Structure (v.17, #11)

In This Issue (ix-x).

TFIID: A Closer Look Highlights Its Complexity by Francisco J. Asturias (1423-1424).
A shrewd cryo-EM study of the TFIID complex in this issue of Structure (Elmlund et al., 2009) has generated a markedly improved understanding of its structure and conformational dynamics. Accurate localization of TBP and other critical components, and a new understanding of TFIID interaction with promoter DNA answer some significant questions and pose interesting new ones.

Divergence of the Flagellar Hook and Filament by Edward H. Egelman (1425-1426).
Advances in electron cryo-microscopic imaging and three-dimensional helical reconstruction have allowed for the rapid structure determination of the bacterial flagellar hook by Fujii et al. (2009). The resulting model helps explain the unusual mechanical properties of the hook.

Watching CO Enmeshed in Hemoglobin by Dominique Bourgeois (1427-1428).
In this issue of Structure, Knapp et al. show that allosteric changes in dimeric hemoglobin are retarded in the crystal, using time-resolved Laue diffraction. They observe that photo-dissociated carbon monoxide, after exploring a web of cavities, rebinds to the heme before it has a chance to exit the protein.

Through Ancient Rings Thread Programming Strings by Martyn F. Symmons; Ben F. Luisi (1429-1431).
A new crystal structure of assembled subunits from the eukaryotic exosome complex gives insight into the interactions underpinning its various functions (Bonneau et al., 2009). Here, we focus on what the emerging structures tell us about the regulation of the exosome interactions with, and actions on, RNA.

Optically Resolving Individual Microtubules in Live Axons by Harsha V. Mudrakola; Kai Zhang; Bianxiao Cui (1433-1441).
Microtubules are essential cytoskeletal tracks for cargo transportation in axons and also serve as the primary structural scaffold of neurons. Structural assembly, stability, and dynamics of axonal microtubules are of great interest for understanding neuronal functions and pathologies. However, microtubules are so densely packed in axons that their separations are well below the diffraction limit of light, which precludes using optical microscopy for live-cell studies. Here, we present a single-molecule imaging method capable of resolving individual microtubules in live axons. In our method, unlabeled microtubules are revealed by following individual axonal cargos that travel along them. We resolved more than six microtubules in a 1 μm diameter axon by real-time tracking of endosomes containing quantum dots. Our live-cell study also provided direct evidence that endosomes switch between microtubules while traveling along axons, which has been proposed to be the primary means for axonal cargos to effectively navigate through the crowded axoplasmic environment.

Cryo-EM Reveals Promoter DNA Binding and Conformational Flexibility of the General Transcription Factor TFIID by Hans Elmlund; Vera Baraznenok; Tomas Linder; Zsolt Szilagyi; Reza Rofougaran; Anders Hofer; Hans Hebert; Martin Lindahl; Claes M. Gustafsson (1442-1452).
The general transcription factor IID (TFIID) is required for initiation of RNA polymerase II-dependent transcription at many eukaryotic promoters. TFIID comprises the TATA-binding protein (TBP) and several conserved TBP-associated factors (TAFs). Recognition of the core promoter by TFIID assists assembly of the preinitiation complex. Using cryo-electron microscopy in combination with methods for ab initio single-particle reconstruction and heterogeneity analysis, we have produced density maps of two conformational states of Schizosaccharomyces pombe TFIID, containing and lacking TBP. We report that TBP-binding is coupled to a massive histone-fold domain rearrangement. Moreover, docking of the TBP-TAF1N-terminus atomic structure to the TFIID map and reconstruction of a TAF-promoter DNA complex helps to account for TAF-dependent regulation of promoter-TBP and promoter-TAF interactions.
Keywords: PROTEINS; DNA;

Regulation of the Protein-Conducting Channel by a Bound Ribosome by James Gumbart; Leonardo G. Trabuco; Eduard Schreiner; Elizabeth Villa; Klaus Schulten (1453-1464).
During protein synthesis, it is often necessary for the ribosome to form a complex with a membrane-bound channel, the SecY/Sec61 complex, in order to translocate nascent proteins across a cellular membrane. Structural data on the ribosome-channel complex are currently limited to low-resolution cryo-electron microscopy maps, including one showing a bacterial ribosome bound to a monomeric SecY complex. Using that map along with available atomic-level models of the ribosome and SecY, we have determined, through molecular dynamics flexible fitting (MDFF), an atomic-resolution model of the ribosome-channel complex. We characterized computationally the sites of ribosome-SecY interaction within the complex and determined the effect of ribosome binding on the SecY channel. We also constructed a model of a ribosome in complex with a SecY dimer by adding a second copy of SecY to the MDFF-derived model. The study involved 2.7-million-atom simulations over altogether nearly 50 ns.

