A selection of this week’s interesting papers, brought to you via the Furman Lab
A Crucial Role of Protein Flexibility in Formation of a Stable Reaction Transition State in an ?-Amylase Catalysis.
Journal of the American Chemical Society (2 April 2012), doi:10.1021/ja212117m
Conformational flexibility of proteins provides enzymes with high catalytic activity. Although the conformational flexibility is known to be pivotal for the ligand binding and release, its role in the chemical reaction process of the reactive substrate remains unclear. We determined a transition state of an enzymatic reaction in a psychrophilic ?-amylase by a hybrid molecular simulation that allows one to identify the optimal chemical state in an extensive conformational ensemble of protein. The molecular simulation uncovered that formation of the reaction transition state accompanies a large and slow movement of a loop adjacent to the catalytic site. Free energy calculations revealed that, although catalytic electrostatic potentials on the reactive moiety are formed by local and fast reorganization around the catalytic site, reorganization of the large and slow movement of the loop significantly contributes to reduction of the free energy barrier by stabilizing the local reorganization.
Takahiro Kosugi, Shigehiko Hayashi
Disorder-to-order transition of an intrinsically disordered region of sortase revealed by multiscale enhanced sampling.
Journal of the American Chemical Society (2 April 2012), doi:10.1021/ja3008402
Molecular functions of intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs), such as molecular recognition and cellular signaling, are ascribed to dynamic changes in the conformational space in response to binding of target molecules. Sortase, a transpeptitase in Gram-positive bacteria, has an IDR in a loop which undergoes a disordered-to-ordered transition (called “disordered loop”), accompanying a tilt of another loop (“dynamic loop”), upon binding of a signal peptide and a calcium ion. In this study, all-atom conformational ensembles of sortase were calculated for the four different binding states (with/without the peptide and with/without a calcium ion) by the multiscale-enhanced-sampling (MSES) simulation to examine how the binding of the peptide and/or calcium influences the conformational ensemble. The MSES is a multiscale and multicopy simulation method that allows an enhanced sampling of the all-atom model of large proteins including explicit solvent. A 100-ns MSES simulation of the ligand-free sortase using 20 replicas (in total 2 ?s) demonstrated large flexibility in both the disordered and dynamic loops; however, their distributions were not random but had a clear preference which populates the N-terminal part of the disordered loop near the bound form. The MSES simulations of the three binding states clarified the allosteric mechanism of sortase: the N-terminal and C-terminal parts of the disordered loop undergo a disorder-to-order transition independently of each other upon binding of the peptide and a calcium ion, respectively; however, upon binding of both ligands, the two parts work cooperatively to stabilize the bound peptide.
Kei Moritsugu, Tohru Terada, Akinori Kidera
Journal of molecular biology (1 April 2012), doi:10.1016/j.jmb.2012.03.021
The functional importance of protein-protein interactions indicates that there should be strong evolutionary constraint on their interaction interfaces. However, binding interfaces are frequently affected by amino acid replacements. Change due to coevolution within interfaces can contribute to variability but is not ubiquitous. An alternative explanation for the ability of surfaces to accept replacements may be that many residues can be changed without affecting the interaction. Candidates for these types of residues are those that make interchain interaction only through the protein main chain, ?-carbon, or associated hydrogen atoms. Since almost all residues have these atoms, we hypothesize that this subset of interface residues may be more easily substituted than those that make interactions through other atoms. We term such interactions “residue type independent.” Investigating this hypothesis, we find that nearly a quarter of residues in protein interaction interfaces make exclusively interchain residue-type-independent contacts. These residues are less structurally constrained and less conserved than residues making residue-type-specific interactions. We propose that residue-type-independent interactions allow substitutions in binding interfaces while the specificity of binding is maintained. Copyright © 2012. Published by Elsevier Ltd.
David Talavera, Simon Williams, Matthew Norris, David Robertson, Simon Lovell
Role of Arginine 293 and Glutamine 288 in Communication between Catalytic and Allosteric Sites in Yeast Ribonucleotide Reductase.
