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Structure (v.14, #8)

Sirtuins Caught in the Act by Brian C. Smith; John M. Denu (pp. 1207-1208).
Sirtuins catalyze the NAD+-dependent protein deacetylation of lysine residues. In this issue of Structure, report the structure of a sirtuin homolog with both substrates bound, providing new insight into the catalytic mechanism.

Gaining Access to ERp57 Function by Lloyd W. Ruddock (pp. 1209-1210).
ERp57, in complex with the lectins calreticulin and calnexin, catalyzes disulphide bond formation in N-glycosylated proteins. In this issue of Structure, report the structure of the noncatalytic domains of ERp57 and characterize the calnexin interaction site.

Outcome of a Workshop on Archiving Structural Models of Biological Macromolecules by Helen M. Berman; Stephen K. Burley; Wah Chiu; Andrej Sali; Alexei Adzhubei; Philip E. Bourne; Stephen H. Bryant; Roland L. Dunbrack Jr.; Krzysztof Fidelis; Joachim Frank; Adam Godzik; Kim Henrick; Andrzej Joachimiak; Bernard Heymann; David Jones; John L. Markley; John Moult; Gaetano T. Montelione; Christine Orengo; Michael G. Rossmann; Burkhard Rost; Helen Saibil; Torsten Schwede; Daron M. Standley; John D. Westbrook (pp. 1211-1217).
This paper describes the outcome of a “Workshop on Biological Macromolecular Structure Models? held in November 2005 in which experimentalists and modelers discussed the best way to archive models of biological macromolecules.

A Steric Antagonism of Actin Polymerization by a Salmonella Virulence Protein by S. Mariana Margarit; Walter Davidson; Lee Frego; C. Erec Stebbins (pp. 1219-1229).
Salmonella spp. require the ADP-ribosyltransferase activity of the SpvB protein for intracellular growth and systemic virulence. SpvB covalently modifies actin, causing cytoskeletal disruption and apoptosis. We report here the crystal structure of the catalytic domain of SpvB, and we show by mass spectrometric analysis that SpvB modifies actin at Arg177, inhibiting its ATPase activity. We also describe two crystal structures of SpvB-modified, polymerization-deficient actin. These structures reveal that ADP-ribosylation does not lead to dramatic conformational changes in actin, suggesting a model in which this large family of toxins inhibits actin polymerization primarily through steric disruption of intrafilament contacts.

Insights into the Sirtuin Mechanism from Ternary Complexes Containing NAD+ and Acetylated Peptide by Kevin G. Hoff; José L. Avalos; Kristin Sens; Cynthia Wolberger (pp. 1231-1240).
Sirtuin proteins comprise a unique class of NAD+-dependent protein deacetylases. Although several structures of sirtuins have been determined, the mechanism by which NAD+ cleavage occurs has remained unclear. We report the structures of ternary complexes containing NAD+ and acetylated peptide bound to the bacterial sirtuin Sir2Tm and to a catalytic mutant (Sir2TmH116Y). NAD+ in these structures binds in a conformation different from that seen in previous structures, exposing the α face of the nicotinamide ribose to the carbonyl oxygen of the acetyl lysine substrate. The NAD+ conformation is identical in both structures, suggesting that proper coenzyme orientation is not dependent on contacts with the catalytic histidine. We also present the structure of Sir2TmH116A bound to deacteylated peptide and 3′- O-acetyl ADP ribose. Taken together, these structures suggest a mechanism for nicotinamide cleavage in which an invariant phenylalanine plays a central role in promoting formation of the O-alkylamidate reaction intermediate and preventing nicotinamide exchange.

The Open State Gating Mechanism of Gramicidin A Requires Relative Opposed Monomer Rotation and Simultaneous Lateral Displacement by Gennady V. Miloshevsky; Peter C. Jordan (pp. 1241-1249).
The gating mechanism of the open state of the gramicidin A (gA) channel is studied by using a new Monte Carlo Normal Mode Following (MC-NMF) technique, one applicable even without a target structure. The results demonstrate that the lowest-frequency normal mode (NM) at ∼6.5 cm−1 is the crucial mode that initiates dissociation. Perturbing the gA dimer in either direction along this NM leads to opposed, nearly rigid-body rotations of the gA monomers around the central pore axis. Tracking this NM by using the eigenvector-following technique reveals the channel's gating mechanism: dissociation via relative opposed monomer rotation and simultaneous lateral displacement. System evolution along the lowest-frequency eigenvector shows that the large-amplitude motions required for gating (dissociation) are not simple relative rigid-body motions of the monomers. Gating involves coupling intermonomer hydrogen bond breaking, backbone realignment, and relative monomer tilt with complex side chain reorganization at the intermonomer junction.

