Structure (v.16, #8)
Single-Molecule Tour de Force: Teasing Apart a Signaling Complex
by Wouter D. Hoff; John L. Spudich (pp. 1149-1150).
The phototaxis receptor sensory rhodopsin II communicates with its transducer in a membrane-embedded complex. The first application of single-molecule force spectroscopy to receptor-transducer interaction by reveals new structural features in the signaling complex.
Heads Up S-Layer Display: The Power of Many
by John Smit (pp. 1151-1153).
Many microbes produce surface protein layers (S-layers) that are organized as geometric arrays. These S-layers offer unique applications in nanotechnology, but are a challenge for atomic structure analysis. have made inroads, however, with an S-layer of Geobacillus stearothermophilus.
Exchange We Can Believe in
by Wayne A. Hendrickson; Qinglian Liu (pp. 1153-1155).
Newly published papers by Polier et al. in Cell and by Schuermann et al. in Molecular Cell present structures of Hsp110:Hsp70 complexes that reveal a compelling picture for the mechanism of nucleotide exchange in Hsp70s of the eukaryotic cytosol.
Synchrotron Protein Footprinting Supports Substrate Translocation by ClpA via ATP-Induced Movements of the D2 Loop
by Jen Bohon; Laura D. Jennings; Christine M. Phillips; Stuart Licht; Mark R. Chance (pp. 1157-1165).
Synchrotron X-ray protein footprinting is used to study structural changes upon formation of the ClpA hexamer. Comparative solvent accessibilities between ClpA monomer and ClpA hexamer samples are in agreement throughout most of the sequence, with calculations based on two previously proposed hexameric models. The data differ substantially from the proposed models in two parts of the structure: the D1 sensor 1 domain and the D2 loop region. The results suggest that these two regions can access alternate conformations in which their solvent protection is greater than that in the structural models based on crystallographic data. In combination with previously reported structural data, the footprinting data provide support for a revised model in which the D2 loop contacts the D1 sensor 1 domain in the ATP-bound form of the complex. These data provide the first direct experimental support for the nucleotide-dependent D2 loop conformational change previously proposed to mediate substrate translocation.
Structure of the E. coli DNA Glycosylase AlkA Bound to the Ends of Duplex DNA: A System for the Structure Determination of Lesion-Containing DNA
by Brian R. Bowman; Seongmin Lee; Shuyu Wang; Gregory L. Verdine (pp. 1166-1174).
The constant attack on DNA by endogenous and exogenous agents gives rise to nucleobase modifications that cause mutations, which can lead to cancer. Visualizing the effects of these lesions on the structure of duplex DNA is key to understanding their biologic consequences. The most definitive method of obtaining such structures, X-ray crystallography, is troublesome to employ owing to the difficulty of obtaining diffraction-quality crystals of DNA. Here, we present a crystallization system that uses a protein, the DNA glycosylase AlkA, as a scaffold to mediate the crystallization of lesion-containing duplex DNA. We demonstrate the use of this system to facilitate the rapid structure determination of DNA containing the lesion 8-oxoguanine in several different sequence contexts, and also deoxyinosine and 1,N6-ethenoadenine, each stabilized as the corresponding 2′-flouro analog. The structures of 8-oxoguanine provide a correct atomic-level view of this important endogenous lesion in DNA.
Keywords: DNA; PROTEIN
The Atomistic Mechanism of Conformational Transition in Adenylate Kinase: A TEE-REX Molecular Dynamics Study
by Marcus B. Kubitzki; Bert L. de Groot (pp. 1175-1182).
We report on an atomistic molecular dynamics simulation of the complete conformational transition of Escherichia coli adenylate kinase (ADK) using the recently developed TEE-REX algorithm. Two phases characterize the transition pathway of ADK, which folds into the domains CORE and LID and the AMP binding domain AMPbd. Starting from the closed conformation, half-opening of the AMPbd precedes a partially correlated opening of the LID and AMPbd, defining the second phase. A highly stable salt bridge D118-K136 at the LID-CORE interface, contributing substantially to the total nonbonded LID-CORE interactions, was identified as a major factor that stabilizes the open conformation. Alternative transition pathways, such as AMPbd opening following LID opening, seem unlikely, as full transition events were not observed along this pathway. The simulation data indicate a high enthalpic penalty, possibly obstructing transitions along this route.
