A selection of this week’s interesting papers, brought to you via the Furman Lab
Structure (London, England : 1993), Vol. 20, No. 3. (7 March 2012), pp. 464-478, doi:10.1016/j.str.2012.01.023
Electron density maps of membrane proteins or large macromolecular complexes are frequently only determined at medium resolution between 4 Å and 10 Å, either by cryo-electron microscopy or X-ray crystallography. In these density maps, the general arrangement of secondary structure elements (SSEs) is revealed, whereas their directionality and connectivity remain elusive. We demonstrate that the topology of proteins with up to 250 amino acids can be determined from such density maps when combined with a computational protein folding protocol. Furthermore, we accurately reconstruct atomic detail in loop regions and amino acid side chains not visible in the experimental data. The EM-Fold algorithm assembles the SSEs de novo before atomic detail is added using Rosetta. In a benchmark of 27 proteins, the protocol consistently and reproducibly achieves models with root mean square deviation values <3 Å. Copyright Â© 2012 Elsevier Ltd. All rights reserved.
Steffen Lindert, Nathan Alexander, Nils Wötzel, Mert Karaka?, Phoebe Stewart, Jens Meiler
Posttranslational modifications of Rab GTPases help their insertion into membranes.
Proceedings of the National Academy of Sciences of the United States of America (26 March 2012), doi:10.1073/pnas.1202494109
Olena Pylypenko, Bruno Goud
Proceedings of the National Academy of Sciences of the United States of America (27 March 2012), doi:10.1073/pnas.1118640109
Surface charges of proteins have in several cases been found to function as “structural gatekeepers,” which avoid unwanted interactions by negative design, for example, in the control of protein aggregation and binding. The question is then if side-chain charges, due to their desolvation penalties, play a corresponding role in protein folding by avoiding competing, misfolded traps? To find out, we removed all 32 side-chain charges from the 101-residue protein S6 from Thermus thermophilus. The results show that the charge-depleted S6 variant not only retains its native structure and cooperative folding transition, but folds also faster than the wild-type protein. In addition, charge removal unleashes pronounced aggregation on longer timescales. S6 provides thus an example where the bias toward native contacts of a naturally evolved protein sequence is independent of charges, and point at a fundamental difference in the codes for folding and intermolecular interaction: specificity in folding is governed primarily by hydrophobic packing and hydrogen bonding, whereas solubility and binding relies critically on the interplay of side-chain charges.
Martin Kurnik, Linda Hedberg, Jens Danielsson, Mikael Oliveberg
Crystal structure of the S100A4-nonmuscle myosin IIA tail fragment complex reveals an asymmetric target binding mechanism.
Proceedings of the National Academy of Sciences of the United States of America (28 March 2012), doi:10.1073/pnas.1114732109
S100A4 is a member of the S100 family of calcium-binding proteins that is directly involved in tumor metastasis. It binds to the nonmuscle myosin IIA (NMIIA) tail near the assembly competence domain (ACD) promoting filament disassembly, which could be associated with increasing metastatic potential of tumor cells. Here, we investigate the mechanism of S100A4-NMIIA interaction based on binding studies and the crystal structure of S100A4 in complex with a 45-residue-long myosin heavy chain fragment. Interestingly, we also find that S100A4 binds as strongly to a homologous heavy chain fragment of nonmuscle myosin IIC as to NMIIA. The structure of the S100A4-NMIIA complex reveals a unique mode of interaction in the S100 family: A single, predominantly ?-helical myosin chain is wrapped around the Ca(2+)-bound S100A4 dimer occupying both hydrophobic binding pockets. Thermal denaturation experiments of coiled-coil forming NMIIA fragments indicate that the coiled-coil partially unwinds upon S100A4 binding. Based on these results, we propose a model for NMIIA filament disassembly: Part of the random coil tailpiece and the C-terminal residues of the coiled-coil are wrapped around an S100A4 dimer disrupting the ACD and resulting in filament dissociation. The description of the complex will facilitate the design of specific drugs that interfere with the S100A4-NMIIA interaction.
