Structure (v.14, #3)
Tight in Titin
by Jeremy C. Smith (pp. 389-390).
The mechanics of a protein joint, gluing two elastic titin molecules together in muscle, are explored using computer simulation in this issue of Structure by . Maybe the glue itself also has another, more subtle, sensory role?
Molecular Mechanics of Single Molecules
by Paul K. Hansma (pp. 390-391).
The mechanics of single molecules of bacteriorhodopsin interacting with purple membrane have been investigated from both sides of the membrane by in this issue of Structure. Remarkably, barriers can be associated with specific amino acid sequences to an accuracy of ±3 amino acids.
A Tale of the Unexpected
by Martin R. Webb (pp. 391-392).
A human homolog of the prokaryotic phosphate binding protein has been described and its structure determined ( [this issue of Structure]). This protein's discovery in plasma was unexpected and leads to questions as to what function this type of protein might have in eukaryotes.
Simulation of Diffusion Time of Small Molecules in Protein Crystals
by Silvano Geremia; Mara Campagnolo; Nicola Demitri; Louise N. Johnson (pp. 393-400).
A simple model for evaluation of diffusion times of small molecule into protein crystals has been developed, which takes into account the physical and chemical properties both of protein crystal and the diffusing molecules. The model also includes consideration of binding and the binding affinity of a ligand to the protein. The model has been validated by simulation of experimental set-ups of several examples found in the literature. These experiments cover a wide range of situations: from small to relatively large diffusing molecules, crystals having low, medium, or high protein density, and different size. The reproduced experiments include ligand exchange in protein crystals by soaking techniques. Despite the simplifying assumptions of the model, theoretical and experimental data are in agreement with available data, with experimental diffusion times ranging from a few seconds to several hours. The method has been used successfully for planning intermediate cryotrapping experiments in maltodextrin phosphorylase crystals.
Structural Model of the mAb 806-EGFR Complex Using Computational Docking followed by Computational and Experimental Mutagenesis
by Arvind Sivasubramanian; Ginger Chao; Heather M. Pressler; K. Dane Wittrup; Jeffrey J. Gray (pp. 401-414).
In this work, we combined computational protein-protein docking with computational and experimental mutagenesis to predict the structure of the complex formed by monoclonal antibody 806 (mAb 806) and the epidermal growth factor receptor (EGFR). We docked mAb 806, an antitumor antibody, to its epitope of EGFR residues 287â€“302. Potential mAb 806-EGFR orientations were generated, and computational mutagenesis was used to filter them according to their agreement with experimental mutagenesis data. Further computational mutagenesis suggested additional mutations, which were tested to arrive at a final structure that was most consistent with experimental mutagenesis data. We propose that this is the EGFR-mAb 806 structure, in which mAb 806 binds to an untethered form of the receptor, consistent with published experimental results. The steric hindrance created by the antibody near the EGFR dimer interface interferes with receptor dimerization, and we postulate this as the structural origin for the antitumor effect of mAb 806.
Conformational Diversity in the TPR Domain-Mediated Interaction of Protein Phosphatase 5 with Hsp90
by Matthew J. Cliff; Richard Harris; David Barford; John E. Ladbury; Mark A. Williams (pp. 415-426).
Protein phosphatase 5 (Ppp5) is one of several proteins that bind to the Hsp90 chaperone via a tetratricopeptide repeat (TPR) domain. We report the solution structure of a complex of the TPR domain of Ppp5 with the C-terminal pentapeptide of Hsp90. This structure has the â€œtwo-carboxylate clampâ€? mechanism of peptide binding first seen in the Hop-TPR domain complexes with Hsp90 and Hsp70 peptides. However, NMR data reveal that the Ppp5 clamp is highly dynamic, and that there are multiple modes of peptide binding and mobility throughout the complex. Although this interaction is of very high affinity, relatively few persistent contacts are found between the peptide and the Ppp5-TPR domain, thus explaining its promiscuity in binding both Hsp70 and Hsp90 in vivo. We consider the possible implications of this dynamic structure for the mechanism of relief of autoinhibition in Ppp5 and for the mechanisms of TPR-mediated recognition of Hsp90 by other proteins.