Solution and Crystal Structures of a Sugar Binding Site Mutant of Cyanovirin-N: No Evidence of Domain Swapping
by Elena Matei; William Furey; Angela M. Gronenborn (pp. 1183-1194).
The cyanobacterial lectin Cyanovirin-N (CV-N) exhibits antiviral activity against HIV at a low nanomolar concentration by interacting with high-mannose oligosaccharides on the virus surface envelope glycoprotein gp120. Atomic structures of wild-type CV-N revealed a monomer in solution and a domain-swapped dimer in the crystal, with the monomer comprising two independent carbohydrate binding sites that individually bind with micromolar affinity to di- and trimannoses. In the mutant CVNmutDB, the binding site on domain B was abolished and the protein was found to be completely inactive against HIV. We determined the solution NMR and crystal structures of this variant and characterized its sugar binding properties. In solution and the crystal, CVNmutDB is a monomer and no domain-swapping was observed. The protein binds to Man-3 and Man-9 with similar dissociation constants (∼4 μM). This confirms that the nanomolar activity of wild-type CV-N is related to the multisite nature of the protein carbohydrate interaction.
Dynamic Properties of a Type II Cadherin Adhesive Domain: Implications for the Mechanism of Strand-Swapping of Classical Cadherins
by Vesselin Z. Miloushev; Fabiana Bahna; Carlo Ciatto; Goran Ahlsen; Barry Honig; Lawrence Shapiro; Arthur G. Palmer III (pp. 1195-1205).
Cadherin-mediated cell adhesion is achieved through dimerization of cadherin N-terminal extracellular (EC1) domains presented from apposed cells. The dimer state is formed by exchange of N-terminal β strands and insertion of conserved tryptophan indole side chains from one monomer into hydrophobic acceptor pockets of the partner molecule. The present work characterizes individual monomer and dimer states and the monomer-dimer equilibrium of the mouse Type II cadherin-8 EC1 domain using NMR spectroscopy. Limited picosecond-to-nanosecond timescale dynamics of the tryptophan indole moieties for both monomer and dimer states are consistent with well-ordered packing of the N-terminal β strands intramolecularly and intermolecularly, respectively. However, pronounced microsecond-to-millisecond timescale dynamics of the side chains are observed for the monomer but not the dimer state, suggesting that monomers transiently sample configurations in which the indole moieties are exposed. The results suggest possible kinetic mechanisms for EC1 dimerization.
Transducer Binding Establishes Localized Interactions to Tune Sensory Rhodopsin II
by David A. Cisneros; Leoni Oberbarnscheidt; Angela Pannier; Johann P. Klare; Jonne Helenius; Martin Engelhard; Filipp Oesterhelt; Daniel J. Muller (pp. 1206-1213).
In haloarchaea, sensory rhodopsin II (SRII) mediates a photophobic response to avoid photo-oxidative damage in bright light. Upon light activation the receptor undergoes a conformational change that activates a tightly bound transducer molecule (HtrII), which in turn by a chain of homologous reactions transmits the signal to the chemotactic eubacterial two-component system. Here, using single-molecule force spectroscopy, we localize and quantify changes to the intramolecular interactions within SRII of Natronomonas pharaonis (NpSRII) upon NpHtrII binding. Transducer binding affected the interactions at transmembrane α helices F and G of NpSRII to which the transducer was in contact. Remarkably, the interactions were distributed asymmetrically and significantly stabilized α helix G entirely but α helix F only at its extracellular tip. These findings provide unique insights into molecular mechanisms that “prime” the complex for signaling, and guide the receptor toward transmitting light-activated structural changes to its cognate transducer.
Keywords: CELLBIO; SIGNALING
The Human Cytomegalovirus UL44 C Clamp Wraps around DNA
by Gloria Komazin-Meredith; Robert J. Petrella; Webster L. Santos; David J. Filman; James M. Hogle; Gregory L. Verdine; Martin Karplus; Donald M. Coen (pp. 1214-1225).
