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Structure (v.15, #3)
Secretion Superfamily ATPases Swing Big by Savvas N. Savvides (pp. 255-257).
In this issue of Structure, present crystallographic snapshots of the bacterial type IV pilus retraction motor PilT and propose a general model for pilus retraction consistent with a growing consensus that secretion superfamily ATPases are dynamic hexameric assemblies.
The THI-box Riboswitch, or How RNA Binds Thiamin Pyrophosphate by Juan Miranda-Ríos (pp. 259-265).
Riboswitches are genetic control elements present mainly in the 5′ untranslated regions of messenger RNAs that, upon binding of a small metabolite (like some vitamins, amino acids, and nucleobases), undergo conformational changes, affecting the expression of downstream genes. Structural studies of riboswitches are important for understanding how they recognize their ligands with high specificity and affinity. The thiamin pyrophosphate binding riboswitch (THI- box) is widely distributed in the three kingdoms of life and is involved in very distinct modes of gene regulation. Three recent THI-box structural analyses revealed how polyanionic RNA is able to bind a molecule with a negatively charged pyrophosphate group like thiamin pyrophosphate (TPP) and how it can discriminate between TPP and monophosphorylated analog molecules. These studies give insight into the genetic regulatory mechanisms in which the THI-box is involved.
Glycoprotein Structural Genomics: Solving the Glycosylation Problem by Veronica T. Chang; Max Crispin; A. Radu Aricescu; David J. Harvey; Joanne E. Nettleship; Janet A. Fennelly; Chao Yu; Kent S. Boles; Edward J. Evans; David I. Stuart; Raymond A. Dwek; E. Yvonne Jones; Raymond J. Owens; Simon J. Davis (pp. 267-273).
Glycoproteins present special problems for structural genomic analysis because they often require glycosylation in order to fold correctly, whereas their chemical and conformational heterogeneity generally inhibits crystallization. We show that the “glycosylation problem” can be solved by expressing glycoproteins transiently in mammalian cells in the presence of the N-glycosylation processing inhibitors, kifunensine or swainsonine. This allows the correct folding of the glycoproteins, but leaves them sensitive to enzymes, such as endoglycosidase H, that reduce the N-glycans to single residues, enhancing crystallization. Since the scalability of transient mammalian expression is now comparable to that of bacterial systems, this approach should relieve one of the major bottlenecks in structural genomic analysis.
Multiprotein Expression Strategy for Structural Biology of Eukaryotic Complexes by Daniel J. Fitzgerald; Christiane Schaffitzel; Philipp Berger; Ralf Wellinger; Christoph Bieniossek; Timothy J. Richmond; Imre Berger (pp. 275-279).
The concept of the cell as a collection of multisubunit protein machines is emerging as a cornerstone of modern biology, and molecular-level study of these machines in most cases will require recombinant production. Here, we present and validate a strategy to rapidly produce, permutate, and posttranslationally modify large, eukaryotic multiprotein complexes by using DNA recombination in a process that is fully automatable. Parallel production of 12 protein complex variants within a period of weeks resulted in specimens of sufficient quantity and homogeneity for structural biology applications.
Structural Roles of Monovalent Cations in the HDV Ribozyme by Ailong Ke; Fang Ding; Joseph D. Batchelor; Jennifer A. Doudna (pp. 281-287).
The hepatitis delta virus (HDV) ribozyme catalyzes viral RNA self-cleavage through general acid-base chemistry in which an active-site cytidine and at least one metal ion are involved. Monovalent metal ions support slow catalysis and were proposed to substitute for structural, but not catalytic, divalent metal ions in the RNA. To investigate the role of monovalent cations in ribozyme structure and function, we determined the crystal structure of the precursor HDV ribozyme in the presence of thallium ions (Tl+). Two Tl+ ions can occupy a previously observed divalent metal ion hexahydrate-binding site located near the scissile phosphate, but are easily competed away by cobalt hexammine, a magnesium hexahydrate mimic and potent reaction inhibitor. Intriguingly, a third Tl+ ion forms direct inner-sphere contacts with the ribose 2′-OH nucleophile and the pro-Sp scissile phosphate oxygen. We discuss possible structural and catalytic implications of monovalent cation binding for the HDV ribozyme mechanism.
Keywords: RNA; MICROBIO
A Snapshot of the 30S Ribosomal Subunit Capturing mRNA via the Shine-Dalgarno Interaction by Tatsuya Kaminishi; Daniel N. Wilson; Chie Takemoto; Joerg M. Harms; Masahito Kawazoe; Frank Schluenzen; Kyoko Hanawa-Suetsugu; Mikako Shirouzu; Paola Fucini; Shigeyuki Yokoyama (pp. 289-297).