Bence Kiss, Annette Duelli, László Radnai, Katalin Kékesi, Gergely Katona, László Nyitray
Proceedings of the National Academy of Sciences of the United States of America (29 March 2012), doi:10.1073/pnas.1113248109
Autosomal recessive loss-of-function mutations within the PARK2 gene functionally inactivate the E3 ubiquitin ligase parkin, resulting in neurodegeneration of catecholaminergic neurons and a familial form of Parkinson disease. Current evidence suggests both a mitochondrial function for parkin and a neuroprotective role, which may in fact be interrelated. The antiapoptotic effects of parkin have been widely reported, and may involve fundamental changes in the threshold for apoptotic cytochrome c release, but the substrate(s) involved in parkin dependent protection had not been identified. Here, we demonstrate the parkin-dependent ubiquitination of endogenous Bax comparing primary cultured neurons from WT and parkin KO mice and using multiple parkin-overexpressing cell culture systems. The direct ubiquitination of purified Bax was also observed in vitro following incubation with recombinant parkin. We found that parkin prevented basal and apoptotic stress-induced translocation of Bax to the mitochondria. Moreover, an engineered ubiquitination-resistant form of Bax retained its apoptotic function, but Bax KO cells complemented with lysine-mutant Bax did not manifest the antiapoptotic effects of parkin that were observed in cells expressing WT Bax. These data suggest that Bax is the primary substrate responsible for the antiapoptotic effects of parkin, and provide mechanistic insight into at least a subset of the mitochondrial effects of parkin.
Bethann Johnson, Alison Berger, Giuseppe Cortese, Matthew Lavoie
Computational investigation of the HIV-1 Rev multimerization using molecular dynamics simulations and binding free energy calculations.
Proteins (20 February 2012), doi:10.1002/prot.24057
The HIV Rev protein mediates the nuclear export of viral mRNA, and is thereby essential for the production of late viral proteins in the replication cycle. Rev forms a large organized multimeric protein-protein complex for proper functioning. Recently, the three-dimensional structures of a Rev dimer and tetramer have been resolved and provide the basis for a thorough structural analysis of the binding interaction. Here, molecular dynamics (MD) and binding free energy calculations were performed to elucidate the forces thriving dimerization and higher order multimerization of the Rev protein. It is found that despite the structural differences between each crystal structure, both display a similar behavior according to our calculations. Our analysis based on a molecular mechanics-generalized Born surface area (MM/GBSA) and a configurational entropy approach demonstrates that the higher order multimerization site is much weaker than the dimerization site. In addition, a quantitative hot spot analysis combined with a mutational analysis reveals the most contributing amino acid residues for protein interactions in agreement with experimental results. Additional residues were found in each interface, which are important for the protein interaction. The investigation of the thermodynamics of the Rev multimerization interactions performed here could be a further step in the development of novel antiretrovirals using structure based drug design. Moreover, the variability of the angle between each Rev monomer as measured during the MD simulations suggests a role of the Rev protein in allowing flexibility of the arginine rich domain (ARM) to accommodate RNA binding. Proteins 2012; © 2012 Wiley Periodicals, Inc. Copyright © 2012 Wiley Periodicals, Inc.
Tom Venken, Dirk Daelemans, Marc De Maeyer, Arnout Voet
Proteins (20 February 2012), pp. n/a-n/a, doi:10.1002/prot.24058
The knowledge of conserved sequences in proteins is valuable in identifying functionally or structurally important residues. Generating the conservation profile of a sequence requires aligning families of homologous sequences and having knowledge of their evolutionary relationships. Here, we report that the conservation profile at the residue level can be quantitatively derived from a single protein structure with only backbone information. We found that the reciprocal packing density profiles of protein structures closely resemble their sequence conservation profiles. For a set of 554 nonhomologous enzymes, 74% (408/554) of the proteins have a correlation coefficient > 0.5 between these two profiles. Our results indicate that the three-dimensional structure, instead of being a mere scaffold for positioning amino acid residues, exerts such strong evolutionary constraints on the residues of the protein that its profile of sequence conservation essentially reflects that of its structural characteristics. Proteins 2012;. © 2012 Wiley Periodicals, Inc. Copyright © 2012 Wiley Periodicals, Inc.