Processivity factors tether the catalytic subunits of DNA polymerases to DNA so that continuous synthesis of long DNA strands is possible. The human cytomegalovirus DNA polymerase subunit UL44 forms a C clamp-shaped dimer intermediate in structure between monomeric herpes simplex virus UL42, which binds DNA directly via a basic surface, and the trimeric sliding clamp PCNA, which encircles DNA. To investigate how UL44 interacts with DNA, calculations were performed in which a 12 bp DNA oligonucleotide was docked to UL44. The calculations suggested that UL44 encircles DNA, which interacts with basic residues both within the cavity of the C clamp and in flexible loops of UL44 that complete the “circle.” The results of mutational and crosslinking studies were consistent with this model. Thus, UL44 is a “hybrid” of UL42 and PCNA: its structure is intermediate between the two and its mode of interaction with DNA has elements of both.
The Structure and Binding Behavior of the Bacterial Cell Surface Layer Protein SbsC
by Tea Pavkov; Eva M. Egelseer; Manfred Tesarz; Dmitri I. Svergun; Uwe B. Sleytr; Walter Keller (pp. 1226-1237).
Surface layers (S-layers) comprise the outermost cell envelope component of most archaea and many bacteria. Here we present the structure of the bacterial S-layer protein SbsC from Geobacillus stearothermophilus, showing a very elongated and flexible molecule, with strong and specific binding to the secondary cell wall polymer (SCWP). The crystal structure of rSbsC(31–844) revealed a novel fold, consisting of six separate domains, which are connected by short flexible linkers. The N-terminal domain exhibits positively charged residues regularly spaced along the putative ligand binding site matching the distance of the negative charges on the extended SCWP. Upon SCWP binding, a considerable stabilization of the N-terminal domain occurs. These findings provide insight into the processes of S-layer attachment to the underlying cell wall and self-assembly, and also accommodate the observed mechanical strength, the polarity of the S-layer, and the pronounced requirement for surface flexibility inherent to cell growth and division.
The Crystal Structure of the Escherichia coli RNase E Apoprotein and a Mechanism for RNA Degradation
by Daniel J. Koslover; Anastasia J. Callaghan; Maria J. Marcaida; Elspeth F. Garman; Monika Martick; William G. Scott; Ben F. Luisi (pp. 1238-1244).
RNase E is an essential bacterial endoribonuclease involved in the turnover of messenger RNA and the maturation of structured RNA precursors in Escherichia coli. Here, we present the crystal structure of the E. coli RNase E catalytic domain in the apo-state at 3.3 Å. This structure indicates that, upon catalytic activation, RNase E undergoes a marked conformational change characterized by the coupled movement of two RNA-binding domains to organize the active site. The structural data suggest a mechanism of RNA recognition and cleavage that explains the enzyme's preference for substrates possessing a 5′-monophosphate and accounts for the protective effect of a triphosphate cap for most transcripts. Internal flexibility within the quaternary structure is also observed, a finding that has implications for recognition of structured RNA substrates and for the mechanism of internal entry for a subset of substrates that are cleaved without 5′-end requirements.
Keywords: RNA; PROTEIN
Structural Insight into the Recognition of the H3K4me3 Mark by the TFIID Subunit TAF3
by Hugo van Ingen; Frederik M.A. van Schaik; Hans Wienk; Joost Ballering; Holger Rehmann; Annemarie C. Dechesne; John A.W. Kruijzer; Rob M.J. Liskamp; H.Th. Marc Timmers; Rolf Boelens (pp. 1245-1256).
Trimethylation of lysine residue K4 of histone H3 (H3K4me3) strongly correlates with active promoters for RNA polymerase II-transcribed genes. Several reader proteins, including the basal transcription factor TFIID, for this nucleosomal mark have been identified. Its TAF3 subunit specifically binds the H3K4me3 mark via its conserved plant homeodomain (PHD) finger. Here, we report the solution structure of the TAF3-PHD finger and its complex with an H3K4me3 peptide. Using a combination of NMR, mutagenesis, and affinity measurements, we reveal the structural basis of binding affinity, methylation-state specificity, and crosstalk with asymmetric dimethylation of R2. A unique local structure rearrangement in the K4me3-binding pocket of TAF3 due to a conserved sequence insertion underscores the requirement for cation-π interactions by two aromatic residues. Interference by asymmetric dimethylation of arginine 2 suggests that a H3R2/K4 “methyl-methyl” switch in the histone code dynamically regulates TFIID-promoter association.
