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Structure (v.14, #9)


A DNA-Centric Look at Protein-DNA Complexes by Taner Z. Sen; Andrzej Kloczkowski; Robert L. Jernigan (pp. 1341-1342).
The availability of many DNA-protein structures makes their classification timely and important. In this issue of Structure, the method of Akinori Sarai and his collaborators () utilizes aspects of the binding interactions and DNA properties to identify seven clusters of structures with a classification scheme that differs significantly from previous approaches.

A New GNAT in Bacterial Signaling? by Mair E.A. Churchill (pp. 1342-1344).
Acyl-amino acids were discovered in the search for novel therapeutic agents produced by uncultured microorganisms. In this issue of Structure, find that the N-acyl-amino acid synthase FeeM bound to acyl-tyrosine is the latest member of the GCN5-related N-acyltransferase superfamily.

PcrA Helicase, a Prototype ATP-Driven Molecular Motor by Markus Dittrich; Klaus Schulten (pp. 1345-1353).
Despite extensive studies, the mechanisms underlying molecular motor function are still poorly understood. Key to the mechanisms is the coupling of ATP hydrolysis to conformational changes of the motor protein. To investigate this coupling, we have conducted combined quantum mechanical/molecular mechanical simulations of PcrA helicase, a strikingly simple motor that translocates unidirectionally along single-stranded DNA (ssDNA). Our results reveal a close similarity in catalytic site structure and reaction pathway to those of F1-ATPase, and these similarities include a proton relay mechanism important for efficient ATP hydrolysis and an “arginine finger? residue that is key to the coupling of the chemical reaction to protein conformational changes. By means of in silico mutation studies, we identified the residue Q254 as being crucial for the coupling of ssDNA translocation to the actual catalytic event. Based on the present result for PcrA helicase and previous findings for F1-ATPase, we propose a general mechanism of ATP-driven molecular motor function.

Classification of Protein-DNA Complexes Based on Structural Descriptors by Ponraj Prabakaran; Jörg G. Siebers; Shandar Ahmad; M. Michael Gromiha; Maria G. Singarayan; Akinori Sarai (pp. 1355-1367).
We attempt to classify protein-DNA complexes by using a set of 11 descriptors, mainly characterizing protein-DNA interactions, including the number of atomic contacts at major and minor grooves, conformational deviations from standard B- and A-DNA forms, widths of DNA grooves, GC content, specificity measures of direct and indirect readouts, and buried surface area at the complex interface. The cluster analyses were carried out for a unique set of 62 complexes including a variety of protein motifs, and 7 distinct clusters were revealed from the analyses. We found that some proteins with the same motif are classified into different clusters, whereas different proteins with distinct motifs are classified into the same cluster. These results suggest that the conventional motif-based classification of DNA binding proteins may not necessarily correspond to structural and functional properties of protein-DNA complexes, and that the present classification will help to identify common properties and rules that govern protein-DNA recognition.

Amyloid Formation May Involve α- to β Sheet Interconversion via Peptide Plane Flipping by James E. Milner-White; James D. Watson; Guoying Qi; Steven Hayward (pp. 1369-1376).
The toxic component of amyloid is not the mature fiber but a soluble prefibrillar intermediate. It has been proposed, from molecular dynamics simulations, that the precursor is composed of α sheet, which converts into the β sheet of mature amyloid via peptide plane flipping. α sheet, not seen in proteins, occurs as isolated stretches of polypeptide. We show that the α- to β sheet transition can occur by the flipping of alternate peptide planes. The flip can be described asαRαL↔ββ. A search conducted within sets of closely related protein crystal structures revealed that these flips are common, occurring in 8.5% of protein families. The average “αL? conformation found is in an adjacent and less populated region of the Ramachandran plot, as expected if the flanking peptide planes, being hydrogen bonded, are restricted in their movements. This work provides evidence for flips allowing direct α- to β sheet interconversion.

Crystal Structure of Ethylbenzene Dehydrogenase from Aromatoleum aromaticum by Daniel P. Kloer; Corina Hagel; Johann Heider; Georg E. Schulz (pp. 1377-1388).
Anaerobic degradation of hydrocarbons was discovered a decade ago, and ethylbenzene dehydrogenase was one of the first characterized enzymes involved. The structure of the soluble periplasmic 165 kDa enzyme was established at 1.88 Å resolution. It is a heterotrimer. The α subunit contains the catalytic center with a molybdenum held by two molybdopterin-guanine dinucleotides, one with an open pyran ring, and an iron-sulfur cluster with a histidine ligand. During catalysis, electrons produced by substrate oxidation are transferred to a heme in the γ subunit and then presumably to a separate cytochrome involved in nitrate respiration. The β subunit contains four iron-sulfur clusters and is structurally related to ferredoxins. The γ subunit is the first known protein with a methionine and a lysine as axial heme ligands. The catalytic product was modeled into the active center, showing the reaction geometry. A mechanism consistent with activity and inhibition data of ethylbenzene-related compounds is proposed.