Chien-Hua Shih, Chih-Min Chang, Yeong-Shin Lin, Wei-Cheng Lo, Jenn-Kang Hwang
Proteins (22 February 2012), doi:10.1002/prot.24059
RNA helicase A (RHA) is a highly conserved protein with multifaceted functions in the gene expression of cellular and viral mRNAs. RHA recognizes highly structured nucleotides and catalytically rearranges the various interactions between RNA, DNA, and protein molecules to provide a platform for the ribonucleoprotein complex. We present the first solution structures of the double-stranded RNA-binding domains (dsRBDs), dsRBD1 and dsRBD2, from mouse RHA. We discuss the binding mode of the dsRBDs of RHA, in comparison with the known dsRBD structures in their complexes. Our structural data provide important information for the elucidation of the molecular reassembly mediated by RHA. Proteins 2012;. © 2012 Wiley Periodicals, Inc. Copyright © 2012 Wiley Periodicals, Inc.
Takashi Nagata, Kengo Tsuda, Naohiro Kobayashi, Mikako Shirouzu, Takanori Kigawa, Peter Güntert, Shigeyuki Yokoyama, Yutaka Muto
Current opinion in chemical biology (5 March 2012), doi:10.1016/j.cbpa.2012.02.001
The enormous pool of chemical diversity found in nature serves as an excellent inventory for accessing biologically active compounds. This chemical inventory, primarily found in microorganisms and plants, is generated by a broad range of enzymatic pathways under precise genetic and protein-level control. In vitro pathway reconstruction can be used to characterize individual pathway enzymes, identify pathway intermediates, and gain an increased understanding of how pathways can be manipulated to generate natural product analogs. Moreover, through in vitro approaches, it is possible to achieve a diversification that is not restricted by toxicity, limited availability of intracellular precursors, or preconceived (by nature) regulatory controls. Additionally, combinatorial biosynthesis and high-throughput techniques can be used to generate both known natural products and analogs that would not likely be generated naturally. This current opinion review will focus on recent advances made in performing in vitro pathway-driven natural product diversification and opportunities for exploiting this approach for elucidating and entering this new chemical biology space. Copyright Â© 2012 Elsevier Ltd. All rights reserved.
Seok Joon Kwon, Mauricio Mora-Pale, Moo-Yeal Lee, Jonathan Dordick
Ribosomal production and in vitro selection of natural product-like peptidomimetics: The FIT and RaPID systems.
Current opinion in chemical biology (6 March 2012), doi:10.1016/j.cbpa.2012.02.014
Bioactive natural product peptides have diverse architectures such as non-standard sidechains and a macrocyclic backbone bearing modifications. In vitro translation of peptides bearing these features would provide the research community with a diverse collection of natural product peptide-like molecules with a potential for drug development. The ordinary in vitro translation system, however, is not amenable to the incorporation of non-proteinogenic amino acids or genetic encoding of macrocyclic backbones. To circumvent this problem, flexible tRNA-acylation ribozymes (flexizymes) were combined with a custom-made reconstituted translation system to produce the flexible in vitro translation (FIT) system. The FIT system was integrated with mRNA display to devise an in vitro selection technique, referred to as the random non-standard peptide integrated discovery (RaPID) system. It has recently yielded an N-methylated macrocyclic peptide having high affinity (K(d)=0.60nM) for its target protein, E6AP. Copyright Â© 2012. Published by Elsevier Ltd.
Christopher Hipolito, Hiroaki Suga
Visualisation of variable binding pockets on protein surfaces by probabilistic analysis of related structure sets.
