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Structure (v.13, #9)


IRF-3 Releases Its Inhibitions by John Hiscott; Rongtuan Lin (pp. 1235-1236).
Interferon regulatory factor (IRF) 3 plays a critical role in triggering the activation of interferon antiviral genes. The structure of IRF-3 in association with the CBP/p300 coactivator by in this issue of Structure illuminates the mechanism of IRF activation and the structural flexibilities inherent in CBP/p300.

Liberating Crystallographers by Randy J. Read (pp. 1236-1237).
In this issue of Structure, DePristo et al. describe a new program, RAPPER, that should automate some of the most time-consuming tasks associated with rebuilding, refining, and completing protein crystal structures at moderate resolutions

No Phobias about PhoB Activation by William R. McCleary (pp. 1238-1239).
Structures of the inactive and activated forms of the receiver domain of PhoB reported in this issue of Structure () suggest that the OmpR/PhoB subclass of transcription factors becomes active by dimerization about a symmetric axis utilizing the α4-β5-α5 surface.

Evolution of NAD Biosynthetic Enzymes by Charles Brenner (pp. 1239-1240).
Two research groups have solved crystal structures of nicotinic acid phosphoribosyltransferase (PRTase) and made the argument that PRTases in three distinct pathways of nicotinamide adenine dinucleotide (NAD) biosynthesis evolved from a common ancestor ().

Insights into the Catalytic Mechanism of Glutathione S-Transferase: The Lesson from Schistosoma haematobium by Francesco Angelucci; Paola Baiocco; Maurizio Brunori; Louise Gourlay; Veronica Morea; Andrea Bellelli (pp. 1241-1246).
Glutathione S-transferases (GSTs) are involved in detoxification of xenobiotic compounds and in the biosynthesis of important metabolites. All GSTs activate glutathione (GSH) to GS; in many GSTs, this is accomplished by a Tyr at H-bonding distance from the sulfur of GSH. The high-resolution structure of GST from Schistosoma haematobium revealed that the catalytic Tyr occupies two alternative positions, one external, involving a π-cation interaction with the conserved Arg21, and the other inside the GSH binding site. The interaction with Arg21 lowers the pKa of the catalytic Tyr10, as required for catalysis. Examination of several other GST structures revealed the presence of an external pocket that may accommodate the catalytic Tyr, and suggested that the change in conformation and acidic properties of the catalytic Tyr may be shared by other GSTs. Arginine and two other residues of the external pocket constitute a conserved structural motif, clearly identified by sequence comparison.

Visualization of Single Receptor Molecules Bound to Human Rhinovirus under Physiological Conditions by Ferry Kienberger; Christian Rankl; Vassili Pastushenko; Rong Zhu; Dieter Blaas; Peter Hinterdorfer (pp. 1247-1253).
Dynamic force microscopy (DFM) was used to image human rhinovirus HRV2 alone and complexed with single receptor molecules under near physiological conditions. Specific and site-directed immobilization of HRV2 on a model cell membrane resulted in a crystalline arrangement of virus particles with hexagonal symmetry and 35 nm spacing. High-resolution imaging of the virus capsid revealed about 20 resolvable structural features with 3 nm diameters; this finding is in agreement with protrusions seen by cryo-electron microscopy. Binding of receptor molecules to individual virus particles was observed after injection of soluble receptors into the liquid cell. Virus-receptor complexes with zero, one, two, or three attached receptor molecules were resolved. The number of receptor molecules associated to virions increased over time. Occasionally, dissociation of single receptor molecules from viral particles was also observed.

Structure and Dynamics of an RNA Tetraloop: A Joint Molecular Dynamics and NMR Study by Jessica Koplin; Yuguang Mu; Christian Richter; Harald Schwalbe; Gerhard Stock (pp. 1255-1267).
Molecular dynamics simulations of the RNA tetraloop 5′-CGCUUUUGCG-3′ with high melting temperature and significant conformational heterogeneity in explicit water solvent are presented and compared to NMR studies. The NMR data allow for a detailed test of the theoretical model, including the quality of the force field and the conformational sampling. Due to the conformational heterogeneity of the tetraloop, high temperature (350 K) and locally enhanced sampling simulations need to be invoked. The Amber98 force field leads to a good overall agreement with experimental data. Based on NMR data and a principal component analysis of the 350 K trajectory, the dynamic structure of the tetraloop is revealed. The principal component free energy surface exhibits four minima, which correspond to well-defined conformational structures that differ mainly by their base stacking in the loop region. No correlation between the motion of the sugar rings and the stacking dynamics of the loop bases is found.

