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Structure (v.15, #7)

F-BAR Proteins Join the BAR Family Fold by Adam Frost; Pietro De Camilli; Vinzenz M. Unger (pp. 751-753).
Expanding the range of curvature generating and curvature stabilizing protein modules, the first F-BAR domain structures support their assignment to the BAR domain superfamily and emphasize how modifications to a basic structural frame can generate a broad spectrum of properties.

Shooting Blanks: Ca2+-free Signaling by Brian D. Sykes (pp. 753-754).
Insect flight muscle is capable of very high oscillatory frequencies. In this issue of Structure, De Nicola and colleagues () describe the structure of the Ca2+ binding protein that regulates asynchronous contraction, casting light on the mechanism of stretch activation.

The Third CAPRI Assessment Meeting Toronto, Canada, April 20–21, 2007 by Joël Janin; Shoshana Wodak (pp. 755-759).
CAPRI is a community-wide experiment to test protein-protein docking methods in blind predictions. The Toronto meeting assessed structure predictions made from 2005–2007 on nine target protein-protein complexes or homodimers, and reported new developments in functions used to score predicted interactions, in treatment of conformational flexibility, and in taking nonstructural information into account in the predictions.

A General Strategy to Solve the Phase Problem in RNA Crystallography by Amanda Y. Keel; Robert P. Rambo; Robert T. Batey; Jeffrey S. Kieft (pp. 761-772).
X-ray crystallography of biologically important RNA molecules has been hampered by technical challenges, including finding heavy-atom derivatives to obtain high-quality experimental phase information. Existing techniques have drawbacks, limiting the rate at which important new structures are solved. To address this, we have developed a reliable means to localize heavy atoms specifically to virtually any RNA. By solving the crystal structures of thirteen variants of the G•U wobble pair cation binding motif, we have identified a version that when inserted into an RNA helix introduces a high-occupancy cation binding site suitable for phasing. This “directed soaking” strategy can be integrated fully into existing RNA crystallography methods, potentially increasing the rate at which important structures are solved and facilitating routine solving of structures using Cu-Kα radiation. This method already has been used to solve several crystal structures.

Keywords: RNA

FASTDXL: A Generalized Screen to Trap Disulfide-Stabilized Complexes for Use in Structural Studies by Jacob E. Corn; James M. Berger (pp. 773-780).
Structural studies of macromolecular complexes have produced extraordinary insights into a wide variety of biological processes. Unfortunately, as structural biologists pursue larger and more challenging assemblies, weakly stable and/or nonspecific interactions can become significant roadblocks to structure determination. We have developed a rapid and effective pool-based screen, termed FASTDXL (focused array screening technique for disulfide X-linking), to produce and identify disulfide-stabilized protein-nucleic acid assemblies. A significant strength of FASTDXL is that it can take advantage of prior structural knowledge about molecular interactions, but does not necessarily rely upon it. A detailed application of the approach to the difficult problem of trapping a bacterial primase-ssDNA complex is described, validating the method as a route toward obtaining diffracting crystals suitable for structure determination.

Structural and Functional Insights into a Peptide Bond-Forming Bidomain from a Nonribosomal Peptide Synthetase by Stefan A. Samel; Georg Schoenafinger; Thomas A. Knappe; Mohamed A. Marahiel; Lars-Oliver Essen (pp. 781-792).
The crystal structure of the bidomain PCP-C from modules 5 and 6 of the nonribosomal tyrocidine synthetase TycC was determined at 1.8 Å resolution. The bidomain structure reveals a V-shaped condensation domain, the canyon-like active site groove of which is associated with the preceding peptidyl carrier protein (PCP) domain at its donor side. The relative arrangement of the PCP and the peptide bond-forming condensation (C) domain places the active sites ∼50 Å apart. Accordingly, this PCP-C structure represents a conformational state prior to peptide transfer from the donor-PCP to the acceptor-PCP domain, implying the existence of additional states of PCP-C domain interaction during catalysis. Additionally, PCP-C exerts a mode of cyclization activity that mimics peptide bond formation catalyzed by C domains. Based on mutational data and pK value analysis of active site residues, it is suggested that nonribosomal peptide bond formation depends on electrostatic interactions rather than on general acid/base catalysis.