Prokaryotic Type II and Type III Pantothenate Kinases: The Same Monomer Fold Creates Dimers with Distinct Catalytic Properties by Bum Soo Hong; Mi Kyung Yun; Yong-Mei Zhang; Shigeru Chohnan; Charles O. Rock; Stephen W. White; Suzanne Jackowski; Hee-Won Park; Roberta Leonardi (pp. 1251-1261).
Three distinct isoforms of pantothenate kinase (CoaA) in bacteria catalyze the first step in coenzyme A biosynthesis. The structures of the type II ( Staphylococcus aureus, SaCoaA) and type III ( Pseudomonas aeruginosa, PaCoaA) enzymes reveal that they assemble nearly identical subunits with actin-like folds into dimers that exhibit distinct biochemical properties. PaCoaA has a fully enclosed pantothenate binding pocket and requires a monovalent cation to weakly bind ATP in an open cavity that does not interact with the adenine nucleotide. Pantothenate binds to an open pocket in SaCoaA that strongly binds ATP by using a classical P loop architecture coupled with specific interactions with the adenine moiety. The PaCoaA•Pan binary complex explains the resistance of bacteria possessing this isoform to the pantothenamide antibiotics, and the similarity between SaCoaA and human pantothenate kinase 2 explains the molecular basis for the development of the neurodegenerative phenotype in three mutations in the human protein.

Origin of Exopolyphosphatase Processivity: Fusion of an ASKHA Phosphotransferase and a Cyclic Nucleotide Phosphodiesterase Homolog by Johnjeff Alvarado; Anita Ghosh; Tyler Janovitz; Andrew Jauregui; Miriam S. Hasson; David Avram Sanders (pp. 1263-1272).
The Escherichia coli Ppx protein is an exopolyphosphatase that degrades long-chain polyphosphates in a highly processive reaction. It also hydrolyzes the terminal 5′ phosphate of the modified nucleotide guanosine 5′ triphosphate 3′ diphosphate (pppGpp). The structure of Ppx has been determined to 1.9 Å resolution by X-ray crystallography. The exopolyphosphatase is an ASKHA ( acetate and sugar kinases, Hsp70, actin) phosphotransferase with an active site found in a cleft between the two amino-terminal domains. Analysis of the active site indicates that among the ASKHA phosphotranferases of known structure, Ppx is the closest to the ectonucleoside triphosphate diphosphohydrolases. A third domain forms a six-helix claw that is similar to the catalytic core of the eukaryotic cyclic nucleotide phosphodiesterases. Most of the 29 sulfate ions bound to the Ppx dimer occupy sites where the polyP chain likely binds. An aqueduct that passes through the enzyme provides a physical basis for the enzyme's high processivity.

Crystal Structure of Rab11 in Complex with Rab11 Family Interacting Protein 2 by William N. Jagoe; Andrew J. Lindsay; Randy J. Read; Airlie J. McCoy; Mary W. McCaffrey; Amir R. Khan (pp. 1273-1283).
The small GTPase Rab11 regulates the recycling of endosomes to the plasma membrane via interactions with the Rab11 family of interacting proteins (FIPs). FIPs contain a highly conserved Rab binding domain (RBD) at their C termini whose structure is unknown. Here, we have determined the crystal structure of the RBD of FIP2 in complex with Rab11(GTP) by single wavelength anomalous diffraction methods. The overall structure is a heterotetramer with dyad symmetry, arranged as a Rab11-(FIP2)2-Rab11 complex. FIP2 forms a central α-helical coiled coil, with both helices contributing to the Rab11 binding patch on equivalent and opposite sides of the homodimer. Switch 1 of Rab11 is embedded between the two helices, while switch 2 remains flexible and is peripherally associated with the effector. The complex reveals the structural basis for Rab11 recognition by FIPs and suggests the molecular mechanisms underlying endocytic recycling pathways.

Structural Basis for Phosphotyrosine Recognition by Suppressor of Cytokine Signaling-3 by Elisa Bergamin; Jinhua Wu; Stevan R. Hubbard (pp. 1285-1292).
Suppressor of cytokine signaling (SOCS) proteins are indispensable negative regulators of cytokine-stimulated Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathways. SOCS proteins (SOCS1–7 and CIS) consist of a variable N-terminal region, a central Src homology-2 (SH2) domain, and a C-terminal SOCS box. The N-terminal region in SOCS1 and SOCS3 includes the so-called kinase inhibitory region that has been shown to inhibit the catalytic activity of JAK2. Here, we present a crystal structure at 2.0 Å resolution of the N-terminally extended SH2 domain of SOCS3 in complex with its phosphopeptide target on the cytokine receptor gp130. The structure reveals that major insertions in the EF and BG loops of the SOCS3 SH2 domain are responsible for binding to gp130 with high affinity and specificity. In addition, the structure provides insights into the possible mechanisms by which SOCS3 and SOCS1 inhibit JAK2 kinase activity.