BMC bioinformatics, Vol. 13, No. 1. (14 March 2012), 39, doi:10.1186/1471-2105-13-39
ABSTRACT: BACKGROUND: Protein structures provide a valuable resource for rational drug design. For a protein with no known ligand, computational tools can predict surface pockets that are of suitable size and shape to accommodate a complementary small-molecule drug. However, pocket prediction against single static structures may miss features of pockets that arise from proteins’ dynamic behaviour. In particular, ligand-binding conformations can be observed as transiently populated states of the apo protein, so it is possible to gain insight into ligand-bound forms by considering conformational variation in apo proteins. This variation can be explored by considering sets of related structures: computationally generated conformers, solution NMR ensembles, multiple crystal structures, homologues or homology models. It is non-trivial to compare pockets, either from different programs or across sets of structures. For a single structure, difficulties arise in defining a particular pocket’s boundaries. For a set of conformationally distinct structures the challenge is how to make reasonable comparisons between them given that a perfect structural alignment is not possible. RESULTS: We have developed a computational method, Provar, that provides a consistent representation of predicted binding pockets across sets of related protein structures. The outputs are probabilities that each atom or residue of the protein borders a predicted pocket. These probabilities can be readily visualised on a protein using existing molecular graphics software. We show how Provar simplifies comparison of the outputs of different pocket prediction algorithms, of pockets across multiple simulated conformations and between homologous structures. We demonstrate the benefits of use of multiple structures for protein-ligand and protein-protein interface analysis on a set of complexes and consider three case studies in detail: i) analysis of a kinase superfamily highlights the conserved occurrence of surface pockets at the active and regulatory sites; ii) a simulated ensemble of unliganded Bcl-2 structures reveals extensions of a known ligand-binding pocket not apparent in the apo crystal structure; iii) visualisations of interleukin-2 and its homologues highlight conserved pockets at the known receptor interfaces and regions whose conformation is known to change on inhibitor binding. CONCLUSIONS: Through post-processing of the output of a variety of pocket prediction software, Provar provides a flexible approach to visualization of the persistence or variability of pockets in sets of related protein structures.
Paul Ashford, David Moss, Alexander Alex, Siew Yeap, Alice Povia, Irene Nobeli, Mark Williams
BMC bioinformatics, Vol. 13, No. 1. (18 March 2012), 41, doi:10.1186/1471-2105-13-41
ABSTRACT: BACKGROUND: Identification of the residues in protein-protein interaction sites has a significant impact in problems such as drug discovery. Motivated by the observation that the set of interface residues of a protein tend to be conserved even among remote structural homologs, we introduce PrISE, a family of local structural similarity-based computational methods for predicting protein-protein interface residues. RESULTS: We present a novel representation of the surface residues of a protein in the form of structural elements. Each structural element consists of a central residue and its surface neighbors. The PrISE family of interface prediction methods use a representation of structural elements that captures the atomic composition and accessible surface area of the residues that make up each structural element. Each of the members of the PrISE methods identifies for each structural element in the query protein, a collection of similar structural elements in its repository of structural elements and weights them according to their similarity with the structural element of the query protein. PrISE_L relies on the similarity between structural elements (i.e. local structural similarity). PrISE_G relies on the similarity between protein surfaces (i.e. general structural similarity). PrISE_C, combines local structural similarity and general structural similarity to predict interface residues. These predictors label the central residue of a structural element in a query protein as an interface residue if a weighted majority of the structural elements that are similar to it are interface residues, and as a non-interface residue otherwise. The results of our experiments using three representative benchmark datasets show that the PrISE_C outperforms PrISE_L and PrISE_G; and that PrISE_C is highly competitive with state-of-the-art structure-based methods for predicting protein-protein interface residues. Our comparison of PrISE_C with PredUs , a recently developed method for predicting interface residues of a query protein based on the known interface residues of its (global) structural homologs, shows that performance superior or comparable to that of PredUs can be obtained using only local surface structural similarity. PrISE_C is available as a Web server at http://prise.cs.iastate.edu/ CONCLUSIONS: Local surface structural similarity based methods offer a simple, efficient, and effective approach to predict protein-protein interface residues.
Rafael Jordan, Yasser El-Manzalawy, Drena Dobbs, Vasant Honavar
Activity, stability, and structure of metagenome-derived LC11-RNase H1, a homolog of Sulfolobus tokodaii RNase H1.