Crystal Structure of IRF-3 in Complex with CBP by Bin Y. Qin; Cheng Liu; Hema Srinath; Suvana S. Lam; John J. Correia; Rik Derynck; Kai Lin (pp. 1269-1277).
Transcriptional activation of interferon β (IFN-β), an antiviral cytokine, requires the assembly of IRF-3 and CBP/p300 at the promoter region of the IFN-β gene. The crystal structure of IRF-3 in complex with CBP reveals that CBP interacts with a hydrophobic surface on IRF-3, which in latent IRF-3 is covered by its autoinhibitory elements. This structural organization suggests that virus-induced phosphoactivation of IRF-3 triggers unfolding of the autoinhibitory elements and exposes the same hydrophobic surface for CBP interaction. The structure also reveals that the interacting CBP segment can exist in drastically different conformations, depending on the identity of the associating transcription cofactor. The finding suggests a possible regulatory mechanism in CBP/p300, by which the interacting transcription factor can specify the coactivator’s conformation and influence the transcriptional outcome.

A Twisted Four-Sheeted Model for an Amyloid Fibril by Jimin Wang; Susanne Glich; Catharine Bradford; Marina Ramirez-Alvarado; Lynne Regan (pp. 1279-1288).
The formation of amyloid fibers and their deposition in the body is a characteristic of a number of devastating human diseases. Here, we propose a structural model, based on X-ray diffraction data, for the basic structure of an amyloid fibril formed by using the variants of the B1 domain of IgG binding protein G of Streptococcus. The model for the fibril incorporates four β sheets in a bundle with a diameter of 45 Å. Its cross-section, or layer, consists of four strands, one strand from each sheet. Layers stack on top of each other to form the fibril, which has an overall helical twist with a periodicity of about 154 Å. Each strand interacts in a parallel fashion with the strands in the layers above and below it, in an infinite β sheet. Some geometric features of this model and the logic behind it may be applicable for constructing other related cross-β amyloid fibrils.

An Active-like Structure in the Unphosphorylated StyR Response Regulator Suggests a Phosphorylation- Dependent Allosteric Activation Mechanism by Mario Milani; Livia Leoni; Giordano Rampioni; Elisabetta Zennaro; Paolo Ascenzi; Martino Bolognesi (pp. 1289-1297).
StyR belongs to the FixJ subfamily of signal transduction response regulators; it controls transcription of the styABCD operon coding for styrene catabolism in Pseudomonas fluorescens ST. The crystal structure of unphosphorylated StyR is reported at 2.2 Å resolution. StyR is composed of an N-terminal regulatory domain (StyR-N) and a C-terminal DNA binding domain (StyR-C). The two domains are separated by an elongated linker α helix (34 residues), a new feature in known response regulator structures. StyR-C is structured similarly to the DNA binding domain of the response regulator NarL. StyR-N shows structural reorganization of the phosphate receiving region involved in activation/homodimerization: specific residues adopt an “active-like? conformation, and the α4 helix, involved in dimerization of the homologous FixJ response regulator, is trimmed to just one helical turn. Overall, structural considerations suggest that phosphorylation may act as an allosteric switch, shifting a preexisting StyR equilibrium toward the active, dimeric, DNA binding form.

The Monomeric dUTPase from Epstein-Barr Virus Mimics Trimeric dUTPases by Nicolas Tarbouriech; Marlyse Buisson; Jean-Marie Seigneurin; Stephen Cusack; Wim P. Burmeister (pp. 1299-1310).
Deoxyuridine 5′-triphosphate pyrophosphatases (dUTPases) are ubiquitous enzymes cleaving dUTP into dUMP and pyrophosphate. They occur as monomeric, dimeric, or trimeric molecules. The trimeric and monomeric enzymes both contain the same five characteristic sequence motifs but in a different order, whereas the dimeric enzymes are not homologous. Monomeric dUTPases only occur in herpesviruses, such as Epstein-Barr virus (EBV). Here, we describe the crystal structures of EBV dUTPase in complex with the product dUMP and a substrate analog α,β-imino-dUTP. The molecule consists of three domains forming one active site that has a structure extremely similar to one of the three active sites of trimeric dUTPases. The three domains functionally correspond to the subunits of the trimeric form. Domains I and II have the dUTPase fold, but they differ considerably in the regions that are not involved in the formation of the unique active site, whereas domain III has only little secondary structure.