Keywords: PROTEINS

Functional and Crystal Structure Analysis of Active Site Adaptations of a Potent Anti-Angiogenic Human tRNA Synthetase by Xiang-Lei Yang; Min Guo; Mili Kapoor; Karla L. Ewalt; Francella J. Otero; Robert J. Skene; Duncan E. McRee; Paul Schimmel (pp. 793-805).
Higher eukaryote tRNA synthetases have expanded functions that come from enlarged, more differentiated structures that were adapted to fit aminoacylation function. How those adaptations affect catalytic mechanisms is not known. Presented here is the structure of a catalytically active natural splice variant of human tryptophanyl-tRNA synthetase (TrpRS) that is a potent angiostatic factor. This and related structures suggest that a eukaryote-specific N-terminal extension of the core enzyme changed substrate recognition by forming an active site cap. At the junction of the extension and core catalytic unit, an arginine is recruited to replace a missing landmark lysine almost 200 residues away. Mutagenesis, rapid kinetic, and substrate binding studies support the functional significance of the cap and arginine recruitment. Thus, the enzyme function of human TrpRS has switched more to the N terminus of the sequence. This switch has the effect of creating selective pressure to retain the N-terminal extension for functional expansion.

Keywords: RNA

Packaging of DNA by Bacteriophage Epsilon15: Structure, Forces, and Thermodynamics by Anton S. Petrov; Krista Lim-Hing; Stephen C. Harvey (pp. 807-812).
The structure of bacteriophage epsilon15 has recently been determined by 3D reconstruction of single particle cryo-electron microscopy images. Although this study revealed that the viral genome inside the bacteriophage is on average coaxially spooled, individual DNA conformations inside the capsid could not be determined. In the current study, we present the results of 40 independent simulations of DNA packaging into epsilon15 using the previously described low-resolution model for bacteriophages. In addition to coaxially spooled conformations, we also observe a number of folded-toroidal patterns, but the density averaged over all conformations closely resembles the experimental density. Thermodynamic analysis of the simulations predicts that a force of ∼125 pN would be required to package DNA into epsilon15. We also show that the origin of this force is predominantly due to electrostatic and entropic contributions. However, the DNA conformation is determined primarily by the need to minimize the DNA bending energy.

Keywords: DNA

The Structure of Lethocerus Troponin C: Insights into the Mechanism of Stretch Activation in Muscles by Gianfelice De Nicola; Christoph Burkart; Feng Qiu; Bogos Agianian; Siegfried Labeit; Stephen Martin; Belinda Bullard; Annalisa Pastore (pp. 813-824).
To gain a molecular description of how muscles can be activated by mechanical stretch, we have solved the structure of the calcium-loaded F1 isoform of troponin C (TnC) from Lethocerus and characterized its interactions with troponin I (TnI). We show that the presence of only one calcium cation in the fourth EF hand motif is sufficient to induce an open conformation in the C-terminal lobe of F1 TnC, in contrast with what is observed in vertebrate muscle. This lobe interacts in a calcium-independent way both with the N terminus of TnI and, with lower affinity, with a region of TnI equivalent to the switch and inhibitory peptides of vertebrate muscles. Using both synthetic peptides and recombinant proteins, we show that the N lobe of F1 TnC is not engaged in interactions with TnI, excluding a regulatory role of this domain. These findings provide insights into mechanically stimulated muscle contraction.

Keywords: CELLBIO

The Structural Coupling between ATPase Activation and Recovery Stroke in the Myosin II Motor by Sampath Koppole; Jeremy C. Smith; Stefan Fischer (pp. 825-837).
Before the myosin motor head can perform the next power stroke, it undergoes a large conformational transition in which the converter domain, bearing the lever arm, rotates ∼65°. Simultaneous with this “recovery stroke,” myosin activates its ATPase function by closing the Switch-2 loop over the bound ATP. This coupling between the motions of the converter domain and of the 40 Å-distant Switch-2 loop is essential to avoid unproductive ATP hydrolysis. The coupling mechanism is determined here by finding a series of optimized intermediates between crystallographic end structures of the recovery stroke ( Dictyostelium discoideum), yielding movies of the transition at atomic detail. The successive formation of two hydrogen bonds by the Switch-2 loop is correlated with the successive see-saw motions of the relay and SH1 helices that hold the converter domain. SH1 helix and Switch-2 loop communicate via a highly conserved loop that wedges against the SH1-helix upon Switch-2 closing.

