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Structure (v.13, #7)

Ufd1 Exhibits Dual Ubiquitin Binding Modes by Kylie J. Walters (pp. 943-944).
Ubiquitin recognition proteins orchestrate the delivery of ubiquitylated substrates to the proteasome for their degradation. Park et al. (2005), in this issue of Structure, provide the first glimpses of how Ufd1 begins this process in endoplasmic reticulum-associated degradation.

Viruses Rock and Roll with Their Receptors by Deena A. Oren; Eddy Arnold (pp. 944-945).
Cryo-EM and X-ray crystallography combine powerfully for imaging large macromolecular structures and assemblies. The article by Xiao et al. (2005) in this issue of Structure is as illuminating in its biological insights as it is a tour-de-force in both X-ray crystallography and cryo-electron microscopy.

Amphiphilic Helices Drive Signaling by Masashi Miyano (pp. 946-947).
In this issue of Structure, Johnston et al. (2005) report the characterization of Gαi1:GDP in complex with a new GEF peptide KB-752, revealing the atomic detail of G protein activation and providing a clue to the energetics of the process.
Frontiers in Computational Biophysics: A Symposium in Honor of Martin Karplus by Carol Beth Post; Christopher M. Dobson (pp. 949-952).

The Solution Structure of a Transient Photoreceptor Intermediate: Δ25 Photoactive Yellow Protein by Cdric Bernard; Klaartje Houben; Nocky M. Derix; David Marks; Michael A. van der Horst; Klaas J. Hellingwerf; Rolf Boelens; Robert Kaptein; Nico A.J. van Nuland (pp. 953-962).
The N-terminally truncated variant of photoactive yellow protein (Δ25-PYP) undergoes a very similar photocycle as the corresponding wild-type protein (WT-PYP), although the lifetime of its light-illuminated (pB) state is much longer. This has allowed determination of the structure of both its dark- (pG) as well as its pB-state in solution by nuclear magnetic resonance (NMR) spectroscopy. The pG structure shows a well-defined fold, similar to WT-PYP and the X-ray structure of the pG state of Δ25-PYP. In the long-lived photocycle intermediate pB, the central β sheet is still intact, as well as a small part of one α helix. The remainder of pB is unfolded and highly flexible, as evidenced by results from proton-deuterium exchange and NMR relaxation studies. Thus, the partially unfolded nature of the presumed signaling state of PYP in solution, as suggested previously, has now been structurally demonstrated.

Obligate Heterodimerization of the Archaeal Alba2 Protein with Alba1 Provides a Mechanism for Control of DNA Packaging by Clare Jelinska; Matthew J. Conroy; C. Jeremy Craven; Andrea M. Hounslow; Per A. Bullough; Jonathan P. Waltho; Garry L. Taylor; Malcolm F. White (pp. 963-971).
Organisms growing at elevated temperatures face a particular challenge to maintain the integrity of their genetic material. All thermophilic and hyperthermophilic archaea encode one or more copies of the Alba (Sac10b) gene. Alba is an abundant, dimeric, highly basic protein that binds cooperatively and at high density to DNA. Sulfolobus solfataricus encodes a second copy of the Alba gene, and the Alba2 protein is expressed at ∼5% of the level of Alba1. We demonstrate by NMR, ITC, and crystallography that Alba2 exists exclusively as a heterodimer with Alba1 at physiological concentrations and that heterodimerization exerts a clear effect upon the DNA packaging, as observed by EM, potentially by changing the interface between adjacent Alba dimers in DNA complexes. A functional role for Alba2 in modulation of higher order chromatin structure and DNA condensation is suggested.

Crystal Structure of Escherichia coli RNase D, an Exoribonuclease Involved in Structured RNA Processing by Yuhong Zuo; Yong Wang; Arun Malhotra (pp. 973-984).
RNase D (RND) is one of seven exoribonucleases identified in Escherichia coli. RNase D has homologs in many eubacteria and eukaryotes, and has been shown to contribute to the 3′ maturation of several stable RNAs. Here, we report the 1.6 Å resolution crystal structure of E. coli RNase D. The conserved DEDD residues of RNase D fold into an arrangement very similar to the Klenow fragment exonuclease domain. Besides the catalytic domain, RNase D also contains two structurally similar α-helical domains with no discernible sequence homology between them. These closely resemble the HRDC domain previously seen in RecQ-family helicases and several other proteins acting on nucleic acids. More interestingly, the DEDD catalytic domain and the two helical domains come together to form a ring-shaped structure. The ring-shaped architecture of E. coli RNase D and the HRDC domains likely play a major role in determining the substrate specificity of this exoribonuclease.