Crystallographic Refinement by Knowledge-Based Exploration of Complex Energy Landscapes by Mark A. DePristo; Paul I.W. de Bakker; Russell J.K. Johnson; Tom L. Blundell (pp. 1311-1319).
Although X-ray crystallography remains the most versatile method to determine the three-dimensional atomic structure of proteins and much progress has been made in model building and refinement techniques, it remains a challenge to elucidate accurately the structure of proteins in medium-resolution crystals. This is largely due to the difficulty of exploring an immense conformational space to identify the set of conformers that collectively best fits the experimental diffraction pattern. We show here that combining knowledge-based conformational sampling in RAPPER with molecular dynamics/simulated annealing (MD/SA) vastly improves the quality and power of refinement compared to MD/SA alone. The utility of this approach is highlighted by the automated determination of a lysozyme mutant from a molecular replacement solution that is in congruence with a model prepared independently by crystallographers. Finally, we discuss the implications of this work on structure determination in particular and conformational sampling and energy minimization in general.

Finding Gas Diffusion Pathways in Proteins: Application to O2 and H2 Transport in CpI [FeFe]-Hydrogenase and the Role of Packing Defects by Jordi Cohen; Kwiseon Kim; Paul King; Michael Seibert; Klaus Schulten (pp. 1321-1329).
We report on a computational investigation of the passive transport of H2 and O2 between the external solution and the hydrogen-producing active site of CpI [FeFe]-hydrogenase from Clostridium pasteurianum. Two distinct methodologies for studying gas access are discussed and applied: (1) temperature-controlled locally enhanced sampling, and (2) volumetric solvent accessibility maps, providing consistent results. Both methodologies confirm the existence and function of a previously hypothesized pathway and reveal a second major pathway that had not been detected by previous analyses of CpI’s static crystal structure. Our results suggest that small hydrophobic molecules, such as H2 and O2, diffusing inside CpI, take advantage of well-defined preexisting packing defects that are not always apparent from the protein’s static structure, but that can be predicted from the protein’s dynamical motion. Finally, we describe two contrasting modes of intraprotein transport for H2 and O2, which in our model are differentiated only by their size.

Crystal Structure of Human CD38 Extracellular Domain by Qun Liu; Irina A. Kriksunov; Richard Graeff; Cyrus Munshi; Hon Cheung Lee; Quan Hao (pp. 1331-1339).
Human CD38 is a multifunctional protein involved in diverse functions. As an enzyme, it is responsible for the synthesis of two Ca2+ messengers, cADPR and NAADP; as an antigen, it is involved in regulating cell adhesion, differentiation, and proliferation. Besides, CD38 is a marker of progression of HIV-1 infection and a negative prognostic marker of B-CLL. We have determined the crystal structure of the soluble extracellular domain of human CD38 to 1.9 Å resolution. The enzyme’s overall topology is similar to the related proteins CD157 and the Aplysia ADP-ribosyl cyclase, except with large structural changes at the two termini. The extended positively charged N terminus has lateral associations with the other CD38 molecule in the crystallographic asymmetric unit. The analysis of the CD38 substrate binding models revealed two key residues that may be critical in controlling CD38’s multifunctionality of NAD hydrolysis, ADP-ribosyl cyclase, and cADPR hydrolysis activities.

The Structure of Bacillus subtilis RecU Holliday Junction Resolvase and Its Role in Substrate Selection and Sequence-Specific Cleavage by Natalie McGregor; Sylvia Ayora; Svetlana Sedelnikova; Begona Carrasco; Juan C. Alonso; Paul Thaw; John Rafferty (pp. 1341-1351).
We have determined the structure of the enzyme RecU from Bacillus subtilis, that is the general Holliday junction resolving enzyme in Gram-positive bacteria. The enzyme fold reveals a striking similarity to a class of resolvase enzymes found in archaeal sources and members of the type II restriction endonuclease family to which they are related. The structure confirms the presence of active sites formed around clusters of acidic residues that we have also shown to bind divalent cations. Mutagenesis data presented here support the key role of certain residues. The RecU structure suggests a basis for Holliday junction selectivity and suggests how sequence-specific cleavage might be achieved. Models for a resolvase-DNA complex address how the enzyme might organize junctions into an approximately 4-fold symmetric form.