Structure and Analysis of FCHo2 F-BAR Domain: A Dimerizing and Membrane Recruitment Module that Effects Membrane Curvature by William Mike Henne; Helen M. Kent; Marijn G.J. Ford; Balachandra G. Hegde; Oliver Daumke; P. Jonathan G. Butler; Rohit Mittal; Ralf Langen; Philip R. Evans; Harvey T. McMahon (pp. 839-852).
A spectrum of membrane curvatures exists within cells, and proteins have evolved different modules to detect, create, and maintain these curvatures. Here we present the crystal structure of one such module found within human FCHo2. This F-BAR (extended FCH) module consists of two F-BAR domains, forming an intrinsically curved all-helical antiparallel dimer with a Kd of 2.5 μM. The module binds liposomes via a concave face, deforming them into tubules with variable diameters of up to 130 nm. Pulse EPR studies showed the membrane-bound dimer is the same as the crystal dimer, although the N-terminal helix changed conformation on membrane binding. Mutation of a phenylalanine on this helix partially attenuated narrow tubule formation, and resulted in a gain of curvature sensitivity. This structure shows a distant relationship to curvature-sensing BAR modules, and suggests how similar coiled-coil architectures in the BAR superfamily have evolved to expand the repertoire of membrane-sculpting possibilities.

Crystallographic Snapshots of Oxalyl-CoA Decarboxylase Give Insights into Catalysis by Nonoxidative ThDP-Dependent Decarboxylases by Catrine L. Berthold; Cory G. Toyota; Patricia Moussatche; Martin D. Wood; Finian Leeper; Nigel G.J. Richards; Ylva Lindqvist (pp. 853-861).
Despite more than five decades of extensive studies of thiamin diphosphate (ThDP) enzymes, there remain many uncertainties as to how these enzymes achieve their rate enhancements. Here, we present a clear picture of catalysis for the simple nonoxidative decarboxylase, oxalyl-coenzyme A (CoA) decarboxylase, based on crystallographic snapshots along the catalytic cycle and kinetic data on active site mutants. First, we provide crystallographic evidence that, upon binding of oxalyl-CoA, the C-terminal 13 residues fold over the substrate, aligning the substrate α-carbon for attack by the ThDP-C2 atom. The second structure presented shows a covalent reaction intermediate after decarboxylation, interpreted as being nonplanar. Finally, the structure of a product complex is presented. In accordance with mutagenesis data, no side chains of the enzyme are implied to directly participate in proton transfer except the glutamic acid (Glu-56), which promotes formation of the 1′,4′-iminopyrimidine tautomer of ThDP needed for activation.

Keywords: PROTEINS

Insights into the Catalytic Mechanism of PPM Ser/Thr Phosphatases from the Atomic Resolution Structures of a Mycobacterial Enzyme by Marco Bellinzoni; Annemarie Wehenkel; William Shepard; Pedro M. Alzari (pp. 863-872).
Serine/threonine-specific phosphatases (PPs) represent, after protein tyrosine phosphatases, the second major class of enzymes that catalyze the dephosphorylation of proteins. They are classed in two large families, known as PPP and PPM, on the basis of sequence similarities, metal ion dependence, and inhibitor sensitivity. Despite their wide species distribution and broad physiological roles, the catalytic mechanism of PPM phosphatases has been primarily inferred from studies of a single enzyme, human PP2Cα. Here, we report the biochemical characterization and the atomic resolution structures of a soluble PPM phosphatase from the saprophyte Mycobacterium smegmatis in complex with different ligands. The structures provide putative snapshots along the catalytic cycle, which support an associative reaction mechanism that differs in some important aspects from the currently accepted model and reinforces the hypothesis of convergent evolution in PPs.

Keywords: PROTEINS

Conformational Change in an MFS Protein: MD Simulations of LacY by John Holyoake; Mark S.P. Sansom (pp. 873-884).
Molecular dynamics simulations of lactose permease (LacY) in a phospholipid bilayer reveal the conformational dynamics of the protein. In inhibitor-bound simulations (i.e., those closest to the X-ray structure) the protein was stable, showing little conformational change over a 50 ns timescale. Movement of the bound inhibitor, TDG, to an alternative binding mode was observed, so that it interacted predominantly with the N-terminal domain and with residue E269 from the C-terminal domain. In multiple ligand-free simulations, a degree of domain closure occurred. This switched LacY to a state with a central cavity closed at both the intracellular and periplasmic ends. This may resemble a possible intermediate in the transport mechanism. Domain closure occurs by a combination of rigid-body movements of domains and of intradomain motions of helices, especially TM4, TM5, TM10, and TM11. A degree of intrahelix flexibility appears to be important in the conformational change.

Keywords: PROTEINS

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