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Structure (v.14, #7)

Common Scaffolds, Diverse Recognition Profiles by Nagasuma Chandra (pp. 1093-1094).
A paper in this issue of Structure reports the crystal structure of griffithsin, a lectin from red algae, and demonstrates its ability to bind and neutralize the SARS coronavirus, providing a link in understanding the evolution of lectins in this family. ().

When Size Matters by Marvin L. Hackert; Austen F. Riggs (pp. 1094-1096).
Erythrocruorins are very large extracellular hemoglobins found in some invertebrates. In this issue of Structure, report the structure of the 3.6 MDa, 180-mer of Lumbricus (earthworm) hemoglobin consisting of 144 globin and 36 linker chains.

PHinDing a New Histone “Effector? Domain by Michael S. Cosgrove (pp. 1096-1098).
Lysine methylation on histones represents an important epigenetic indexing system for active and repressed chromatin states. Four papers published recently by Nature highlight the discovery of the PHD finger as a new histone methyl-lysine recognition domain.

How to Avoid Premature Decay of Your Macromolecular Crystal: A Quick Soak for Long Life by Brice Kauffmann; Manfred S. Weiss; Victor S. Lamzin; Andrea Schmidt (pp. 1099-1105).
Radiation damage to biological samples is currently one of the major limiting factors in macromolecular X-ray crystallography, since it severely and irreversibly affects the quality of the data that can be obtained from a diffraction experiment. However, radiation damage can effectively be reduced by utilizing the electron and radical scavenging potential of certain small-molecule compounds. We propose an approach to protect macromolecular crystals prior to data collection by quick soaking with scavengers. This, in favorable cases, can more than double crystal lifetime in the X-ray beam. The approach has the potential to yield diffraction data of superior quality and hence to increase the amount of high-quality diffraction data and of structural information attainable from a single crystal.

The Structure of Bovine Viral Diarrhea Virus RNA-Dependent RNA Polymerase and Its Amino-Terminal Domain by Kyung H. Choi; Andreas Gallei; Paul Becher; Michael G. Rossmann (pp. 1107-1113).
Viral RNA-dependent RNA polymerases (RdRp) differ from DNA-dependent RNA polymerases, DNA-dependent DNA polymerases, and reverse transcriptases in that RdRps contain “fingertips? consisting of several polypeptide strands in the fingers domain interacting with the thumb domain. The crystal structure of bovine viral diarrhea virus (BVDV) RdRp containing an Asn438 duplication shows that the “N-terminal domain,? which occurs only in pestiviruses such as BVDV, interacts with the polymerase component of the same polypeptide chain. This contrasts with the domain swapping observed in the previously determined structure of the BVDV NADL strain RdRp. By comparison with the NADL structure and through the use of biochemical data, it is possible that the N-terminal domain, in conjunction with the fingertips, is required to bind and assist the translocation of the RNA template. The partial disorder of the loop containing the additional Asn438 residue may explain the low replication rate of the recombinant compared with the wild-type virus.

Flexible Fitting in 3D-EM Guided by the Structural Variability of Protein Superfamilies by Javier-?ngel Velazquez-Muriel; Mikel Valle; Alberto Santamaría-Pang; Ioannis A. Kakadiaris; José-María Carazo (pp. 1115-1126).
A method for flexible fitting of molecular models into three-dimensional electron microscopy (3D-EM) reconstructions at a resolution range of 8–12 Å is proposed. The approach uses the evolutionarily related structural variability existing among the protein domains of a given superfamily, according to structural databases such as CATH. A structural alignment of domains belonging to the superfamily, followed by a principal components analysis, is performed, and the first three principal components of the decomposition are explored. Using rigid body transformations for the secondary structure elements (SSEs) plus the cyclic coordinate descent algorithm to close the loops, stereochemically correct models are built for the structure to fit. All of the models are fitted into the 3D-EM map, and the best one is selected based on crosscorrelation measures. This work applies the method to both simulated and experimental data and shows that the flexible fitting was able to produce better results than rigid body fitting.