Structural Basis of Ubiquitin Recognition by the Deubiquitinating Protease USP2 by Martin Renatus; Shirley Gil Parrado; Allan D'Arcy; Ulf Eidhoff; Bernd Gerhartz; Ulrich Hassiepen; Benoit Pierrat; Ralph Riedl; Daniela Vinzenz; Susanne Worpenberg; Markus Kroemer (pp. 1293-1302).
Deubiquitinating proteases reverse protein ubiquitination and rescue their target proteins from destruction by the proteasome. USP2, a cysteine protease and a member of the ubiquitin specific protease family, is overexpressed in prostate cancer and stabilizes fatty acid synthase, which has been associated with the malignancy of some aggressive prostate cancers. Here, we report the structure of the human USP2 catalytic domain in complex with ubiquitin. Ubiquitin uses two major sites for the interaction with the protease. Both sites are required simultaneously, as shown by USP2 inhibition assays with peptides and ubiquitin mutants. In addition, a layer of ordered water molecules mediates key interactions between ubiquitin and USP2. As several of those molecules are found at identical positions in the previously solved USP7/ubiquitin-aldehyde complex structure, we suggest a general mechanism of water-mediated ubiquitin recognition by USPs.

The Notch Ankyrin Domain Folds via a Discrete, Centralized Pathway by Christina Marchetti Bradley; Doug Barrick (pp. 1303-1312).
The Notch ankyrin repeat domain contains seven ankyrin sequence repeats, six of which adopt very similar structures. To determine if folding proceeds along parallel pathways and the order in which repeats become structured during folding, we examined the effect of analogous destabilizing Ala→Gly substitutions in each repeat on folding kinetics. We find that folding proceeds to an on-pathway kinetic intermediate through a transition state ensemble containing structure in repeats three through five. Repeats two, six, and seven remain largely unstructured in this intermediate, becoming structured in a second kinetic step that leads to the native state. These data suggest that the Notch ankyrin domain folds according to a discrete kinetic pathway despite structural redundancy in the native state and highlight the importance of sequence-specific interactions in controlling pathway selection. This centralized pathway roughly corresponds to a low energy channel through the experimentally determined energy landscape.

Knowledge-Based Real-Space Explorations for Low-Resolution Structure Determination by Nicholas Furnham; Andrew S. Doré; Dimitri Y. Chirgadze; Paul I.W. de Bakker; Mark A. DePristo; Tom L. Blundell (pp. 1313-1320).
The accurate and effective interpretation of low-resolution data in X-ray crystallography is becoming increasingly important as structural initiatives turn toward large multiprotein complexes. Substantial challenges remain due to the poor information content and ambiguity in the interpretation of electron density maps at low resolution. Here, we describe a semiautomated procedure that employs a restraint-based conformational search algorithm, RAPPER, to produce a starting model for the structure determination of ligase interacting factor 1 in complex with a fragment of DNA ligase IV at low resolution. The combined use of experimental data and a priori knowledge of protein structure enabled us not only to generate an all-atom model but also to reaffirm the inferred sequence registry. This approach provides a means to extract quickly from experimental data useful information that would otherwise be discarded and to take into account the uncertainty in the interpretation—an overriding issue for low-resolution data.

The Crystal Structure of the Costimulatory OX40-OX40L Complex by Deanne M. Compaan; Sarah G. Hymowitz (pp. 1321-1330).
OX40 is a T cell costimulator activated by OX40L. Blockade of the OX40L-OX40 interaction has ameliorative effects in animal models of T cell pathologies. In order to better understand the interaction between OX40 and OX40L, we have determined the crystal structure of murine OX40L and of the human OX40-OX40L complex at 1.45 and 2.4 Å, respectively. These structures show that OX40L is an unusually small member of the tumor necrosis factor superfamily (TNFSF). The arrangement of the OX40L protomers forming the functional trimer is atypical and differs from that of other members by a 15° rotation of each protomer with respect to the trimer axis, resulting in an open assembly. Site-directed changes of the interfacial residues of OX40L suggest this interface lacks a single “hot spot? and that instead, binding energy is dispersed over at least two distinct areas. These structures demonstrate the structural plasticity of TNFSF members and their interactions with receptors.

Crystal Structure of the bb′ Domains of the Protein Disulfide Isomerase ERp57 by Guennadi Kozlov; Pekka Maattanen; Joseph D. Schrag; Stephanie Pollock; Miroslaw Cygler; Bhushan Nagar; David Y. Thomas; Kalle Gehring (pp. 1331-1339).
The synthesis of proteins in the endoplasmic reticulum (ER) is limited by the rate of correct disulfide bond formation. This process is carried out by protein disulfide isomerases, a family of ER proteins which includes general enzymes such as PDI that recognize unfolded proteins and others that are selective for specific proteins or classes. Using small-angle X-ray scattering and X-ray crystallography, we report the structure of a selective isomerase, ERp57, and its interactions with the lectin chaperone calnexin. Using isothermal titration calorimetry and NMR spectroscopy, we show that the b′ domain of ERp57 binds calnexin with micromolar affinity through a conserved patch of basic residues. Disruption of this binding site by mutagenesis abrogates folding of RNase B in an in vitro assay. The relative positions of the ERp57 catalytic sites and calnexin binding site suggest that activation by calnexin is due to substrate recruitment rather than a direct stimulation of ERp57 oxidoreductase activity.
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