Structure of a Transient Intermediate for GTP Hydrolysis by Ras
by Bradley Ford; Viktor Hornak; Holly Kleinman; Nicolas Nassar (pp. 427-436).
The flexibility of the conserved57DTAGQ61 motif is essential for Ras proper cycling in response to growth factors. Here, we increase the flexibility of the57DTAGQ61 motif by mutating Gln61 to Gly. The crystal structure of the RasQ61G mutant reveals a new conformation of switch 2 that bears remarkable structural homology to an intermediate for GTP hydrolysis revealed by targeted molecular dynamics simulations. The mutation increased retention of GTP and inhibited Ras binding to the catalytic site, but not to the distal site of Sos. Most importantly, the thermodynamics of RafRBD binding to Ras are altered even though the structure of switch 1 is not affected by the mutation. Our results suggest that interplay and transmission of structural information between the switch regions are important factors for Ras function. They propose that initiation of GTP hydrolysis sets off the separation of the Ras/effector complex even before the GDP conformation is reached.
Molecular Dynamics Simulations of the Complete Satellite Tobacco Mosaic Virus
by Peter L. Freddolino; Anton S. Arkhipov; Steven B. Larson; Alexander McPherson; Klaus Schulten (pp. 437-449).
This work presents an all-atom molecular dynamics simulation of a complete virus, the satellite tobacco mosaic virus. Simulations with up to 1 million atoms for over 50 ns demonstrate the stability of the entire virion and of the RNA core alone, while the capsid without RNA exhibits a pronounced instability. Physical properties of the simulated virus particle including electrostatic potential, radial distribution of viral components, and patterns of correlated motion are analyzed, and the implications for the assembly and infection mechanism of the virus are discussed.
Crystal Structure of the Boronic Acid-Based Proteasome Inhibitor Bortezomib in Complex with the Yeast 20S Proteasome
by Michael Groll; Celia R. Berkers; Hidde L. Ploegh; Huib Ovaa (pp. 451-456).
The dipeptide boronic acid bortezomib, also termed VELCADE®, is a proteasome inhibitor now in use for the treatment of multiple myeloma, and its use for the treatment of other malignancies is being explored. We determined the crystal structure of the yeast 20S proteasome in complex with bortezomib to establish the specificity and binding mode of bortezomib to the proteasome's different catalytically active sites. This structure should enable the rational design of new boronic acid derivatives with improved affinities and specificities for individual active subunits.
Solution Structure of the SWIRM Domain of Human Histone Demethylase LSD1
by Naoya Tochio; Takashi Umehara; Seizo Koshiba; Makoto Inoue; Takashi Yabuki; Masaaki Aoki; Eiko Seki; Satoru Watanabe; Yasuko Tomo; Masaru Hanada; Masaomi Ikari; Miyuki Sato; Takaho Terada; Takahiro Nagase; Osamu Ohara; Mikako Shirouzu; Akiko Tanaka; Takanori Kigawa; Shigeyuki Yokoyama (pp. 457-468).
SWIRM is an evolutionarily conserved domain involved in several chromatin-modifying complexes. Recently, the LSD1 protein, which bears a SWIRM domain, was found to be a demethylase for Lys4-methylated histone H3. Here, we report a solution structure of the SWIRM domain of human LSD1. It forms a compact fold composed of 6 Î± helices, in which a 20 amino acid long helix (Î±4) is surrounded by 5 other short helices. The SWIRM domain structure could be divided into the N-terminal part (Î±1â€“Î±3) and the C-terminal part (Î±4â€“Î±6), which are connected to each other by a salt bridge. While the N-terminal part forms a SWIRM-specific structure, the C-terminal part adopts a helix-turn-helix (HTH)-related fold. We discuss a model in which the SWIRM domain acts as an anchor site for a histone tail.
The Structural Basis of Actin Interaction with Multiple WH2/Î²-Thymosin Motif-Containing Proteins
by Adeleke H. Aguda; Bo Xue; Edward Irobi; Thomas Préat; Robert C. Robinson (pp. 469-476).