Protein science : a publication of the Protein Society, Vol. 21, No. 4. (April 2012), pp. 553-561, doi:10.1002/pro.2043
Metagenome-derived LC11-RNase H1 is a homolog of Sulfolobus tokodaii RNase H1 (Sto-RNase H1). It lacks a C-terminal tail, which is responsible for hyperstabilization of Sto-RNase H1. Sto-RNase H1 is characterized by its ability to cleave not only an RNA/DNA hybrid but also a double-stranded RNA (dsRNA). To examine whether LC11-RNase H1 also exhibits both RNase H and dsRNase activities, LC11-RNase H1 was overproduced in Escherichia coli, purified, and characterized. LC11-RNase H1 exhibited RNase H activity with similar metal ion preference, optimum pH, and cleavage mode of substrate with those of Sto-RNase H1. However, LC11-RNase H1 did not exhibit dsRNase activity at any condition examined. LC11-RNase H1 was less stable than Sto-RNases H1 and its derivative lacking the C-terminal tail (Sto-RNase H1?C6) by 37 and 13°C in T(m) , respectively. To understand the structural bases for these differences, the crystal structure of LC11-RNase H1 was determined at 1.4 Å resolution. The LC11-RNase H1 structure is highly similar to the Sto-RNase H1 structure. However, LC11-RNase H1 has two grooves on protein surface, one containing the active site and the other containing DNA-phosphate binding pocket, while Sto-RNase H1 has one groove containing the active site. In addition, LC11-RNase H1 contains more cavities and buried charged residues than Sto-RNase H1. We propose that LC11-RNase H1 does not exhibit dsRNase activity because dsRNA cannot fit to the two grooves on protein surface and that LC11-RNase H1 is less stable than Sto-RNase H1?C6 because of the increase in cavity volume and number of buried charged residues. Copyright © 2012 The Protein Society.
Tri-Nhan Nguyen, Clement Angkawidjaja, Eiko Kanaya, Yuichi Koga, Kazufumi Takano, Shigenori Kanaya
Protein science : a publication of the Protein Society (9 March 2012), doi:10.1002/pro.2059
Enzyme catalysts of a retro-aldol reaction have been generated by computational design using a motif that combines a lysine in a non-polar environment with water-mediated stabilization of the carbinolamine hydroxyl and ?-hydroxyl groups. Here we show that the design process is robust and repeatable, with 33 new active designs constructed on 13 different protein scaffold backbones. The initial activities are not high but are increased through site-directed mutagenesis and laboratory evolution. Mutational data highlight areas for improvement in design. Different designed catalysts give different borohydride-reduced reaction intermediates, suggesting a distribution of properties of the designed enzymes that may be further explored and exploited. Copyright © 2012 The Protein Society.
Eric Althoff, Ling Wang, Lin Jiang, Lars Giger, Jonathan Lassila, Zhizhi Wang, Matthew Smith, Sanjay Hari, Peter Kast, Daniel Herschlag, Donald Hilvert, David Baker
GLU121-LYS319 salt-bridge between catalytic and N-terminal domains is pivotal for the activity and stability of E. coli aminopeptidase N.
Protein science : a publication of the Protein Society (12 March 2012), doi:10.1002/pro.2060
E. coli aminopeptidase N (ePepN) belongs to the gluzincin family of M1 class metalloproteases that share a common primary structure with consensus zinc binding motif (HEXXH-(X18)-E) and an exopeptidase motif (GXMEN) in the active site. There is one amino acid, E121 in Domain I that blocks the extended active site grove of the thermolysin like catalytic Domain II limiting the substrate to S1 pocket. E121 forms a part of the S1 pocket while making critical contact with the amino-terminus of the substrate. In addition, the carboxylate of E121 forms a salt-bridge with K319 in Domain II. Both these residues are absolutely conserved in ePepN homologs. Analogous Glu-Asn pair in tricon interacting factor F3 (F3) and Gln-Asn pair in human leukotriene A(4) hydrolase (LTA(4) H) are also conserved in respective homologs. Mutation of either of these residues individually or together substantially reduced or entirely eliminated enzymatic activity. In addition, thermal denaturation studies suggest that the mutation at K319 destabilizes the protein as much as by 3.7 °C while E121 mutants were insensitive. Crystal structure of E121Q mutant reveals that the enzyme is inactive due to the reduced S1 subsite volume. Together, data presented here suggests that ePepN, F3 and LTA(4) H homologs adopted a divergent evolution that includes E121-K319 or its analogous pairs, and these cannot be interchanged. Copyright © 2012 The Protein Society.