Journal of molecular biology (29 March 2012), doi:10.1016/j.jmb.2012.03.014
Ribonucleotide reductases (RRs) catalyze the rate-limiting step of de novo deoxynucleotide (dNTP) synthesis. Eukaryotic RRs consist of two proteins, RR1 (?) that contains the catalytic site and RR2 (?) that houses a diferric-tyrosyl radical essential for ribonucleoside diphosphate reduction. Biochemical analysis has been combined with isothermal titration calorimetry (ITC), X-ray crystallography and yeast genetics to elucidate the roles of two loop 2 mutations R293A and Q288A in Saccharomyces cerevisiae RR1 (ScRR1). These mutations, R293A and Q288A, cause lethality and severe S phase defects, respectively, in cells that use ScRR1 as the sole source of RR1 activity. Compared to the wild-type enzyme activity, R293A and Q288A mutants show 4% and 15%, respectively, for ADP reduction, whereas they are 20% and 23%, respectively, for CDP reduction. ITC data showed that R293A ScRR1 is unable to bind ADP and binds CDP with 2-fold lower affinity compared to wild-type ScRR1. With the Q288A ScRR1 mutant, there is a 6-fold loss of affinity for ADP binding and a 2-fold loss of affinity for CDP compared to the wild type. X-ray structures of R293A ScRR1 complexed with dGTP and AMPPNP-CDP [AMPPNP, adenosine 5-(?,?-imido)triphosphate tetralithium salt] reveal that ADP is not bound at the catalytic site, and CDP binds farther from the catalytic site compared to wild type. Our in vivo functional analyses demonstrated that R293A cannot support mitotic growth, whereas Q288A can, albeit with a severe S phase defect. Taken together, our structure, activity, ITC and in vivo data reveal that the arginine 293 and glutamine 288 residues of ScRR1 are crucial in facilitating ADP and CDP substrate selection. Copyright © 2012 Elsevier Ltd. All rights reserved.
Faiz Ahmad, Prem Singh Kaushal, Qun Wan, Sanath Wijerathna, Xiuxiang An, Mingxia Huang, Chris Godfrey Dealwis
Structural and functional discussion of the tetra-trico-Peptide repeat, a protein interaction module.
Structure (London, England : 1993), Vol. 20, No. 3. (7 March 2012), pp. 397-405, doi:10.1016/j.str.2012.01.006
Tetra-trico-peptide repeat (TPR) domains are found in numerous proteins, where they serve as interaction modules and multiprotein complex mediators. TPRs can be found in all kingdoms of life and regulate diverse biological processes, such as organelle targeting and protein import, vesicle fusion, and biomineralization. This review considers the structural features of TPR domains that permit the great ligand-binding diversity of this motif, given that TPR-interacting partners display variations in both sequence and secondary structure. In addition, tools for predicting TPR-interacting partners are discussed, as are the abilities of TPR domains to serve as protein-protein interaction scaffolds in biotechnology and therapeutics. Copyright Â© 2012 Elsevier Ltd. All rights reserved.
Natalie Zeytuni, Raz Zarivach
Structure (London, England : 1993), Vol. 20, No. 3. (7 March 2012), pp. 414-428, doi:10.1016/j.str.2011.12.013
The endosomal sorting complexes required for transport (ESCRTs) facilitate endosomal sorting of ubiquitinated cargo, MVB biogenesis, late stages of cytokinesis, and retroviral budding. Here we show that ubiquitin associated protein 1 (UBAP1), a subunit of human ESCRT-I, coassembles in a stable 1:1:1:1 complex with Vps23/TSG101, VPS28, and VPS37. The X-ray crystal structure of the C-terminal region of UBAP1 reveals a domain that we describe as a solenoid of overlapping UBAs (SOUBA). NMR analysis shows that each of the three rigidly arranged overlapping UBAs making up the SOUBA interact with ubiquitin. We demonstrate that UBAP1-containing ESCRT-I is essential for degradation of antiviral cell-surface proteins, such as tetherin (BST-2/CD317), by viral countermeasures, namely, the HIV-1 accessory protein Vpu and the Kaposi sarcoma-associated herpesvirus (KSHV) ubiquitin ligase K5. Copyright Â© 2012 Elsevier Ltd. All rights reserved.
Monica Agromayor, Nicolas Soler, Anna Caballe, Tonya Kueck, Stefan Freund, Mark Allen, Mark Bycroft, Olga Perisic, Yu Ye, Bethan McDonald, Hartmut Scheel, Kay Hofmann, Stuart Neil, Juan Martin-Serrano, Roger Williams
Variable Lymphocyte Receptor Recognition of the Immunodominant Glycoprotein of Bacillus anthracis Spores.