Participation of actin in cellular processes relies on the dynamics of filament assembly. Filament elongation is fed by monomeric actin in complex with either profilin or a Wiscott-Aldrich syndrome protein (WASP) homology domain 2 (WH2)/Î²-thymosin (Î²T) domain. WH2/Î²T motif repetition (typified by ciboulot) or combination with nonrelated domains (as found in N-WASP) results in proteins that yield their actin to filament elongation. Here, we report the crystal structures of actin bound hybrid proteins, constructed between gelsolin and WH2/Î²T domains from ciboulot or N-WASP. We observe the C-terminal half of ciboulot domain 2 bound to actin. In solution, we show that cibolout domains 2 and 3 bind to both G- and F-actin, and that whole ciboulot forms a complex with two actin monomers. In contrast, the analogous portion of N-WASP WH2 domain 2 is detached from actin, indicating that the C-terminal halves of the Î²T and WH2 motifs are not functionally analogous.
Structure and Dimerization of the Kinase Domain from Yeast Snf1, a Member of the Snf1/AMPK Protein Family
by Vinod Nayak; Kehao Zhao; Anastasia Wyce; Marc F. Schwartz; Wan-Sheng Lo; Shelley L. Berger; Ronen Marmorstein (pp. 477-485).
The Snf1/AMPK kinases are intracellular energy sensors, and the AMPK pathway has been implicated in a variety of metabolic human disorders. Here we report the crystal structure of the kinase domain from yeast Snf1, revealing a bilobe kinase fold with greatest homology to cyclin-dependant kinase-2. Unexpectedly, the crystal structure also reveals a novel homodimer that we show also forms in solution, as demonstrated by equilibrium sedimentation, and in yeast cells, as shown by coimmunoprecipitation of differentially tagged intact Snf1. A mapping of sequence conservation suggests that dimer formation is a conserved feature of the Snf1/AMPK kinases. The conformation of the conserved Î±C helix, and the burial of the activation segment and substrate binding site within the dimer, suggests that it represents an inactive form of the kinase. Taken together, these studies suggest another layer of kinase regulation within the Snf1/AMPK family, and an avenue for development of AMPK-specific activating compounds.
Structural Basis of RNA Binding Discrimination between Bacteriophages QÎ² and MS2
by Wilf T. Horn; Kaspars Tars; Elin Grahn; Charlotte Helgstrand; Andrew J. Baron; Hugo Lago; Chris J. Adams; David S. Peabody; Simon E.V. Phillips; Nicola J. Stonehouse; Lars Liljas; Peter G. Stockley (pp. 487-495).
Sequence-specific interactions between RNA stem-loops and coat protein (CP) subunits play vital roles in the life cycles of the RNA bacteriophages, e.g., by allowing translational repression of their replicase cistrons and tagging their own RNA genomes for encapsidation. The CPs of bacteriophages QÎ² and MS2 each discriminate in favor of their cognate translational operators, even in the presence of closely related operators from other phages in vivo. Discrete mutations within the MS2 CP have been shown to relax this discrimination in vitro. We have determined the structures of eight complexes between such mutants and both MS2 and QÎ² stem-loops with X-ray crystallography. In conjunction with previously determined in vivo repression data, the structures enable us to propose the molecular basis for the discrimination mechanism.
Keywords: MICROBIO; RNA
Mechanical Strength of the Titin Z1Z2-Telethonin Complex
by Eric H. Lee; Mu Gao; Nikos Pinotsis; Matthias Wilmanns; Klaus Schulten (pp. 497-509).