Rajesh Gumpena, Chandan Kishor, Roopa Jones Ganji, Nishant Jain, Anthony Addlagatta
The extension peptide of plant ferritin from sea lettuce contributes to shell stability and surface hydrophobicity.
Protein science : a publication of the Protein Society (14 March 2012), doi:10.1002/pro.2061
Plant ferritins have some unique structural and functional features. Most of these features can be related to the plant-specific ‘extension peptide’ (EP), which exists in the N-terminus of the mature region of a plant ferritin. Recent crystallographic analysis of a plant ferritin revealed the structure of the EP, however, two points remain unclear: (i) whether the structures of well-conserved EP of plant ferritins are common in all plants, and (ii) whether the EP truly contributes to the shell stability of the plant ferritin oligomer. To clarify these matters, we have cloned a green-plant-type ferritin cDNA from a green alga, Ulva pertusa, and investigated its crystal structure. Ulva pertusa ferritin (UpFER) has a plant-ferritin-specific extension peptide composed of 28 amino acid residues. In the crystal structure of UpFER, the EP lay on and interacted with the neighboring three-fold symmetry-related subunit. The amino acid residues involved in the interaction were very highly conserved among plant ferritins. The EPs masked the hydrophobic pockets on the ferritin shell surface by lying on them, and this made the ferritin oligomer more hydrophilic. Furthermore, differential scanning calorimetric analysis of the native and its EP-deletion mutant suggested that the EP contributed to the thermal stability of the plant ferritin shell. Thus, the shell stability and surface hydrophobicity of plant ferritin were controlled by the presence or absence of the plant-ferritin-specific EP. This regulation can account for those processes such as shell stability, degradation and association of plant ferritin, which are significantly related to iron utilization in plants. Copyright © 2012 The Protein Society.
Taro Masuda, Shin-Ichiro Morimoto, Bunzo Mikami, Haruhiko Toyohara
Current opinion in structural biology (17 March 2012), doi:10.1016/j.sbi.2012.02.006
Death domain (DD) superfamily members play a central role in apoptotic and inflammatory signaling through formation of oligomeric molecular scaffolds. These scaffolds promote the activation of proinflammatory and apoptotic initiator caspases, as well as Ser/Thr kinases. Interactions between DDs are facilitated by a conserved set of interaction surfaces, type I, type II, and type III. Recently structural information on a ternary complex containing the DDs of MyD88, IRAK4, and IRAK2 and a binary complex containing Fas and FADD DDs has become available. This review will focus on how the three DD interaction surfaces cooperate to facilitate the assembly of these oligomeric signaling complexes. Copyright Â© 2012 Elsevier Ltd. All rights reserved.
Ryan Ferrao, Hao Wu
Current opinion in structural biology (22 March 2012), doi:10.1016/j.sbi.2012.02.004
Membrane proteins regulate many cellular processes including signaling cascades, ion transport, membrane fusion, and cell-to-cell communications. Understanding the architecture and conformational fluctuations of these proteins is critical to understanding their regulation and functions. Fluorescence methods including intensity mapping, fluorescence resonance energy transfer (FRET), and photo-induced electron transfer, allow for targeted measurements of domains within membrane proteins. These methods can reveal how a protein is structured and how it transitions between different conformational states. Here, I will review recent work done using fluorescence to map the structures of membrane proteins, focusing on how each of these methods can be applied to understanding the dynamic nature of individual membrane proteins and protein complexes. Copyright Â© 2012. Published by Elsevier Ltd.
Current opinion in structural biology (22 March 2012), doi:10.1016/j.sbi.2012.03.002
?-Helical coiled coils are ubiquitous protein-protein-interaction domains. They share a relatively straightforward sequence repeat, which directs the folding and assembly of amphipathic ?-helices. The helices can combine in a number of oligomerisation states and topologies to direct a wide variety of protein assemblies. Although in nature parallel dimers, trimers and tetramers dominate, the potential to form larger oligomers and more-complex assemblies has long been recognised. In particular, complexes above pentamer are interesting because they are barrel-like, having central channels or pores with well-defined dimensions and chemistry. Recent empirical and rational design experiments are beginning to chart this potential new territory in coiled-coil space, leading to intriguing new structures, and possibilities for functionalisation and applications. Copyright Â© 2012 Elsevier Ltd. All rights reserved.