Structure (London, England : 1993), Vol. 20, No. 3. (7 March 2012), pp. 479-486, doi:10.1016/j.str.2012.01.009
Variable lymphocyte receptors (VLRs) are the adaptive immune receptors of jawless fish, which evolved adaptive immunity independent of other vertebrates. In lieu of the immunoglobulin fold-based T and B cell receptors, lymphocyte-like cells of jawless fish express VLRs (VLRA, VLRB, or VLRC) composed of leucine-rich repeats and are similar to toll-like receptors (TLRs) in structure, but antibodies (VLRB) and T cell receptors (VLRA and VLRC) in function. Here, we present the structural and biochemical characterization of VLR4, a VLRB, in complex with BclA, the immunodominant glycoprotein of Bacillus anthracis spores. Using a combination of crystallography, mutagenesis, and binding studies, we delineate the mode of antigen recognition and binding between VLR4 and BclA, examine commonalities in VLRB recognition of antigens, and demonstrate the potential of VLR4 as a diagnostic tool for the identification of B. anthracis spores. Copyright Â© 2012 Elsevier Ltd. All rights reserved.
Robert Kirchdoerfer, Brantley Herrin, Byung Woo Han, Charles Turnbough, Max Cooper, Ian Wilson
Structure of the human obesity receptor leptin-binding domain reveals the mechanism of leptin antagonism by a monoclonal antibody.
Structure (London, England : 1993), Vol. 20, No. 3. (7 March 2012), pp. 487-497, doi:10.1016/j.str.2012.01.019
Leptin regulates energy homeostasis, fertility, and the immune system, making it an important drug target. However, due to a complete lack of structural data for the obesity receptor (ObR), leptin’s mechanism of receptor activation remains poorly understood. We have crystallized the Fab fragment of a leptin-blocking monoclonal antibody (9F8), both in its uncomplexed state and bound to the leptin-binding domain (LBD) of human ObR. We describe the structure of the LBD-9F8 Fab complex and the conformational changes in 9F8 associated with LBD binding. A molecular model of the putative leptin-LBD complex reveals that 9F8 Fab blocks leptin binding through only a small (10%) overlap in their binding sites, and that leptin binding is likely to involve an induced fit mechanism. This crystal structure of the leptin-binding domain of the obesity receptor will facilitate the design of therapeutics to modulate leptin signaling. Copyright Â© 2012 Elsevier Ltd. All rights reserved.
Byron Carpenter, Glyn Hemsworth, Zida Wu, Mabrouka Maamra, Christian Strasburger, Richard Ross, Peter Artymiuk
Structure (London, England : 1993), Vol. 20, No. 3. (7 March 2012), pp. 504-512, doi:10.1016/j.str.2012.01.012
In conformational diseases, native protein conformers convert to pathological intermediates that polymerize. Structural characterization of these key intermediates is challenging. They are unstable and minimally populated in dynamic equilibria that may be perturbed by many analytical techniques. We have characterized a forme fruste deficiency variant of ?(1)-antitrypsin (Lys154Asn) that forms polymers recapitulating the conformer-specific neo-epitope observed in polymers that form in vivo. Lys154Asn ?(1)-antitrypsin populates an intermediate ensemble along the polymerization pathway at physiological temperatures. Nuclear magnetic resonance spectroscopy was used to report the structural and dynamic changes associated with this. Our data highlight an interaction network likely to regulate conformational change and do not support the recent contention that the disease-relevant intermediate is substantially unfolded. Conformational disease intermediates may best be defined using powerful but minimally perturbing techniques, mild disease mutants, and physiological conditions. Copyright Â© 2012 Elsevier Ltd. All rights reserved.
Mun Peak Nyon, Lakshmi Segu, Lisa Cabrita, Géraldine Lévy, John Kirkpatrick, Benoit Roussel, Anathe Patschull, Tracey Barrett, Ugo Ekeowa, Richard Kerr, Christopher Waudby, Noor Kalsheker, Marian Hill, Konstantinos Thalassinos, David Lomas, John Christodoulou, Bibek Gooptu
Recognition pliability is coupled to structural heterogeneity: a calmodulin intrinsically disordered binding region complex.