Crystal and Solution Structures of a Prokaryotic M16B Peptidase: an Open and Shut Case by Alexander E. Aleshin; Svetlana Gramatikova; Gregory L. Hura; Andrey Bobkov; Alex Y. Strongin; Boguslaw Stec; John A. Tainer; Robert C. Liddington; Jeffrey W. Smith (1465-1475).
The M16 family of zinc peptidases comprises a pair of homologous domains that form two halves of a “clam-shell” surrounding the active site. The M16A and M16C subfamilies form one class (“peptidasomes”): they degrade 30–70 residue peptides, and adopt both open and closed conformations. The eukaryotic M16B subfamily forms a second class (“processing proteases”): they adopt a single partly-open conformation that enables them to cleave signal sequences from larger proteins. Here, we report the solution and crystal structures of a prokaryotic M16B peptidase, and demonstrate that it has features of both classes: thus, it forms stable “open” homodimers in solution that resemble the processing proteases; but the clam-shell closes upon binding substrate, a feature of the M16A/C peptidasomes. Moreover, clam-shell closure is required for proteolytic activity. We predict that other prokaryotic M16B family members will form dimeric peptidasomes, and propose a model for the evolution of the M16 family.
Keywords: PROTEINS;

A Structural Explanation for the Antithrombotic Activity of ARC1172, a DNA Aptamer that Binds von Willebrand Factor Domain A1 by Ren-Huai Huang; Daved H. Fremont; John L. Diener; Robert G. Schaub; J. Evan Sadler (1476-1484).
ARC1172 is a 41-mer DNA aptamer selected to bind the A1 domain of von Willebrand factor (VWF). A derivative of ARC1172 with modifications to increase intravascular survival inhibits carotid artery thrombosis in a Cynomolgus macaque model and inhibits VWF-dependent platelet aggregation in humans, suggesting that such aptamers may be useful to prevent or treat thrombosis. In the crystal structure of a VWF A1-ARC1172 complex, the aptamer adopts a three-stem structure of mainly B-form DNA with three noncanonical base pairs and 9 unpaired residues, 6 of which are stabilized by base-base or base-deoxyribose stacking interactions. The aptamer-protein interface is characterized by cation-π interactions involving Arg, Lys, and Gln residues, often stabilized by H-bonds with adjacent bases. The ARC1172 binding site on the A1 domain overlaps with that of botrocetin and clashes with glycoprotein Ibα binding at an adjacent site, which accounts for the antithrombotic activity of ARC1172 and related aptamers.
Keywords: DNA;

The bacterial flagellar hook is a short, highly curved tubular structure connecting the rotary motor to the filament acting as a helical propeller. The bending flexibility of the hook allows it to work as a universal joint. A partial atomic model of the hook revealed a sliding intersubunit domain interaction along the protofilament to produce bending flexibility. However, it remained unclear how the tightly packed inner core domains can still permit axial extension and compression. We report advances in cryoEM image analysis for high-resolution, high-throughput structural analysis and a density map of the hook that reveals most of the secondary structures, including the terminal α helices forming a coiled coil. The orientations and axial packing interactions of these two α helices are distinctly different from those of the filament, allowing them to have a room for axial compression and extension for bending flexibility without impairing the mechanical stability of the hook.

Ligand Migration and Cavities within Scapharca Dimeric HbI: Studies by Time-Resolved Crystallo- graphy, Xe Binding, and Computational Analysis by James E. Knapp; Reinhard Pahl; Jordi Cohen; Jeffry C. Nichols; Klaus Schulten; Quentin H. Gibson; Vukica Šrajer; William E. Royer (1494-1504).
As in many other hemoglobins, no direct route for migration of ligands between solvent and active site is evident from crystal structures of Scapharca inaequivalvis dimeric HbI. Xenon (Xe) and organic halide binding experiments, along with computational analysis presented here, reveal protein cavities as potential ligand migration routes. Time-resolved crystallographic experiments show that photodissociated carbon monoxide (CO) docks within 5 ns at the distal pocket B site and at more remote Xe4 and Xe2 cavities. CO rebinding is not affected by the presence of dichloroethane within the major Xe4 protein cavity, demonstrating that this cavity is not on the major exit pathway. The crystal lattice has a substantial influence on ligand migration, suggesting that significant conformational rearrangements may be required for ligand exit. Taken together, these results are consistent with a distal histidine gate as one important ligand entry and exit route, despite its participation in the dimeric interface.
Keywords: PROTEINS;

