scale studies (e.g. interactomes), and other time-critical scenarios. We present here a simple similarity measure for structural clustering based on atomic contacts – the fraction of common contacts – and compare it with the most used similarity measure of the protein docking community – interface backbone RMSD. We show that this method produces very compact clusters in remarkably short time when applied to a collection of binary and multi-component protein-protein and protein-DNA complexes. Furthermore, it allows easy clustering of similar conformations of multi-component symmetrical assemblies in which chain permutations can occur. Simple contact-based metrics should be applicable to other structural biology clustering problems, in particular for time-critical or large-scale endeavors.
João Pglm Rodrigues, Mikaël Trellet, Christophe Schmitz, Panagiotis Kastritis, Ezgi Karaca, Adrien Sj Melquiond, Alexandre Mjj Bonvin
Proteins (13 April 2012), doi:10.1002/prot.24092
We introduce Toleranced Models (TOM), a generic and versatile framework meant to handle models of macro-molecular assemblies featuring uncertainties on the shapes and the positions of proteins. A TOM being a continuum of nested shapes, the inner (resp. outer) ones representing high (low) confidence regions, we present topological and geometric statistics assessing features of this continuum at multiple scales. While the topological statistics qualify contacts between instances of protein types and complexes involving prescribed protein types, the geometric statistics scale the geometric accuracy of these complexes. We validate the TOM framework on recent average models of the entire Nuclear Pore Complex (NPC) obtained from reconstruction by data integration, and confront our quantitative analysis against experimental findings related to complexes of the NPC, namely the Y-complex, the T-complex, and the Nsp1-Nup82-Nup159 complex. In the three cases, our analysis bridges the gap between global qualitative models of the entire NPC, and atomic resolution models or putative models of the aforementioned complexes. In a broader perspective, the quantitative assessments provided by the TOM framework should prove instrumental to implement a virtuous loop model reconstruction – model selection, in the context of reconstruction by data integration.
Tom Dreyfus, Valérie Doye, Frédéric Cazals
Structural and Functional Organization of the Ska Complex, a Key Component of the Kinetochore-Microtubule Interface.
Molecular cell (5 April 2012), doi:10.1016/j.molcel.2012.03.005
The Ska complex is an essential mitotic component required for accurate cell division in human cells. It is composed of three subunits that function together to establish stable kinetochore-microtubule interactions in concert with the Ndc80 network. We show that the structure of the Ska core complex is a W-shaped dimer of coiled coils, formed by intertwined interactions between Ska1, Ska2, and Ska3. The C-terminal domains of Ska1 and Ska3 protrude at each end of the homodimer, bind microtubules in vitro when connected to the central core, and are essential in vivo. Mutations disrupting the central coiled coil or the dimerization interface result in chromosome congression failure followed by cell death. The Ska complex is thus endowed with bipartite and cooperative tubulin-binding properties at the ends of a 350 Å-long molecule. We discuss how this symmetric architecture might complement and stabilize the Ndc80-microtubule attachments with analogies to the yeast Dam1/DASH complex.
Arockia Jeyaprakash, Anna Santamaria, Uma Jayachandran, Ying Wai Chan, Christian Benda, Erich Nigg, Elena Conti
Functional determinants of temperature adaptation in enzymes of cold- vs. warm-adapted mussels (genus Mytilus).
Molecular biology and evolution (6 April 2012), doi:10.1093/molbev/mss111
Temperature is a strong selective force on the evolution of proteins due to its effects on higher orders of protein structure and, thereby, on critical protein functions like ligand binding and catalysis. Comparisons among orthologous proteins from differently thermally adapted species show consistent patterns of adaptive variation in function, but few studies have examined functional adaptation among multiple structural families of proteins. Thus, with our present state of knowledge, it is difficult to predict what fraction of the proteome will exhibit adaptive variation in the face of temperature increases of a few to several degrees Celsius, i.e., temperature increases of the magnitude predicted by models of global warming. Here, we compared orthologous enzymes of the warm-adapted Mediterranean mussel Mytilus galloprovincialis and the cold-adapted Mytilus trossulus, a native of the North Pacific Ocean, species whose physiologies exhibit significantly different responses to temperature. We measured the effects of temperature on the kinetics (Michaelis-Menten constant – K(m)) of five enzymes that are important for ATP-generation and that represent distinct protein structural families. Among phosphoglucomutase (PGM), phosphoglucose isomerase (PGI), pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (GTP) (PEPCK), and isocitrate dehydrogenase (NADP) (IDH), only IDH orthologs showed significantly different thermal responses of K(m) between the two species. The K(m) of isocitrate of M. galloprovincialis-IDH was intrinsically lower and more thermally stable than that of M. trossulus-IDH, and thus had higher substrate affinity at high temperatures. Two amino acid substitutions account for the functional differences between IDH orthologs, one of which allows for more hydrogen bonds to form near the mobile region of the active site in M. galloprovincialis-IDH. Taken together, our findings cast light on the targets of adaptive evolution in the context of climate change; only a minority of proteins might adapt to small changes in temperature and these adaptations may involve only small changes in sequence.
Brent Lockwood, George Somero
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