Using molecular dynamics simulations, we have explored the mechanical strength of the titin Z1Z2-telethonin complex, namely, its ability to bear strong forces such as those encountered during passive muscle stretch. Our results show that not only does this complex resist considerable mechanical force through Î² strand crosslinking, suggesting that telethonin is an important component of the N-terminal titin anchor, but also that telethonin distributes these forces between its two joined titin Z2 domains to protect the proximal Z1 domains from bearing too much stress. Our simulations also reveal that without telethonin, apo-titin Z1Z2 exhibits significantly decreased resistance to mechanical stress, and that the N-terminal segment of telethonin (residues 1â€“89) does not exhibit a stable fold conformation when it is unbound from titin Z1Z2. Consequently, our study sheds light on a key but little studied architectural feature of biological cellsâ€”the existence of strong mechanical links that glue separate proteins together.
Keywords: CELLBIO; HUMDISEASE
Cryo-Electron Microscopy Studies of Human TFIID: Conformational Breathing in the Integration of Gene Regulatory Cues
by Patricia Grob; Michael J. Cruse; Carla Inouye; Marian Peris; Pawel A. Penczek; Robert Tjian; Eva Nogales (pp. 511-520).
The multisubunit transcription factor TFIID is essential for directing eukaryotic promoter recognition and mediating interactions with activators/cofactors during assembly of the preinitiation complex. Despite its central role in transcription initiation and regulation, structural knowledge of the TFIID complex has so far been largely limited to electron microscopy studies of negatively stained samples. Here, we present a cryo-electron microscopy 3D reconstruction of the large endogenous human TFIID complex. The improved cryopreservation has allowed for a more detailed definition of the structural elements in the complex and for the detection, by an extensive statistical analysis of the data, of a conformational opening and closing of the cavity central to the TFIID architecture. We propose that these density rearrangements in the structure are a likely reflection of the plasticity of the interactions between TFIID and its many partner proteins.
Unfolding Barriers in Bacteriorhodopsin Probed from the Cytoplasmic and the Extracellular Side by AFM
by Max Kessler; Hermann E. Gaub (pp. 521-527).
Selecting an individual membrane protein and probing its mechanical properties has become possible by AFM-based single-molecule force spectroscopy. In contrast to earlier studies, we extracted and unfolded bacteriorhodopsin monomers from the purple membrane not only from the cytoplasmic side, but also from the extracellular side, and recorded the force extension profiles. This way different pathways through the potential landscape are explored. A map of the 21 most dominant barriers with their positions relative to the amino acid sequences is given at an accuracy of ±3 aa. Most barriers were found to provide resistance to forced unfolding only when extracted toward one of the sides. However, certain barriers have identical positions to within a few amino acids when probed from either of the sides, which typifies them as structural traps.
Conformational and Sequence Signatures in Î² Helix Proteins
by Prathima Iengar; N.V. Joshi; Padmanabhan Balaram (pp. 529-542).
Î² helix proteins are characterized by a repetitive fold, in which the repeating unit is a Î²-helical coil formed by three strand segments linked by three loop segments. Using a data set of left- and right-handed Î² helix proteins, we have examined conformational features at equivalent positions in successive coils. This has provided insights into the conformational rules that the proteins employ to fold into Î² helices. Left-handed Î² helices attain their equilateral prism fold by incorporating â€œcornersâ€? with the conformational sequence PII-PII-Î±L-PII, which imposes sequence restrictions, resulting in the first and third PII residues often being G and a small, uncharged residue (V, A, S, T, C), respectively. Right-handed Î² helices feature mid-sized loops (4, 5, or 6 residues) of conserved conformation, but not of conserved sequence; they also display an Î±-helical residue at the C-terminal end of L2 loops. Backbone conformational parameters (Ï•,Ïˆ) that permit the formation of continuous, loopless Î² helices (Perutz nanotubes) have also been investigated.
Structural Determinants for High-Affinity Binding in a Nedd4 WW3âˆ— Domain-Comm PY Motif Complex
by Voula Kanelis; M. Christine Bruce; Nikolai R. Skrynnikov; Daniela Rotin; Julie D. Forman-Kay (pp. 543-553).