Ryanodine receptors (RyRs) are channels governing the release of Ca2+ from the sarcoplasmic or endoplasmic reticulum. They are required for the contraction of both skeletal (RyR1) and cardiac (RyR2) muscles. Mutations in both RyR1 and RyR2 have been associated with severe genetic disorders, but high-resolution data describing the disease variants in detail have been lacking. Here we present the crystal structures of the N-terminal domains of both RyR2 (1–217) and RyR1 (9–205) at 2.55 Å and 2.9 Å, respectively. The domains map in a hot spot region for disease mutations. Both structures consist of a core beta trefoil domain flanked by an alpha helix. Crystal structures of two RyR2 disease mutants, A77V (2.2 Å) and V186M (1.7 Å), show that the mutations cause distinct local changes in the surface of the protein. A RyR2 deletion mutant causes significant changes in the thermal stability. The disease positions highlight two putative binding interfaces required for normal RyR function.

Local structures predicted from protein sequences are used extensively in every aspect of modeling and prediction of protein structure and function. For more than 50 years, they have been predicted at a low-resolution coarse-grained level (e.g., three-state secondary structure). Here, we combine a two-state classifier with real-value predictor to predict local structure in continuous representation by backbone torsion angles. The accuracy of the angles predicted by this approach is close to that derived from NMR chemical shifts. Their substitution for predicted secondary structure as restraints for ab initio structure prediction doubles the success rate. This result demonstrates the potential of predicted local structure for fragment-free tertiary-structure prediction. It further implies potentially significant benefits from using predicted real-valued torsion angles as a replacement for or supplement to the secondary-structure prediction tools used almost exclusively in many computational methods ranging from sequence alignment to function prediction.
Keywords: PROTEINS;

Structural Basis for Targeting of Human RNA Helicase DDX3 by Poxvirus Protein K7 by Shun-ichiro Oda; Martina Schröder; Amir R. Khan (1528-1537).
Poxviruses are DNA viruses that express numerous proteins to subvert the host immune response. Vaccinia virus protein K7 adopts a Bcl-2 fold and displays structural and functional similarities to Toll-like receptor antagonist A52. Both proteins interact with IRAK2 and TRAF6 and suppress TLR-dependent NF-κB activation. However, unlike A52, K7 also forms a complex with RNA helicase DDX3 and antagonizes interferon-β promoter induction. We have narrowed the K7 binding site to an N-terminal peptide motif of DDX3 ahead of its core RNA-helicase domains. The crystal structure of full-length K7 in complex with the DDX3 peptide reveals a thumblike projection of tandem phenalyalanine residues of DDX3 into a deep hydrophobic cleft. Mutagenesis of these phenylalanines abolishes the effects of DDX3 on interferon-β promoter induction. The structure of K7-DDX3 reveals a novel binding mode by a viral Bcl-2 protein that antagonizes a key pathway in innate immunity.

Structural Basis of the Cross-Reactivity of Genetically Related Human Anti-HIV-1 mAbs: Implications for Design of V3-Based Immunogens by Valicia Burke; Constance Williams; Madhav Sukumaran; Seung-Sup Kim; Huiguang Li; Xiao-Hong Wang; Miroslaw K. Gorny; Susan Zolla-Pazner; Xiang-Peng Kong (1538-1546).
Human monoclonal antibodies 447-52D and 537-10D, both coded by the VH3 gene and specific for the third variable region (V3) of the HIV-1 gp120, were found to share antigen-binding structural elements including an elongated CDR H3 forming main-chain interactions with the N terminus of the V3 crown. However, water-mediated hydrogen bonds and a unique cation-π sandwich stacking allow 447-52D to be broadly reactive with V3 containing both the GPGR and GPGQ crown motifs, while the deeper binding pocket and a buried Glu in the binding site of 537-10D limit its reactivity to only V3 containing the GPGR motif. Our results suggest that the design of immunogens for anti-V3 antibodies should avoid the Arg at the V3 crown, as GPGR-containing epitopes appear to select for B cells making antibodies of narrower specificity than V3 that carry Gln at this position.