Domain-Swapped Structure of the Potent Antiviral Protein Griffithsin and Its Mode of Carbohydrate Binding by Natasza E. Ziółkowska; Barry R. O'Keefe; Toshiyuki Mori; Charles Zhu; Barbara Giomarelli; Fakhrieh Vojdani; Kenneth E. Palmer; James B. McMahon; Alexander Wlodawer (pp. 1127-1135).
The crystal structure of griffithsin, an antiviral lectin from the red alga Griffithsia sp., was solved and refined at 1.3 Å resolution for the free protein and 0.94 Å for a complex with mannose. Griffithsin molecules form a domain-swapped dimer, in which two β strands of one molecule complete a β prism consisting of three four-stranded sheets, with an approximate 3-fold axis, of another molecule. The structure of each monomer bears close resemblance to jacalin-related lectins, but its dimeric structure is unique. The structures of complexes of griffithsin with mannose and N-acetylglucosamine defined the locations of three almost identical carbohydrate binding sites on each monomer. We have also shown that griffithsin is a potent inhibitor of the coronavirus responsible for severe acute respiratory syndrome (SARS). Antiviral potency of griffithsin is likely due to the presence of multiple, similar sugar binding sites that provide redundant attachment points for complex carbohydrate molecules present on viral envelopes.

De Novo Tubular Nanostructure Design Based on Self-Assembly of β-Helical Protein Motifs by Nurit Haspel; David Zanuy; Carlos Alemán; Haim Wolfson; Ruth Nussinov (pp. 1137-1148).
We present an approach for designing self-assembled nanostructures from naturally occurring building block segments obtained from native protein structures. We focus on structural motifs from left-handed β-helical proteins. We selected 17 motifs. Copies of each of the motifs are stacked one atop the other. The obtained structures were simulated for long periods by using Molecular Dynamics to test their ability to retain their organization over time. We observed that a structural model based on the self-assembly of a motif from E. coli galactoside acetyltransferase produced a very stable tube. We studied the interactions that help maintain the conformational stability of the systems, focusing on the role of specific amino acids at specific positions. Analysis of these systems and a mutational study of selected candidates revealed that the presence of proline and glycine residues in the loops of β-helical structures greatly enhances the structural stability of the systems.

An Expanded and Flexible Form of the Vacuolar ATPase Membrane Sector by Daniel K. Clare; Elena V. Orlova; Malcolm A. Finbow; Michael A. Harrison; John B.C. Findlay; Helen R. Saibil (pp. 1149-1156).
The vacuolar ATPase integral membrane c-ring from Nephrops norvegicus occurs in paired complexes in a double membrane. Using cryo-electron microscopy and single particle image processing of 2D crystals, we have obtained a projection structure of the c-ring of N. norvegicus. The c-ring was found to be very flexible, most likely as a result of an expanded conformation of the c subunits. This structure may support a role for the vacuolar ATPase c-rings in membrane fusion.

The Crystal Structure of ORF-9b, a Lipid Binding Protein from the SARS Coronavirus by Christoph Meier; A. Radu Aricescu; Rene Assenberg; Robin T. Aplin; Robert J.C. Gilbert; Jonathan M. Grimes; David I. Stuart (pp. 1157-1165).
To achieve the greatest output from their limited genomes, viruses frequently make use of alternative open reading frames, in which translation is initiated from a start codon within an existing gene and, being out of frame, gives rise to a distinct protein product. These alternative protein products are, as yet, poorly characterized structurally. Here we report the crystal structure of ORF-9b, an alternative open reading frame within the nucleocapsid (N) gene from the SARS coronavirus. The protein has a novel fold, a dimeric tent-like β structure with an amphipathic surface, and a central hydrophobic cavity that binds lipid molecules. This cavity is likely to be involved in membrane attachment and, in mammalian cells, ORF-9b associates with intracellular vesicles, consistent with a role in the assembly of the virion. Analysis of ORF-9b and other overlapping genes suggests that they provide snapshots of the early evolution of novel protein folds.