In the initiation phase of bacterial translation, the 30S ribosomal subunit captures mRNA in preparation for binding with initiator tRNA. The purine-rich Shine-Dalgarno (SD) sequence, in the 5′ untranslated region of the mRNA, anchors the 30S subunit near the start codon, via base pairing with an anti-SD (aSD) sequence at the 3′ terminus of 16S rRNA. Here, we present the 3.3 Å crystal structure of the Thermus thermophilus 30S subunit bound with an mRNA mimic. The duplex formed by the SD and aSD sequences is snugly docked in a “chamber” between the head and platform domains, demonstrating how the 30S subunit captures and stabilizes the otherwise labile SD helix. This location of the SD helix is suitable for the placement of the start codon AUG in the immediate vicinity of the mRNA channel, in agreement with reported crosslinks between the second position of the start codon and G1530 of 16S rRNA.
c-Src Binds to the Cancer Drug Imatinib with an Inactive Abl/c-Kit Conformation and a Distributed Thermodynamic Penalty by Markus A. Seeliger; Bhushan Nagar; Filipp Frank; Xiaoxian Cao; M. Nidanie Henderson; John Kuriyan (pp. 299-311).
The cancer drug imatinib inhibits the tyrosine kinases c-Abl, c-Kit, and the PDGF receptor. Imatinib is less effective against c-Src, which is difficult to understand because residues interacting with imatinib in crystal structures of Abl and c-Kit are conserved in c-Src. The crystal structure of the c-Src kinase domain in complex with imatinib closely resembles that of Abl•imatinib and c-Kit•imatinib, and differs significantly from the inactive “Src/CDK” conformation of the Src family kinases. Attempts to increase the affinity of c-Src for imatinib by swapping residues with the corresponding residues in Abl have not been successful, suggesting that the thermodynamic penalty for adoption of the imatinib-binding conformation by c-Src is distributed over a broad region of the structure. Two mutations that are expected to destabilize the inactive Src/CDK conformation increase drug sensitivity 15-fold, suggesting that the free-energy balance between different inactive states is a key to imatinib binding.
Keywords: SIGNALING; CELLCYCLE
Substrate Recognition Reduces Side-Chain Flexibility for Conserved Hydrophobic Residues in Human Pin1 by Andrew T. Namanja; Tao Peng; John S. Zintsmaster; Andrew C. Elson; Maria G. Shakour; Jeffrey W. Peng (pp. 313-327).
Pin1 is a peptidyl-prolyl isomerase consisting of a WW domain and a catalytic isomerase (PPIase) domain connected by a flexible linker. Pin1 recognizes phospho-Ser/Thr-Pro motifs in cell-signaling proteins, and is both a cancer and an Alzheimer's disease target. Here, we provide novel insight into the functional motions underlying Pin1 substrate interaction using nuclear magnetic resonance deuterium (2D) and carbon (13C) spin relaxation. Specifically, we compare Pin1 side-chain motions in the presence and absence of a known phosphopeptide substrate derived from the mitotic phosphatase Cdc25. Substrate interaction alters Pin1 side-chain motions on both the microsecond-millisecond (μs-ms) and picosecond-nanosecond (ps-ns) timescales. Alterations include loss of ps-ns flexibility along an internal conduit of hydrophobic residues connecting the catalytic site with the interdomain interface. These residues are conserved among Pin1 homologs; hence, their dynamics are likely important for the Pin1 mechanism.
Modular Structure of the Full-Length DNA Gyrase B Subunit Revealed by Small-Angle X-Ray Scattering by Lionel Costenaro; J. Günter Grossmann; Christine Ebel; Anthony Maxwell (pp. 329-339).
DNA gyrase, the only topoisomerase able to introduce negative supercoils into DNA, is essential for bacterial transcription and replication; absent from humans, it is a successful target for antibacterials. From biophysical experiments in solution, we report a structural model at ∼12–15 Å resolution of the full-length B subunit (GyrB). Analytical ultracentrifugation shows that GyrB is mainly a nonglobular monomer. Ab initio modeling of small-angle X-ray scattering data for GyrB consistently yields a “tadpole”-like envelope. It allows us to propose an organization of GyrB into three domains—ATPase, Toprim, and Tail—based on their crystallographic and modeled structures. Our study reveals the modular organization of GyrB and points out its potential flexibility, needed during the gyrase catalytic cycle. It provides important insights into the supercoiling mechanism by gyrase and suggests new lines of research.
Keywords: MICROBIO; DNA
Similar Binding Sites and Different Partners: Implications to Shared Proteins in Cellular Pathways by Ozlem Keskin; Ruth Nussinov (pp. 341-354).