Interactions between the WW domains of Drosophila Nedd4 (dNedd4) and Commissureless (Comm) PY motifs promote axon crossing at the CNS midline and muscle synaptogenesis. Here we report the solution structure of the dNedd4 WW3âˆ— domain complexed to the second PY motif (227â€²TGLPSYDEALH237â€²) of Comm. Unexpectedly, there are interactions between WW3âˆ— and ligand residues both N- and C-terminal to the PY motif. Residues Y232â€²â€“L236â€² form a helical turn, following the PPII helical PY motif. Mutagenesis and binding studies confirm the importance of these extensive contacts, not simultaneously observed in other WW domain complexes, and identify a variable loop in WW3âˆ— responsible for its high-affinity interaction. These studies expand our general understanding of the molecular determinants involved in WW domain-ligand recognition. In addition, they provide insights into the specific regulation of dNedd4-mediated ubiquitination of Comm and subsequent internalization of Comm or the Comm/Roundabout complex, critical for CNS and muscle development.
Keywords: SIGNALING; MOLNEURO
Principles of Protein-DNA Recognition Revealed in the Structural Analysis of Ndt80-MSE DNA Complexes
by Jason S. Lamoureux; J.N. Mark Glover (pp. 555-565).
The Saccharomyces cerevisiae transcription factor Ndt80 selectively binds a DNA consensus sequence (the middle sporulation element [MSE]) to activate gene expression after the successful completion of meiotic recombination. Here we report the X-ray crystal structures of Ndt80 bound to ten distinct MSE variants. Comparison of these structures with the structure of Ndt80 bound to a consensus MSE reveals structural principles that determine the DNA binding specificity of this transcription factor. The 5â€² GC-rich end of the MSE contains distinct 5â€²-YpG-3â€² steps that are recognized by arginine side chains through a combination of hydrogen bonding and cation-Ï€ interactions. The 3â€² AT-rich region is recognized via minor groove contacts that sterically exclude the N2 atom of GC base pairs. The conformation of the AT-rich region is fixed by interactions with the protein that favor recognition of poly(A)-poly(T) versus mixed AT sequences through an avoidance of major groove steric clashes at 5â€²-ApT-3â€² steps.
Structure-Guided Engineering of Xylitol Dehydrogenase Cosubstrate Specificity
by Andreas H. Ehrensberger; Robert A. Elling; David K. Wilson (pp. 567-575).
Xylitol dehydrogenase (XDH) is one of several enzymes responsible for assimilating xylose into eukaryotic metabolism and is useful for fermentation of xylose contained in agricultural byproducts to produce ethanol. For efficient xylose utilization at high flux rates, cosubstrates should be recycled between the NAD+-specific XDH and the NADPH-preferring xylose reductase, another enzyme in the pathway. To understand and alter the cosubstrate specificity of XDH, we determined the crystal structure of the Gluconobacter oxydans holoenzyme to 1.9 Ã… resolution. The structure reveals that NAD+ specificity is largely conferred by Asp38, which interacts with the hydroxyls of the adenosine ribose. Met39 stacked under the purine ring and was also located near the 2â€² hydroxyl. Based on the location of these residues and on sequence alignments with related enzymes of various cosubstrate specificities, we constructed a double mutant (D38S/M39R) that was able to exclusively use NADP+, with no loss of activity.
Conformational Flexibility in the Multidrug Efflux System Protein AcrA
by Jonathan Mikolosko; Kostyantyn Bobyk; Helen I. Zgurskaya; Partho Ghosh (pp. 577-587).
Intrinsic resistance to multiple drugs in many gram-negative bacterial pathogens is conferred by resistance nodulation cell division efflux pumps, which are composed of three essential components as typified by the extensively characterized Escherichia coli AcrA-AcrB-TolC system. The inner membrane drug:proton antiporter AcrB and the outer membrane channel TolC export chemically diverse compounds out of the bacterial cell, and require the activity of the third component, the periplasmic protein AcrA. The crystal structures of AcrB and TolC have previously been determined, and we complete the molecular picture of the efflux system by presenting the structure of a stable fragment of AcrA. The AcrA fragment resembles the elongated sickle shape of its homolog Pseudomonas aeruginosa MexA, being composed of three domains: Î²-barrel, lipoyl, and Î±-helical hairpin. Notably, unsuspected conformational flexibility in the Î±-helical hairpin domain of AcrA is observed, which has potential mechanistic significance in coupling between AcrA conformations and TolC channel opening.