Structure (London, England : 1993), Vol. 20, No. 3. (7 March 2012), pp. 522-533, doi:10.1016/j.str.2012.01.021
Protein interactions within regulatory networks should adapt in a spatiotemporal-dependent dynamic environment, in order to process and respond to diverse and versatile cellular signals. However, the principles governing recognition pliability in protein complexes are not well understood. We have investigated a region of the intrinsically disordered protein myelin basic protein (MBP(145-165)) that interacts with calmodulin, but that also promiscuously binds other biomolecules (membranes, modifying enzymes). To characterize this interaction, we implemented an NMR spectroscopic approach that calculates, for each conformation of the complex, the maximum occurrence based on recorded pseudocontact shifts and residual dipolar couplings. We found that the MBP(145-165)-calmodulin interaction is characterized by structural heterogeneity. Quantitative comparative analysis indicated that distinct conformational landscapes of structural heterogeneity are sampled for different calmodulin-target complexes. Such structural heterogeneity in protein complexes could potentially explain the way that transient and promiscuous protein interactions are optimized and tuned in complex regulatory networks. Copyright Â© 2012 Elsevier Ltd. All rights reserved.
Malini Nagulapalli, Giacomo Parigi, Jing Yuan, Joerg Gsponer, George Deraos, Vladimir Bamm, George Harauz, John Matsoukas, Maurits de Planque, Ioannis Gerothanassis, Madan Babu, Claudio Luchinat, Andreas Tzakos
CLIPS-1D: Analysis of multiple sequence alignments to deduce for residue-positions a role in catalysis, ligand-binding, or protein structure.
BMC bioinformatics, Vol. 13, No. 1. (5 April 2012), 55, doi:10.1186/1471-2105-13-55
ABSTRACT: BACKGROUND: One aim of the in silico characterization of proteins is to identify all residue-positions, which are crucial for function or structure. Several sequence-based algorithms exist, which predict functionally important sites. However, with respect to sequence information, many functionally and structurally important sites are hard to distinguish and consequently a large number of incorrectly predicted functional sites have to be expected. This is why we were interested to design a new classifier that differentiates between functionally and structurally important sites and to assess its performance on representative datasets. RESULTS: We have implemented CLIPS-1D, which predicts a role in catalysis, ligand-binding, or protein structure for residue-positions in a mutually exclusive manner. By analyzing a multiple sequence alignment, the algorithm scores conservation as well as abundance of residues at individual sites and their local neighborhood and categorizes by means of a multiclass support vector machine. A cross-validation confirmed that residue-positions involved in catalysis were identified with state-of-the-art quality; the mean MCC-value was 0.34. For structurally important sites, prediction quality was considerably higher (mean MCC = 0.67). For ligand-binding sites, prediction quality was lower (mean MCC = 0.12), because binding sites and structurally important residue-positions share conservation and abundance values, which makes their separation difficult. We show that classification success varies for residues in a class-specific manner. This is why our algorithm computes residue-specific p-values, which allow for the statistical assessment of each individual prediction. CLIPS-1D is available as a Web service at http://www-bioinf.uni-regensburg.de/. CONCLUSIONS: CLIPS-1D is a classifier, whose prediction quality has been determined separately for catalytic sites, ligand-binding sites, and structurally important sites. It generates hypotheses about residue-positions important for a set of homologous proteins and focuses on conservation and abundance signals. Thus, the algorithm can be applied in cases where function cannot be transferred from well-characterized proteins by means of sequence comparison.
Jan-Oliver Janda, Markus Busch, Fabian Kuck, Mikhail Porfenenko, Rainer Merkl
Proceedings of the National Academy of Sciences of the United States of America (2 April 2012), doi:10.1073/pnas.1119366109
Prions are important disease agents and epigenetic regulatory elements. Prion formation involves the structural conversion of proteins from a soluble form into an insoluble amyloid form. In many cases, this structural conversion is driven by a glutamine/asparagine (Q/N)-rich prion-forming domain. However, our understanding of the sequence requirements for prion formation and propagation by Q/N-rich domains has been insufficient for accurate prion propensity prediction or prion domain design. By focusing exclusively on amino acid composition, we have developed a prion aggregation prediction algorithm (PAPA), specifically designed to predict prion propensity of Q/N-rich proteins. Here, we show not only that this algorithm is far more effective than traditional amyloid prediction algorithms at predicting prion propensity of Q/N-rich proteins, but remarkably, also that PAPA is capable of rationally designing protein domains that function as prions in vivo.