Keywords: DNA; PROTEIN
Iterative Assembly of Helical Proteins by Optimal Hydrophobic Packing
by G. Albert Wu; Evangelos A. Coutsias; Ken A. Dill (pp. 1257-1266).
We present a method for the computer-based iterative assembly of native-like tertiary structures of helical proteins from α-helical fragments. For any pair of helices, our method, called MATCHSTIX, first generates an ensemble of possible relative orientations of the helices with various ways to form hydrophobic contacts between them. Those conformations having steric clashes, or a large radius of gyration of hydrophobic residues, or with helices too far separated to be connected by the intervening linking region, are discarded. Then, we attempt to connect the two helical fragments by using a robotics-based loop-closure algorithm. When loop closure is feasible, the algorithm generates an ensemble of viable interconnecting loops. After energy minimization and clustering, we use a representative set of conformations for further assembly with the remaining helices, adding one helix at a time. To efficiently sample the conformational space, the order of assembly generally proceeds from the pair of helices connected by the shortest loop, followed by joining one of its adjacent helices, always proceeding with the shorter connecting loop. We tested MATCHSTIX on 28 helical proteins each containing up to 5 helices and found it to heavily sample native-like conformations. The average rmsd of the best conformations for the 17 helix-bundle proteins that have 2 or 3 helices is less than 2 Å; errors increase somewhat for proteins containing more helices. Native-like states are even more densely sampled when disulfide bonds are known and imposed as restraints. We conclude that, at least for helical proteins, if the secondary structures are known, this rapid rigid-body maximization of hydrophobic interactions can lead to small ensembles of highly native-like structures. It may be useful for protein structure prediction.
Defining Molecular and Domain Boundaries in the Bacteriophage ϕ29 DNA Packaging Motor
by Marc C. Morais; Jaya S. Koti; Valorie D. Bowman; Emilio Reyes-Aldrete; Dwight L. Anderson; Michael G. Rossmann (pp. 1267-1274).
Cryo-electron microscopy (cryo-EM) studies of the bacteriophage ϕ29 DNA packaging motor have delineated the relative positions and molecular boundaries of the 12-fold symmetric head-tail connector, the 5-fold symmetric prohead RNA (pRNA), the ATPase that provides the energy for packaging, and the procapsid. Reconstructions, assuming 5-fold symmetry, were determined for proheads with 174-base, 120-base, and 71-base pRNA; proheads lacking pRNA; proheads with ATPase bound; and proheads in which the packaging motor was missing the connector. These structures are consistent with pRNA and ATPase forming a pentameric motor component around the unique vertex of proheads. They suggest an assembly pathway for the packaging motor and a mechanism for DNA translocation into empty proheads.
Keywords: PROTEIN; MICROBIO
Tetrameric Structure of a Serine Integrase Catalytic Domain
by Peng Yuan; Kushol Gupta; Gregory D. Van Duyne (pp. 1275-1286).
The serine integrases have recently emerged as powerful new chromosome engineering tools in various organisms and show promise for therapeutic use in human cells. The serine integrases are structurally and mechanistically unrelated to the bacteriophage λ integrase but share a similar catalytic domain with the resolvase/invertase enzymes typified by the resolvase proteins from transposons Tn3 and γδ. Here we report the crystal structure and solution properties of the catalytic domain from bacteriophage TP901-1 integrase. The protein is a dimer in solution but crystallizes as a tetramer that is closely related in overall architecture to structures of activated γδ-resolvase mutants. The ability of the integrase tetramer to explain biochemical experiments performed in the resolvase and invertase systems suggests that the TP901 integrase tetramer represents a unique intermediate on the recombination pathway that is shared within the serine recombinase superfamily.
Keywords: PROTEINS; CELLBIO
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