We studied a data set of structurally similar interfaces that bind to proteins with different binding-site structures and different functions. Our multipartner protein interface clusters enable us to address questions like: What makes a given site bind different proteins? How similar/different are the interactions? And, what drives the apparently less-specific association? We find that proteins with common binding-site motifs preferentially use conserved interactions at similar interface locations, despite the different partners. Helices are major vehicles for binding different partners, allowing alternate ways to achieve favorable association. The binding sites are characterized by imperfect packing, planar architectures, bridging water molecules, and, on average, smaller size. Interestingly, analysis of the connectivity of these proteins illustrates that they have more interactions with other proteins. These findings are important in predicting “date hubs,” if we assume that “date hubs” are shared proteins with binding sites capable of transient binding to multipartners, linking higher-order networks.
Crystal Structure of a TOG Domain: Conserved Features of XMAP215/Dis1-Family TOG Domains and Implications for Tubulin Binding by Jawdat Al-Bassam; Nicholas A. Larsen; Anthony A. Hyman; Stephen C. Harrison (pp. 355-362).
Members of the XMAP215/Dis1 family of microtubule-associated proteins (MAPs) are essential for microtubule growth. MAPs in this family contain several 250 residue repeats, called TOG domains, which are thought to bind tubulin dimers and promote microtubule polymerization. We have determined the crystal structure of a single TOG domain from the Caenorhabditis elegans homolog, Zyg9, to 1.9 Å resolution, and from it we describe a structural blueprint for TOG domains. These domains are flat, paddle-like structures, composed of six HEAT-repeat elements stacked side by side. The two wide faces of the paddle contain the HEAT-repeat helices, and the two narrow faces, the intra- and inter-HEAT repeat turns. Solvent-exposed residues in the intrarepeat turns are conserved, both within a particular protein and across the XMAP215/Dis1 family. Mutation of some of these residues in the TOG1 domain from the budding yeast homolog, Stu2p, shows that this face indeed participates in the tubulin contact.
Crystal Structures of the Pilus Retraction Motor PilT Suggest Large Domain Movements and Subunit Cooperation Drive Motility by Kenneth A. Satyshur; Gregory A. Worzalla; Lorraine S. Meyer; Erin K. Heiniger; Kelly G. Aukema; Ana M. Misic; Katrina T. Forest (pp. 363-376).
PilT is a hexameric ATPase required for bacterial type IV pilus retraction and surface motility. Crystal structures of ADP- and ATP-bound Aquifex aeolicus PilT at 2.8 and 3.2 Å resolution show N-terminal PAS-like and C-terminal RecA-like ATPase domains followed by a set of short C-terminal helices. The hexamer is formed by extensive polar subunit interactions between the ATPase core of one monomer and the N-terminal domain of the next. An additional structure captures a nonsymmetric PilT hexamer in which approach of invariant arginines from two subunits to the bound nucleotide forms an enzymatically competent active site. A panel of pilT mutations highlights the importance of the arginines, the PAS-like domain, the polar subunit interface, and the C-terminal helices for retraction. We present a model for ATP binding leading to dramatic PilT domain motions, engagement of the arginine wire, and subunit communication in this hexameric motor. Our conclusions apply to the entire type II/IV secretion ATPase family.
Keywords: CELLBIO; MICROBIO
Structural Basis of Inhibition of the Human NAD+-Dependent Deacetylase SIRT5 by Suramin by Anja Schuetz; Jinrong Min; Tatiana Antoshenko; Chia-Lin Wang; Abdellah Allali-Hassani; Aiping Dong; Peter Loppnau; Masoud Vedadi; Alexey Bochkarev; Rolf Sternglanz; Alexander N. Plotnikov (pp. 377-389).
Sirtuins are NAD+-dependent protein deacetylases and are emerging as molecular targets for the development of pharmaceuticals to treat human metabolic and neurological diseases and cancer. To date, several sirtuin inhibitors and activators have been identified, but the structural mechanisms of how these compounds modulate sirtuin activity have not yet been determined. We identified suramin as a compound that binds to human SIRT5 and showed that it inhibits SIRT5 NAD+-dependent deacetylase activity with an IC50 value of 22 μM. To provide insights into how sirtuin function is altered by inhibitors, we determined two crystal structures of SIRT5, one in complex with ADP-ribose, the other bound to suramin. Our structural studies provide a view of a synthetic inhibitory compound in a sirtuin active site revealing that suramin binds into the NAD+, the product, and the substrate-binding site. Finally, our structures may enable the rational design of more potent inhibitors.