The Crystal Structure of the Rare-Cutting Restriction Enzyme SdaI Reveals Unexpected Domain Architecture by Giedre Tamulaitiene; Arturas Jakubauskas; Claus Urbanke; Robert Huber; Saulius Grazulis; Virginijus Siksnys (pp. 1389-1400).
Rare-cutting restriction enzymes are important tools in genome analysis. We report here the crystal structure of SdaI restriction endonuclease, which is specific for the 8 bp sequence CCTGCA/GG (“/? designates the cleavage site). Unlike orthodox Type IIP enzymes, which are single domain proteins, the SdaI monomer is composed of two structural domains. The N domain contains a classical winged helix-turn-helix (wHTH) DNA binding motif, while the C domain shows a typical restriction endonuclease fold. The active site of SdaI is located within the C domain and represents a variant of the canonical PD-(D/E)XK motif. SdaI determinants of sequence specificity are clustered on the recognition helix of the wHTH motif at the N domain. The modular architecture of SdaI, wherein one domain mediates DNA binding while the other domain is predicted to catalyze hydrolysis, distinguishes SdaI from previously characterized restriction enzymes interacting with symmetric recognition sequences.

Correlation between Protein Stability Cores and Protein Folding Kinetics: A Case Study on Pseudomonas aeruginosa Apo-Azurin by Mingzhi Chen; Corey J. Wilson; Yinghao Wu; Pernilla Wittung-Stafshede; Jianpeng Ma (pp. 1401-1410).
This paper reports a combined computational and experimental study of the correlation between protein stability cores and folding kinetics. An empirical potential function was developed, and it was used for analyzing interaction energies among secondary structure elements. Studies on a β sandwich protein, Pseudomonas aeruginosa azurin, showed that the computationally identified substructure with the strongest interactions in the native state is identical to the “interlocked pair? of β strands, an invariant motif found in most sandwich-like proteins. Moreover, previous and new in vitro folding results revealed that the identified substructure harbors most residues that form native-like interactions in the folding transition state. These observations demonstrate that the potential function is effective in revealing the relative strength of interactions among various protein parts; they also strengthen the suggestion that the most stable regions in native proteins favor stable interactions early during folding.

Mechanism of Gating and Ion Conductivity of a Possible Tetrameric Pore in Aquaporin-1 by Jin Yu; Andrea J. Yool; Klaus Schulten; Emad Tajkhorshid (pp. 1411-1423).
While substrate permeation through monomeric pores of aquaporins is well characterized, little is known about the possible tetrameric pore. AQP1 has been suggested to function as an ion channel upon cGMP activation, although this idea has been controversial. Taking a theoretical and experimental approach, we demonstrate that the current might arise through the tetrameric pore and propose a plausible mechanism for conduction and gating. In response to simulated ion permeation, immediate hydration of the putative central pore was facilitated by moderate conformational changes of pore-lining residues. cGMP is found to interact with an unusually arginine-rich, cytoplasmic loop (loop D) facilitating its outward motion, which is hypothesized to trigger the opening of a cytoplasmic gate. Physiological analyses of wild-type AQP1 and a designed mutant in which two arginines of the gating loop are replaced by alanine provide experimental support for identifying a key component of the proposed mechanism.

FeeM, an N-Acyl Amino Acid Synthase from an Uncultured Soil Microbe: Structure, Mechanism, and Acyl Carrier Protein Binding by Ryan M. Van Wagoner; Jon Clardy (pp. 1425-1435).
Attempts to access antibiotics by capturing biosynthetic genes and pathways directly from environmental DNA, which is overwhelmingly derived from uncultured bacteria, have revealed a large and previously unknown family of N-acyl amino acid synthases (NASs). The structure of the NAS FeeM reveals structural similarity to the GCN5-related N-acyl transferases and acylhomoserine lactone synthases. The overall structure has a central β sheet with α helices on both sides. A bound product at a cleft in the β sheet identifies the active site and the structural basis for catalysis, and sequence conservation in this region indicates a bias for recognition over speed. FeeM interacts with an acyl carrier protein (FeeL), and the structure, mutagenesis, and enzymatic measurements reveal that a small hydrophobic pocket in α helix 5 dominates binding of FeeM to FeeL. The structural and mechanistic analyses suggest that the products of FeeM could be bacterial signaling agents.