Mechanism of Activation for Transcription Factor PhoB Suggested by Different Modes of Dimerization in the Inactive and Active States by Priti Bachhawat; G.V.T. Swapna; Gaetano T. Montelione; Ann M. Stock (pp. 1353-1363).
Response regulators (RRs), which undergo phosphorylation/dephosphorylation at aspartate residues, are highly prevalent in bacterial signal transduction. RRs typically contain an N-terminal receiver domain that regulates the activities of a C-terminal DNA binding domain in a phosphorylation-dependent manner. We present crystallography and solution NMR data for the receiver domain of Escherichia coli PhoB which show distinct 2-fold symmetric dimers in the inactive and active states. These structures, together with the previously determined structure of the C-terminal domain of PhoB bound to DNA, define the conformation of the active transcription factor and provide a model for the mechanism of activation in the OmpR/PhoB subfamily, the largest group of RRs. In the active state, the receiver domains dimerize with 2-fold rotational symmetry using their α4-β5-α5 faces, while the effector domains bind to DNA direct repeats with tandem symmetry, implying a loss of intramolecular interactions.

NF-κB RelB Forms an Intertwined Homodimer by De-Bin Huang; Don Vu; Gourisankar Ghosh (pp. 1365-1373).
The X-ray structure of the RelB dimerization domain (DD) reveals that the RelBDD assumes an unexpected intertwined fold topology atypical of other NF-κB dimers. All typical NF-κB dimers are formed by the association of two independently folded immunoglobulin (Ig) domains. In RelBDD, two polypeptides reconstruct both Ig domains in the dimer with an extra β sheet connecting the two domains. Residues most critical to NF-κB dimer formation are invariant in RelB, and Y300 plays a positive role in RelBDD dimer formation. The presence of RelB-specific nonpolar residues at the surface removes several intradomain surface hydrogen bonds that may render the domain fold unstable. Intertwining may stabilize the RelBDD homodimer by forming the extra β sheet. We show that, as in the crystal, RelB forms an intertwined homodimer in solution. We suggest that the transiently stable RelB homodimer might prevent its rapid degradation, allowing for heterodimer formation with p50 and p52.

RNA Silencing Suppressor p21 of Beet Yellows Virus Forms an RNA Binding Octameric Ring Structure by Keqiong Ye; Dinshaw J. Patel (pp. 1375-1384).
Many plant viruses encode proteins that suppress the antiviral RNA silencing response mounted by the host. The suppressors p19 from tombusvirus and p21 from Beet yellows virus appear to block silencing by directly binding siRNA, a critical mediator in the process. Here, we report the crystal structure of p21, which reveals an octameric ring architecture with a large central cavity of ∼90 Å diameter. The all α-helical p21 monomer consists of N- and C-terminal domains that associate with their neighboring counterparts through symmetric head-to-head and tail-to-tail interactions. A putative RNA binding surface is identified in the conserved, positive-charged inner surface of the ring. In contrast to the specific p19-siRNA duplex interaction, p21 is a general nucleic acid binding protein, interacting with 21 nt or longer single- and double-stranded RNAs in vitro. This study reveals an RNA binding structure adopted by the p21 silencing suppressor.

The Structure of a Eukaryotic Nicotinic Acid Phosphoribosyltransferase Reveals Structural Heterogeneity among Type II PRTases by Joshua S. Chappie; Jaume M. Cnaves; Gye Won Han; Christopher L. Rife; Qingping Xu; Raymond C. Stevens (pp. 1385-1396).
Nicotinamide adenine dinucleotide (NAD) is an essential cofactor for cellular redox reactions and can act as an important substrate in numerous biological processes. As a result, nature has evolved multiple biosynthetic pathways to meet this high chemical demand. In Saccharomyces cerevisiae, the NAD salvage pathway relies on the activity of nicotinic acid phosphoribosyltransferase (NAPRTase), a member of the phosphoribosyltransferase (PRTase) superfamily. Here, we report the structure of a eukaryotic (yeast) NAPRTase at 1.75 resolution (locus name: YOR209C, gene name: NPT1). The structure reveals a two-domain fold that resembles the architecture of quinolinic acid phosphoribosyltransferases (QAPRTases), but with completely different dispositions that provide evidence for structural heterogeneity among the Type II PRTases. The identification of a third domain in NAPRTases provides a structural basis and possible mechanism for the functional modulation of this family of enzymes by ATP.
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