Derek Woolfson, Gail Bartlett, Marc Bruning, Andrew Thomson
Corresponding Functional Dynamics across the Hsp90 Chaperone Family: Insights from a Multiscale Analysis of MD Simulations.
PLoS computational biology, Vol. 8, No. 3. (22 March 2012), e1002433, doi:10.1371/journal.pcbi.1002433
Understanding how local protein modifications, such as binding small-molecule ligands, can trigger and regulate large-scale motions of large protein domains is a major open issue in molecular biology. We address various aspects of this problem by analyzing and comparing atomistic simulations of Hsp90 family representatives for which crystal structures of the full length protein are available: mammalian Grp94, yeast Hsp90 and E.coli HtpG. These chaperones are studied in complex with the natural ligands ATP, ADP and in the Apo state. Common key aspects of their functional dynamics are elucidated with a novel multi-scale comparison of their internal dynamics. Starting from the atomic resolution investigation of internal fluctuations and geometric strain patterns, a novel analysis of domain dynamics is developed. The results reveal that the ligand-dependent structural modulations mostly consist of relative rigid-like movements of a limited number of quasi-rigid domains, shared by the three proteins. Two common primary hinges for such movements are identified. The first hinge, whose functional role has been demonstrated by several experimental approaches, is located at the boundary between the N-terminal and Middle-domains. The second hinge is located at the end of a three-helix bundle in the Middle-domain and unfolds/unpacks going from the ATP- to the ADP-state. This latter site could represent a promising novel druggable allosteric site common to all chaperones.
Giulia Morra, Raffaello Potestio, Cristian Micheletti, Giorgio Colombo
Nature (28 March 2012), doi:10.1038/nature10963
H(+)-translocating pyrophosphatases (H(+)-PPases) are active proton transporters that establish a proton gradient across the endomembrane by means of pyrophosphate (PP(i)) hydrolysis. H(+)-PPases are found primarily as homodimers in the vacuolar membrane of plants and the plasma membrane of several protozoa and prokaryotes. The three-dimensional structure and detailed mechanisms underlying the enzymatic and proton translocation reactions of H(+)-PPases are unclear. Here we report the crystal structure of a Vigna radiata H(+)-PPase (VrH(+)-PPase) in complex with a non-hydrolysable substrate analogue, imidodiphosphate (IDP), at 2.35?Å resolution. Each VrH(+)-PPase subunit consists of an integral membrane domain formed by 16 transmembrane helices. IDP is bound in the cytosolic region of each subunit and trapped by numerous charged residues and five Mg(2+) ions. A previously undescribed proton translocation pathway is formed by six core transmembrane helices. Proton pumping can be initialized by PP(i) hydrolysis, and H(+) is then transported into the vacuolar lumen through a pathway consisting of Arg?242, Asp?294, Lys?742 and Glu?301. We propose a working model of the mechanism for the coupling between proton pumping and PP(i) hydrolysis by H(+)-PPases.