James Toombs, Michelina Petri, Kacy Paul, Grace Kan, Asa Ben-Hur, Eric Ross
Structures of KIX domain of CBP in complex with two FOXO3a transactivation domains reveal promiscuity and plasticity in coactivator recruitment.
Proceedings of the National Academy of Sciences of the United States of America (2 April 2012), doi:10.1073/pnas.1119073109
Forkhead box class O 3a (FOXO3a) is a transcription factor and tumor suppressor linked to longevity that determines cell fate through activating transcription of cell differentiation, survival, and apoptotic genes. Recruitment of the coactivator CBP/p300 is a crucial step in transcription, and we revealed that in addition to conserved region 3 (CR3) of FOXO3a, the C-terminal segment of CR2 (CR2C) binds CBP/p300 and contributes to transcriptional activity. CR2C and CR3 of FOXO3a interact with the KIX domain of CBP/p300 at both “MLL” and “c-Myb” binding sites simultaneously. A FOXO3a CR2C-CR3 peptide in complex with KIX exists in equilibrium between two equally populated conformational states, one of which has CR2C bound to the MLL site and CR3 bound to the c-Myb site, whereas in the other, CR2C and CR3 bind the c-Myb and MLL sites, respectively. This promiscuous interaction between FOXO3a and CBP/p300 is further supported by additional binding sites on CBP/p300, namely, the TAZ1 and TAZ2 domains. In functional studies, our structure-guided mutagenesis showed that both CR2C and CR3 are involved in the activation of certain endogenous FOXO3a target genes. Further, phosphorylation of S626, a known AMP-dependent protein kinase target in CR3, increased affinity for CBP/p300 and the phosphomimetic mutation enhanced transactivation of luciferase. These findings underscore the significance of promiscuous multivalent interactions and posttranslational modification in the recruitment of transcriptional coactivators, which may allow transcription factors to adapt to various gene-specific genomic and chromatin structures and respond to cell signals.
Feng Wang, Christopher Marshall, Kazuo Yamamoto, Guang-Yao Li, Geneviève Gasmi-Seabrook, Hitoshi Okada, Tak Mak, Mitsuhiko Ikura
Journal of the American Chemical Society (27 March 2012), doi:10.1021/ja210588m
Abnormal interactions of Cu and Zn ions with the amyloid ? (A?) peptide are proposed to play an important role in the pathogenesis of Alzheimer’s disease (AD). Disruption of these metal-peptide interactions using chemical agents holds considerable promise as a therapeutic strategy to combat this incurable disease. Reported herein are two bifunctional compounds (BFCs) L1 and L2 that contain both amyloid-binding and metal-chelating molecular motifs. Both L1 and L2 exhibit high stability constants for Cu<sup>2+</sup> and Zn<sup>2+</sup> and thus are good chelators for these metal ions. In addition, L1 and L2 show strong affinity toward A? species. Both compounds are efficient inhibitors of the metal-mediated aggregation of the A?<sub>42</sub> peptide and promote disaggregation of amyloid fibrils, as observed by ThT fluorescence, native gel electrophoresis/Western blotting, and transmission electron microscopy (TEM). Interestingly, the formation of soluble A?<sub>42</sub> oligomers in presence of metal ions and BFCs leads to an increased cellular toxicity. These results suggest that for the A?<sub>42</sub> peptide – in contrast to the A?<sub>40</sub> peptide, the previously employed strategy of inhibiting A? aggregation and promoting amyloid fibril dissagregation may not be optimal for the development of potential AD therapeutics, due to formation of neurotoxic soluble A?<sub>42</sub> oligomers.
Anuj Kumar Sharma, Stephanie Pavlova, Jaekwang Kim, Darren Finkelstein, Nicholas Hawco, Nigam Rath, Jungsu Kim, Liviu Mirica
The Crowded Environment of a Reverse Micelle Induces the Formation of ?-Strand Seed Structures for Nucleating Amyloid Fibril Formation
J. Am. Chem. Soc. In Journal of the American Chemical Society, Vol. 134, No. 14. (26 March 2012), pp. 6061-6063, doi:10.1021/ja3004478
A hallmark of Alzheimer?s disease is the accumulation of insoluble fibrils in the brain composed of amyloid beta (A?) proteins with parallel in-register cross-?-sheet structure. It has been suggested that the aggregation of monomeric A? proteins into fibrils is promoted by ?seeds? that form within compartments of the brain that have limited solvent due to macromolecular crowding. To characterize these seeds, a crowded macromolecular environment was mimicked by encapsulating A?40 monomers into reverse micelles. Fourier-transform infrared spectroscopy revealed that monomeric A? proteins form extended ?-strands in reverse micelles, while an analogue with a scrambled sequence does not. This is a remarkable finding, because the formation of extended ?-strands by monomeric A? proteins suggests a plausible mechanism whereby the formation of amyloid fibrils may be nucleated in the human brain.