Molecular Mechanism for the Regulation of Rho-Kinase by Dimerization and Its Inhibition by Fasudil
by Hiroto Yamaguchi; Miyuki Kasa; Mutsuki Amano; Kozo Kaibuchi; Toshio Hakoshima (pp. 589-600).
Rho-kinase is a key regulator of cytoskeletal events and a promising drug target in the treatment of vascular diseases and neurological disorders. Unlike other protein kinases, Rho-kinase requires both N- and C-terminal extension segments outside the kinase domain for activity, although the details of this requirement have been elusive. The crystal structure of an active Rho-kinase fragment containing the kinase domain and both the extensions revealed a head-to-head homodimer through the N-terminal extension forming a helix bundle that structurally integrates the C-terminal extension. This structural organization enables binding of the C-terminal hydrophobic motif to the N-terminal lobe, which defines the correct disposition of helix Î±C that is important for the catalytic activity. The bound inhibitor fasudil significantly alters the conformation and, consequently, the mode of interaction with the catalytic cleft that contains local structural changes. Thus, both kinase and drug conformational pliability and stability confer selectivity.
Keywords: CELLBIO; SIGNALING
Serendipitous Discovery and X-Ray Structure of a Human Phosphate Binding Apolipoprotein
by Renaud Morales; Anne Berna; Philippe Carpentier; Carlos Contreras-Martel; Frédérique Renault; Murielle Nicodeme; Marie-Laure Chesne-Seck; François Bernier; Jérôme Dupuy; Christine Schaeffer; Hélène Diemer; Alain Van-Dorsselaer; Juan C. Fontecilla-Camps; Patrick Masson; Daniel Rochu; Eric Chabriere (pp. 601-609).
We report the serendipitous discovery of a human plasma phosphate binding protein (HPBP). This 38 kDa protein is copurified with the enzyme paraoxonase. Its X-ray structure is similar to the prokaryotic phosphate solute binding proteins (SBPs) associated with ATP binding cassette transmembrane transporters, though phosphate-SBPs have never been characterized or predicted from nucleic acid databases in eukaryotes. However, HPBP belongs to the family of ubiquitous eukaryotic proteins named DING, meaning that phosphate-SBPs are also widespread in eukaryotes. The systematic absence of complete genes for eukaryotic phosphate-SBP from databases is intriguing, but the astonishing 90% sequence conservation between genes belonging to evolutionary distant species suggests that the corresponding proteins play an important function. HPBP is the only known transporter capable of binding phosphate ions in human plasma and may become a new predictor of or a potential therapeutic agent for phosphate-related diseases such as atherosclerosis.
Structural Insight into Interactions between Dihydrolipoamide Dehydrogenase (E3) and E3 Binding Protein of Human Pyruvate Dehydrogenase Complex
by Chad A. Brautigam; R. Max Wynn; Jacinta L. Chuang; Mischa Machius; Diana R. Tomchick; David T. Chuang (pp. 611-621).
The 9.5 MDa human pyruvate dehydrogenase complex (PDC) utilizes the specific dihydrolipoamide dehydrogenase (E3) binding protein (E3BP) to tether the essential E3 component to the 60-meric core of the complex. Here, we report crystal structures of the binding domain (E3BD) of human E3BP alone and in complex with human E3 at 1.6 Ã… and 2.2 Ã…, respectively. The latter structure shows that residues from E3BD contact E3 across its 2-fold axis, resulting in one E3BD binding site on the E3 homodimer. Negligible conformational changes occur in E3BD upon its high-affinity binding to E3. Modifications of E3BD residues at the center of the E3BD/E3 interface impede E3 binding far more severely than those of residues on the periphery, validating the â€œhot spotâ€? paradigm for protein interactions. A cluster of disease-causing E3 mutations located near the center of the E3BD/E3 interface prevents the efficient recruitment of these E3 variants by E3BP into the PDC, leading to the dysfunction of the PDC catalytic machine.
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