Structural Basis and Kinetics of DsbD-Dependent Cytochrome c Maturation by Christian U. Stirnimann; Anna Rozhkova; Ulla Grauschopf; Markus G. Grtter; Rudi Glockshuber; Guido Capitani (pp. 985-993).
DsbD from Escherichia coli transports two electrons from cytoplasmic thioredoxin to the periplasmic substrate proteins DsbC, DsbG and CcmG. DsbD consists of an N-terminal periplasmic domain (nDsbD), a C-terminal periplasmic domain, and a central transmembrane domain. Each domain possesses two cysteines required for electron transport. Herein, we demonstrate fast (3.9 × 105 M−1s−1) and direct disulfide exchange between nDsbD and CcmG, a highly specific disulfide reductase essential for cytochrome c maturation. We determined the crystal structure of the disulfide-linked complex between nDsbD and the soluble part of CcmG at 1.94 Å resolution. In contrast to the other two known complexes of nDsbD with target proteins, the N-terminal segment of nDsbD contributes to specific recognition of CcmG. This and other features, like the possibility of using an additional interaction surface, constitute the structural basis for the adaptability of nDsbD to different protein substrates.

Ufd1 Exhibits the AAA-ATPase Fold with Two Distinct Ubiquitin Interaction Sites by Sunghyouk Park; Rivka Isaacson; Hyoung Tae Kim; Pamela A. Silver; Gerhard Wagner (pp. 995-1005).
Ufd1 mediates ubiquitin fusion degradation by association with Npl4 and Cdc48/p97. The Ufd1-ubiquitin interaction is essential for transfer of substrates to the proteasome. However, the mechanism and specificity of ubiquitin recognition by Ufd1 are poorly understood due to the lack of detailed structural information. Here, we present the solution structure of yeast Ufd1 N domain and show that it has two distinct binding sites for mono- and polyubiquitin. The structure exhibits striking similarities to the Cdc48/p97 N domain. It contains the double-psi β barrel motif, which is thus identified as a ubiquitin binding domain. Significantly, Ufd1 shows higher affinity toward polyubiquitin than monoubiquitin, attributable to the utilization of separate binding sites with different affinities. Further studies revealed that the Ufd1-ubiquitin interaction involves hydrophobic contacts similar to those in well-characterized ubiquitin binding proteins. Our results provide a structural basis for a previously proposed synergistic binding of polyubiquitin by Cdc48/p97 and Ufd1.

Structural Polymorphism of the Major Capsid Protein of a Double-Stranded RNA Virus: An Amphipathic α Helix as a Molecular Switch by Irene Saugar; Daniel Luque; Ana Oa; Jos F. Rodrguez; Jos L. Carrascosa; Benes L. Trus; Jos R. Castn (pp. 1007-1017).
The infectious bursal disease virus T=13 viral particle is composed of two major proteins, VP2 and VP3. Here, we show that the molecular basis of the conformational flexibility of the major capsid protein precursor, pVP2, is an amphipatic α helix formed by the sequence GFKDIIRAIR. VP2 containing this α helix is able to assemble into the T=13 capsid only when expressed as a chimeric protein with an N-terminal His tag. An amphiphilic α helix, which acts as a conformational switch, is thus responsible for the inherent structural polymorphism of VP2. The His tag mimics the VP3 C-terminal region closely and acts as a molecular triggering factor. Using cryo-electron microscopy difference imaging, both polypeptide elements were detected on the capsid inner surface. We propose that electrostatic interactions between these two morphogenic elements are transmitted to VP2 to acquire the competent conformations for capsid assembly.

The Crystal Structure of Coxsackievirus A21 and Its Interaction with ICAM-1 by Chuan Xiao; Carol M. Bator-Kelly; Elizabeth Rieder; Paul R. Chipman; Alister Craig; Richard J. Kuhn; Eckard Wimmer; Michael G. Rossmann (pp. 1019-1033).
CVA21 and polioviruses both belong to the Enterovirus genus in the family of Picornaviridae, whereas rhinoviruses form a distinct picornavirus genus. Nevertheless, CVA21 and the major group of human rhinoviruses recognize intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor, whereas polioviruses use poliovirus receptor. The crystal structure of CVA21 has been determined to 3.2 resolution. Its structure has greater similarity to poliovirus structures than to other known picornavirus structures. Cryo-electron microscopy (cryo-EM) was used to determine an 8.0 resolution structure of CVA21 complexed with an ICAM-1 variant, ICAM-1Kilifi. The cryo-EM map was fitted with the crystal structures of ICAM-1 and CVA21. Significant differences in the structure of CVA21 with respect to the poliovirus structures account for the inability of ICAM-1 to bind polioviruses. The interface between CVA21 and ICAM-1 has shape and electrostatic complementarity with many residues being conserved among those CVAs that bind ICAM-1.

Structural Basis of FFAT Motif-Mediated ER Targeting by Stephen E. Kaiser; Jason H. Brickner; Amy R. Reilein; Tim D. Fenn; Peter Walter; Axel T. Brunger (pp. 1035-1045).
The FFAT motif is a targeting signal responsible for localizing a number of proteins to the cytosolic surface of the endoplasmic reticulum (ER) and to the nuclear membrane. FFAT motifs bind to members of the highly conserved VAP protein family, which are tethered to the cytoplasmic face of the ER by a C-terminal transmembrane domain. We have solved crystal structures of the rat VAP-A MSP homology domain alone and in complex with an FFAT motif. The co-crystal structure was used to design a VAP mutant that disrupts rat and yeast VAP-FFAT interactions in vitro. The FFAT binding-defective mutant also blocked function of the VAP homolog Scs2p in yeast. Finally, overexpression of the FFAT binding-defective VAP in COS7 cells dramatically altered ER morphology. Our data establish the structural basis of FFAT-mediated ER targeting and suggest that FFAT-targeted proteins play an important role in determining ER morphology.