Priscilla Yeung, Paul Axelsen
Journal of the American Chemical Society, Vol. 134, No. 13. (4 April 2012), pp. 5746-5749, doi:10.1021/ja300523q
Mechanical properties of biological membranes are known to regulate membrane protein function. Despite this, current models of protein communication typically feature only direct protein-protein or protein-small molecule interactions. Here we show for the first time that, by harnessing nanoscale mechanical energy within biological membranes, it is possible to promote controlled communication between proteins. By coupling lipid-protein modules and matching their response to the mechanical properties of the membrane, we have shown that the action of phospholipase A(2) on acyl-based phospholipids triggers the opening of the mechanosensitive channel, MscL, by generating membrane asymmetry. Our findings confirm that the global physical properties of biological membranes can act as information pathways between proteins, a novel mechanism of membrane-mediated protein-protein communication that has important implications for (i) the underlying structure of signaling pathways, (ii) our understanding of in vivo communication networks, and (iii) the generation of building blocks for artificial protein networks.
Kalypso Charalambous, Paula Booth, Rudiger Woscholski, John Seddon, Richard Templer, Robert Law, Laura Barter, Oscar Ces
Journal of molecular biology (15 March 2012), doi:10.1016/j.jmb.2012.03.005
Combinatorial sequence optimization for protein design requires libraries of discrete side-chain conformations. The discreteness of these libraries is problematic, particularly for long, polar side chains, since favorable interactions can be missed. Previously, an approach to loop remodeling where protein backbone movement is directed by side-chain rotamers predicted to form interactions previously observed in native complexes (termed “motifs”) was described. Here, we show how such motif libraries can be incorporated into combinatorial sequence optimization protocols and improve native complex recapitulation. Guided by the motif rotamer searches, we made improvements to the underlying energy function, increasing recapitulation of native interactions. To further test the methods, we carried out a comprehensive experimental scan of amino acid preferences in the I-AniI protein-DNA interface and found that many positions tolerated multiple amino acids. This sequence plasticity is not observed in the computational results because of the fixed-backbone approximation of the model. We improved modeling of this diversity by introducing DNA flexibility and reducing the convergence of the simulated annealing algorithm that drives the design process. In addition to serving as a benchmark, this extensive experimental data set provides insight into the types of interactions essential to maintain the function of this potential gene therapy reagent. Published by Elsevier Ltd.
Summer Thyme, David Baker, Philip Bradley
Journal of molecular biology (15 March 2012), doi:10.1016/j.jmb.2012.03.004
This work demonstrates that all packing in ?-helices can be simplified to repetitive patterns of a single motif: the knob-socket. Using the precision of Voronoi Polyhedra/Delauney Tessellations to identify contacts, the knob-socket is a four-residue tetrahedral motif: a knob residue on one ?-helix packs into the three-residue socket on another ?-helix. The principle of the knob-socket model relates the packing between levels of protein structure: the intra-helical packing arrangements within secondary structure that permit inter-helix tertiary packing interactions. Within an ?-helix, the three-residue sockets arrange residues into a uniform packing lattice. Inter-helix packing results from a definable pattern of interdigitated knob-socket motifs between two ?-helices. Furthermore, the knob-socket model classifies three types of sockets: (1) free, favoring only intra-helical packing; (2) filled, favoring inter-helical interactions; and (3) non, disfavoring ?-helical structure. The amino acid propensities in these three socket classes essentially represent an amino acid code for structure in ?-helical packing. Using this code, we used a novel yet straightforward approach for the design of ?-helical structure to validate the knob-socket model. Unique sequences for three peptides were created to produce a predicted amount of ?-helical structure: mostly helical, some helical, and no helix. These three peptides were synthesized, and helical content was assessed using CD spectroscopy. The measured ?-helicity of each peptide was consistent with the expected predictions. These results and analysis demonstrate that the knob-socket motif functions as the basic unit of packing and presents an intuitive tool to decipher the rules governing packing in protein structure. Copyright Â© 2012 Elsevier Ltd. All rights reserved.