Lumbricus Erythrocruorin at 3.5 Å Resolution: Architecture of a Megadalton Respiratory Complex by William E. Royer Jr.; Hitesh Sharma; Kristen Strand; James E. Knapp; Balaji Bhyravbhatla (pp. 1167-1177).
Annelid erythrocruorins are highly cooperative extracellular respiratory proteins with molecular masses on the order of 3.6 million Daltons. We report here the 3.5 Å crystal structure of erythrocruorin from the earthworm Lumbricus terrestris. This structure reveals details of symmetrical and quasi-symmetrical interactions that dictate the self-limited assembly of 144 hemoglobin and 36 linker subunits. The linker subunits assemble into a core complex with D6 symmetry onto which 12 hemoglobin dodecamers bind to form the entire complex. Although the three unique linker subunits share structural similarity, their interactions with each other and the hemoglobin subunits display striking diversity. The observed diversity includes design features that have been incorporated into the linker subunits and may be critical for efficient assembly of large quantities of this complex respiratory protein.

Proteasome Assembly Triggers a Switch Required for Active-Site Maturation by Susanne Witt; Young Do Kwon; Michal Sharon; Karin Felderer; Mirjam Beuttler; Carol V. Robinson; Wolfgang Baumeister; Bing K. Jap (pp. 1179-1188).
The processing of propeptides and the maturation of 20S proteasomes require the association of β rings from two half proteasomes. We propose an assembly-dependent activation model in which interactions between helix (H3 and H4) residues of the opposing half proteasomes are prerequisite for appropriate positioning of the S2-S3 loop; such positioning enables correct coordination of the active-site residue needed for propeptide cleavage. Mutations of H3 or H4 residues that participate in the association of two half proteasomes inhibit activation and prevent, in nearly all cases, the formation of full proteasomes. In contrast, mutations affecting interactions with residues of the S2-S3 loop allow the assembly of full, but activity impacted, proteasomes. The crystal structure of the inactive H3 mutant, Phe145Ala, shows that the S2-S3 loop is displaced from the position observed in wild-type proteasomes. These data support the proposed assembly-dependent activation model in which the S2-S3 loop acts as an activation switch.

Structural Polymorphism in Bacterial EspA Filaments Revealed by Cryo-EM and an Improved Approach to Helical Reconstruction by Ying A. Wang; Xiong Yu; Calvin Yip; Natalie C. Strynadka; Edward H. Egelman (pp. 1189-1196).
The traditional Fourier-Bessel approach to three-dimensional reconstruction from electron microscopic (EM) images of helical polymers involves averaging over filaments, assuming a homogeneous structure and symmetry. We have used a real-space reconstruction approach to study the EspA filaments formed by enteropathogenic E. coli. In negative stain, the symmetry of these filaments is ambiguous, and we suggest that such ambiguities may be more prevalent than realized. Using cryo-EM of frozen-hydrated filaments, we find that these filaments have a fixed twist with 5.6 subunits per turn but an axial rise per subunit that varies from about 3.6 Å to 5.6 Å. Reconstructions at ∼15 Å resolution show a switching between the more compressed and extended filaments in the packing of putative α helices around the hollow lumen. Outside of a crystal, where there is nothing to maintain long-range order, the structural polymorphism in helical polymers may be much greater than has been assumed.

Multiple Distinct Assemblies Reveal Conformational Flexibility in the Small Heat Shock Protein Hsp26 by Helen E. White; Elena V. Orlova; Shaoxia Chen; Luchun Wang; Athanasios Ignatiou; Brent Gowen; Thusnelda Stromer; Titus M. Franzmann; Martin Haslbeck; Johannes Buchner; Helen R. Saibil (pp. 1197-1204).
Small heat shock proteins are a superfamily of molecular chaperones that suppress protein aggregation and provide protection from cell stress. A key issue for understanding their action is to define the interactions of subunit domains in these oligomeric assemblies. Cryo-electron microscopy of yeast Hsp26 reveals two distinct forms, each comprising 24 subunits arranged in a porous shell with tetrahedral symmetry. The subunits form elongated, asymmetric dimers that assemble via trimeric contacts. Modifications of both termini cause rearrangements that yield a further four assemblies. Each subunit contains an N-terminal region, a globular middle domain, the α-crystallin domain, and a C-terminal tail. Twelve of the C termini form 3-fold assembly contacts which are inserted into the interior of the shell, while the other 12 C termini form contacts on the surface. Hinge points between the domains allow a variety of assembly contacts, providing the flexibility required for formation of supercomplexes with nonnative proteins.
Flexibility and Conformational Entropy in Protein-Protein Binding by Raik Grünberg; Michael Nilges; Johan Leckner (pp. 1205-1205).
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