Shih-Ming Lin, Jia-Yin Tsai, Chwan-Deng Hsiao, Yun-Tzu Huang, Chen-Liang Chiu, Mu-Hsuan Liu, Jung-Yu Tung, Tseng-Huang Liu, Rong-Long Pan, Yuh-Ju Sun
Biophysical journal, Vol. 102, No. 6. (21 March 2012), pp. 1265-1273, doi:10.1016/j.bpj.2012.02.006
Cell surface receptors have been extensively studied because they initiate and regulate signal transduction cascades leading to a variety of functional cellular outcomes. An important class of immune receptors (e.g., T-cell antigen receptors) whose ligands are anchored to the surfaces of other cells remain poorly understood. The mechanism by which ligand binding initiates receptor phosphorylation, a process termed “receptor triggering”, remains controversial. Recently, direct measurements of the (two-dimensional) receptor-ligand complex lifetimes at cell-cell interface were found to be smaller than (three-dimensional) lifetimes in solution but the underlying mechanism is unknown. At the cell-cell interface, the receptor-ligand complex spans a short intermembrane distance (15 nm) compared to long surface molecules (LSMs) whose ectodomains span >40 nm and these LSMs include phosphatases (e.g., CD45) that dephosphorylate the receptor. It has been proposed that size-based segregation of LSMs from a receptor-ligand complex is a mechanism of receptor triggering but it is unclear whether the mechanochemistry supports such small-scale segregation. Here we present a nanometer-scale mathematical model that couples membrane elasticity with the compressional stiffness and lateral mobility of LSMs. We find robust supradiffusive segregation of LSMs from a single receptor-ligand complex. The model predicts that LSM redistribution will result in a time-dependent tension on the complex leading to a decreased two-dimensional lifetime. Interestingly, the model predicts a nonlinear relationship between the three- and two-dimensional lifetimes, which can enhance the ability of receptors to discriminate between similar ligands. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Jun Allard, Omer Dushek, Daniel Coombs, P Anton van der Merwe
Genome-wide Functional Annotation of Dual-Specificity Protein- and Lipid-Binding Modules that Regulate Protein Interactions.
Molecular cell (20 March 2012), doi:10.1016/j.molcel.2012.02.012
Emerging evidence indicates that membrane lipids regulate protein networking by directly interacting with protein-interaction domains (PIDs). As a pilot study to identify and functionally annodate lipid-binding PIDs on a genomic scale, we performed experimental and computational studies of PDZ domains. Characterization of 70 PDZ domains showed that ?40% had submicromolar membrane affinity. Using a computational model built from these data, we predicted the membrane-binding properties of 2,000 PDZ domains from 20 species. The accuracy of the prediction was experimentally validated for 26 PDZ domains. We also subdivided lipid-binding PDZ domains into three classes based on the interplay between membrane- and protein-binding sites. For different classes of PDZ domains, lipid binding regulates their protein interactions by different mechanisms. Functional studies of a PDZ domain protein, rhophilin 2, suggest that all classes of lipid-binding PDZ domains serve as genuine dual-specificity modules regulating protein interactions at the membrane under physiological conditions. Copyright Â© 2012 Elsevier Inc. All rights reserved.
Yong Chen, Ren Sheng, Morten Källberg, Antonina Silkov, Moe Tun, Nitin Bhardwaj, Svetlana Kurilova, Randy Hall, Barry Honig, Hui Lu, Wonhwa Cho
A novel mutation in the spectrin-like repeat 1 of dystrophin central domain causes protein misfolding and mild Becker muscular dystrophy.
The Journal of biological chemistry (27 March 2012), doi:10.1074/jbc.M111.284521
Mutations in the dystrophin gene without disruption of the reading frame often lead to Becker muscular dystrophy (BMD), but a genotype/phenotype correlation is difficult to establish. Amino acid substitutions may disrupt binding capacities of dystrophin and have a major impact on the functionality of this protein. We have identified two brothers (ages 8 and 10 years) with very mild proximal weakness, recurrent abdominal pain and moderately elevated serum creatine kinase levels. Gene sequencing revealed a novel mutation in exon 11 of the dystrophin gene (c.1280T>C) leading to a L427P amino acid substitution in repeat 1 of the central rod domain. Immunostaining of skeletal muscle showed weak staining of the dystrophin region encoded by exons 7 and 8 corresponding to the end of the actin-binding domain 1 and the N terminal part of hinge 1. Spectrofluorescence and circular dichroism analysis of the domain repeat 1-2 (R1-2) revealed partial misfolding of the L427P mutated protein as well as a reduced refolding rate after denaturation. Based on computational homology models of the wild type and mutated R1-2, a molecular dynamics study showed an alteration in the flexibility of the structure, which also strongly affects the conformational space available in the N-terminal region of the fragment. Our results suggest that this missense mutation hinders the dynamic properties of the entire N-terminal region of dystrophin.
Gyulia Ascadi, Steven Moore, Angelique Cheron, Olivier Delalande, Lindsey Benett, William Kupsky, Mohamad El-Baba, Elisabeth Le Rumeur, Jean-Francois Hubert
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