Hyun Joo, Archana Chavan, Jamie Phan, Ryan Day, Jerry Tsai
Journal of molecular biology (7 March 2012), doi:10.1016/j.jmb.2012.02.037
The mechanism of light-triggered conformational change and signaling in light-oxygen-voltage (LOV) domains remains elusive in spite of extensive investigation and their use in optogenetic studies. The LOV2 domain of Avenasativa phototropin 1 (AsLOV2), a member of the Per-Arnt-Sim (PAS) family, contains a flavin mononucleotide chromophore that forms a covalent bond with a cysteine upon illumination. This event leads to the release of the carboxy-terminal J? helix, the biological output signal. Using mutational analysis, circular dichroism, and NMR, we find that the largely ignored amino-terminal helix is a control element in AsLOV2′s light-activated conformational change. We further identify a direct amino-to-carboxy-terminal “input-output” signaling pathway. These findings provide a framework to rationalize the LOV domain architecture, as well as the signaling mechanisms in both isolated and tandem arrangements of PAS domains. This knowledge can be applied in engineering LOV-based photoswitches, opening up new design strategies and improving existing ones. Josiah Zayner, Chloe Antoniou, Tobin Sosnick
Proline isomer-specific antibodies reveal the early pathogenic tau conformation in Alzheimer’s disease.
Cell, Vol. 149, No. 1. (30 March 2012), pp. 232-244, doi:10.1016/j.cell.2012.02.016
cis-trans isomerization of proteins phosphorylated by proline-directed kinases is proposed to control numerous signaling molecules and is implicated in the pathogenesis of Alzheimer’s and other diseases. However, there is no direct evidence for the existence of cis-trans protein isomers in vivo or for their conformation-specific function or regulation. Here we develop peptide chemistries that allow the generation of cis- and trans-specific antibodies and use them to raise antibodies specific for isomers of phosphorylated tau. cis, but not trans, p-tau appears early in the brains of humans with mild cognitive impairment, accumulates exclusively in degenerated neurons, and localizes to dystrophic neurites during Alzheimer’s progression. Unlike trans p-tau, the cis isomer cannot promote microtubule assembly, is more resistant to dephosphorylation and degradation, and is more prone to aggregation. Pin1 converts cis to trans p-tau to prevent Alzheimer’s tau pathology. Isomer-specific antibodies and vaccines may therefore have value for the early diagnosis and treatment of Alzheimer’s disease. Copyright © 2012 Elsevier Inc. All rights reserved.
Kazuhiro Nakamura, Alex Greenwood, Lester Binder, Eileen Bigio, Sarah Denial, Linda Nicholson, Xiao Zhen Zhou, Kun Ping Lu
Transient structure and dynamics in the disordered c-Myc transactivation domain affect Bin1 binding.
Nucleic acids research (28 March 2012), doi:10.1093/nar/gks263
The crucial role of Myc as an oncoprotein and as a key regulator of cell growth makes it essential to understand the molecular basis of Myc function. The N-terminal region of c-Myc coordinates a wealth of protein interactions involved in transformation, differentiation and apoptosis. We have characterized in detail the intrinsically disordered properties of Myc-1-88, where hierarchical phosphorylation of S62 and T58 regulates activation and destruction of the Myc protein. By nuclear magnetic resonance (NMR) chemical shift analysis, relaxation measurements and NOE analysis, we show that although Myc occupies a very heterogeneous conformational space, we find transiently structured regions in residues 22-33 and in the Myc homology box I (MBI; residues 45-65); both these regions are conserved in other members of the Myc family. Binding of Bin1 to Myc-1-88 as assayed by NMR and surface plasmon resonance (SPR) revealed primary binding to the S62 region in a dynamically disordered and multivalent complex, accompanied by population shifts leading to altered intramolecular conformational dynamics. These findings expand the increasingly recognized concept of intrinsically disordered regions mediating transient interactions to Myc, a key transcriptional regulator of major medical importance, and have important implications for further understanding its multifaceted role in gene regulation.
Cecilia Andresen, Sara Helander, Alexander Lemak, Christophe Farès, Veronika Csizmok, Jonas Carlsson, Linda Penn, Julie Forman-Kay, Cheryl Arrowsmith, Patrik Lundström, Maria Sunnerhagen
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