The Ig Doublet Z1Z2: A Model System for the Hybrid Analysis of Conformational Dynamics in Ig Tandems from Titin by Marco Marino; Peijian Zou; Dmitri Svergun; Pilar Garcia; Christian Edlich; Bernd Simon; Matthias Wilmanns; Claudia Muhle-Goll; Olga Mayans (pp. 1437-1447).
Titin is a gigantic elastic filament that determines sarcomere ultrastructure and stretch response in vertebrate muscle. It folds into numerous Ig and FnIII domains connected in tandem. Data on interdomain arrangements and dynamics are essential for understanding the function of this filament. Here, we report a mechanistic analysis of the conformational dynamics of two Ig domains from the N terminus of titin, Z1Z2, by using X-ray crystallography, SAXS, NMR relaxation data, and residual dipolar couplings in combination. Z1Z2 preferentially adopts semiextended conformations in solution, with close-hinge arrangements representing low-probability states. Although interdomain contacts are not observed, the linker appears to acquire moderate rigidity via small, local hydrophobic interactions. Thus, Z1Z2 constitutes an adaptable modular system with restricted dynamics. We speculate that its preexistent conformation contributes to the selective recruitment of the binding partner telethonin onto the repetitive surface of the filament. The structural interconversion of four Z1Z2 conformers is analyzed.

The Crystal Structure of the Venezuelan Equine Encephalitis Alphavirus nsP2 Protease by Andrew T. Russo; Mark A. White; Stanley J. Watowich (pp. 1449-1458).
Alphavirus replication and propagation is dependent on the protease activity of the viral nsP2 protein, which cleaves the nsP1234 polyprotein replication complex into functional components. Thus, nsP2 is an attractive target for drug discovery efforts to combat highly pathogenic alphaviruses. Unfortunately, antiviral development has been hampered by a lack of structural information for the nsP2 protease. Here, we report the crystal structure of the nsP2 protease (nsP2pro) from Venezuelan equine encephalitis alphavirus determined at 2.45 Å resolution. The protease structure consists of two distinct domains. The nsP2pro N-terminal domain contains the catalytic dyad cysteine and histidine residues organized in a protein fold that differs significantly from any known cysteine protease or protein folds. The nsP2pro C-terminal domain displays structural similarity to S-adenosyl-L-methionine-dependent RNA methyltransferases and provides essential elements that contribute to substrate recognition and may also regulate the structure of the substrate binding cleft.

Crystal Structures of the Thi-Box Riboswitch Bound to Thiamine Pyrophosphate Analogs Reveal Adaptive RNA-Small Molecule Recognition by Thomas E. Edwards; Adrian R. Ferré-D'Amaré (pp. 1459-1468).
Riboswitches are noncoding mRNA elements that bind small-molecule metabolites with high affinity and specificity, and they regulate the expression of associated genes. The thi-box riboswitch can exhibit a 1000-fold higher affinity for thiamine pyrophosphate over closely related noncognate compounds such as thiamine monophosphate. To understand the chemical basis of thi-box pyrophosphate specificity, we have determined crystal structures of an E. coli thi-box bound to thiamine pyrophosphate, thiamine monophosphate, and the structural analogs benfotiamine and pyrithiamine. When bound to monophosphorylated compounds, the RNA elements that recognize the thiamine and phosphate moieties of the ligand move closer together. This allows the riboswitch to recognize the monophosphate in a manner similar to how it recognizes the β-phosphate of thiamine pyrophosphate. In the pyrithiamine complex, the pyrophosphate binding site is largely unstructured. These results show how the riboswitch can bind to various metabolites, and why the thi-box preferentially binds thiamine pyrophosphate.

A Highly Strained Nuclear Conformation of the Exportin Cse1p Revealed by Molecular Dynamics Simulations by Ulrich Zachariae; Helmut Grubmüller (pp. 1469-1478).
To investigate the stability of the open nuclear state of the exportin Cse1p and its closing mechanism at the atomic level, we have performed multiple molecular dynamics simulations. The simulations revealed a strikingly fast transition of Cse1p from the open conformation to the closed cytoplasmic form, consistent with the proposal that Cse1p represents a “spring-loaded molecule.? The structure of the ring-shaped state obtained in the simulations is remarkably close to the crystal structure of the cytoplasmic state, though the open nuclear structure was used as the only input. The conformational change is initially driven by release of strain due to RanGTP/importin-α binding. Subsequently, a stable closed state is formed, driven by attraction of electrostatically complementary interfaces. These results are consistent with and extend previous proposals. Reverse-charge and neutral mutants remained in an open state. The simulations predict a detailed reaction pathway and resolve the role of suggested hinge regions.
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