Scaling Behavior and Structure of Denatured Proteins by Feng Ding; Ramesh K. Jha; Nikolay V. Dokholyan (pp. 1047-1054).
An ensemble of random-coil conformations with no persistent structures has long been accepted as the classical model of denatured proteins due to its consistency with the experimentally determined scaling of protein sizes. However, recent NMR spectroscopy studies on proteins at high chemical denaturant concentrations suggest the presence of significant amounts of native-like structures, in contrast to the classical random-coil picture. To reconcile these seemingly controversial observations, we examine thermally denatured states of experimentally characterized proteins by using molecular dynamics simulations. For all studied proteins, we find that denatured states indeed have strong local conformational bias toward native states while a random-coil power law scaling of protein sizes is preserved. In addition, we explain why experimentally determined size of the protein creatine kinase does not follow general scaling. In simulations, we observe that this protein exhibits a stable intermediate state, the size of which is consistent with the reported experimental observation.

X-Ray Crystallographic and NMR Studies of the Third KH Domain of hnRNP K in Complex with Single-Stranded Nucleic Acids by Paul H. Backe; Ana C. Messias; Raimond B.G. Ravelli; Michael Sattler; Stephen Cusack (pp. 1055-1067).
The heterogeneous nuclear ribonucleoprotein (hnRNP) K is implicated in multiple functions in the regulation of gene expression and acts as a hub at the intersection of signaling pathways and processes involving nucleic acids. Central to its function is its ability to bind both ssDNA and ssRNA via its KH (hnRNP K homology) domains. We determined crystal structures of hnRNP K KH3 domain complexed with 15-mer and 6-mer (CTC4) ssDNAs at 2.4 and 1.8 resolution, respectively, and show that the KH3 domain binds specifically to both TCCC and CCCC sequences. In parallel, we used NMR to compare the binding affinity and mode of interaction of the KH3 domain with several ssRNA ligands and CTC4 ssDNA. Based on a structure alignment of the KH3-CTC4 complex with known structures of other KH domains in complex with ssRNA, we discuss recognition of tetranucleotide sequences by KH domains.

Structure of Gαi1 Bound to a GDP-Selective Peptide Provides Insight into Guanine Nucleotide Exchange by Christopher A. Johnston; Francis S. Willard; Mark R. Jezyk; Zoey Fredericks; Erik T. Bodor; Miller B. Jones; Rainer Blaesius; Val J. Watts; T. Kendall Harden; John Sondek; J. Kevin Ramer; David P. Siderovski (pp. 1069-1080).
Heterotrimeric G proteins are molecular switches that regulate numerous signaling pathways involved in cellular physiology. This characteristic is achieved by the adoption of two principal states: an inactive, GDP bound state and an active, GTP bound state. Under basal conditions, G proteins exist in the inactive, GDP bound state; thus, nucleotide exchange is crucial to the onset of signaling. Despite our understanding of G protein signaling pathways, the mechanism of nucleotide exchange remains elusive. We employed phage display technology to identify nucleotide state-dependent Gα binding peptides. Herein, we report a GDP-selective Gα binding peptide, KB-752, that enhances spontaneous nucleotide exchange of Gαi subunits. Structural determination of the Gαi1/peptide complex reveals unique changes in the Gα switch regions predicted to enhance nucleotide exchange by creating a GDP dissociation route. Our results cast light onto a potential mechanism by which Gα subunits adopt a conformation suitable for nucleotide exchange.

Structural Basis for Substrate Specificity of the Human Mitochondrial Deoxyribonucleotidase by Karin Walldn; Benedetta Ruzzenente; Agnes Rinaldo-Matthis; Vera Bianchi; Pr Nordlund (pp. 1081-1088).
The human mitochondrial deoxyribonucleotidase catalyzes the dephosphorylation of thymidine and deoxyuridine monophosphates and participates in the regulation of the dTTP pool in mitochondria. We present seven structures of the inactive D41N variant of this enzyme in complex with thymidine 3′-monophosphate, thymidine 5′-monophosphate, deoxyuridine 5′-monophosphate, uridine 5′-monophosphate, deoxyguanosine 5′-monophosphate, uridine 2′-monophosphate, and the 5′-monophosphate of the nucleoside analog 3′-deoxy 2′3′-didehydrothymidine, and we draw conclusions about the substrate specificity based on comparisons with enzyme activities. We show that the enzyme’s specificity for the deoxyribo form of nucleoside 5′-monophosphates is due to Ile-133, Phe-49, and Phe-102, which surround the 2′ position of the sugar and cause an energetically unfavorable environment for the 2′-hydroxyl group of ribonucleoside 5′-monophosphates. The close binding of the 3′-hydroxyl group of nucleoside 5′-monophosphates to the enzyme indicates that nucleoside analog drugs that are substituted with a bulky group at this position will not be good substrates for this enzyme.
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