Institut de Biologie Structurale

MOTIVATION: The program ESPript (Easy Sequencing in PostScript) allows the rapid visualization, via PostScript output, of sequences aligned with popular programs such as CLUSTAL-W or GCG PILEUP. It can read secondary structure files (such as that created by the program DSSP) to produce a synthesis of both sequence and structural information. RESULTS: ESPript can be run via a command file or a friendly html-based user interface. The program calculates an homology score by columns of residues and can sort this calculation by groups of sequences. It offers a palette of markers to highlight important regions in the alignment. ESPript can also paste information on residue conservation into coordinate files, for subsequent visualization with a graphics program. AVAILABILITY: ESPript can be accessed on its Web site at http://www.ipbs.fr/ESPript. Sources and helpfiles can be downloaded via anonymous ftp from ftp.ipbs.fr. A tar file is held in the directory pub/ESPript.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTADDITION / CORRECTIONThis article has been corrected. View the notice.Structure/Function Relationships of [NiFe]- and [FeFe]-HydrogenasesJuan C. Fontecilla-Camps, Anne Volbeda, Christine Cavazza, and Yvain NicoletView Author Information Laboratoire de Cristallographie et Cristallogenèse des Proteines, Institut de Biologie Structurale J. P. Ebel, CEA, CNRS, Universitè Joseph Fourier, 41 rue J. Horowitz, 38027 Grenoble Cedex 1, France Cite this: Chem. Rev. 2007, 107, 10, 4273–4303Publication Date (Web):September 13, 2007Publication History Received26 March 2007Published online13 September 2007Published inissue 1 October 2007https://pubs.acs.org/doi/10.1021/cr050195zhttps://doi.org/10.1021/cr050195zresearch-articleACS PublicationsCopyright © 2007 American Chemical SocietyRequest reuse permissionsArticle Views11789Altmetric-Citations1185LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose SUBJECTS:Cluster chemistry,Ligands,Monomers,Peptides and proteins,Redox reactions Get e-Alerts

Structural analysis of flexible macromolecular systems such as intrinsically disordered or multidomain proteins with flexible linkers is a difficult task as high-resolution techniques are barely applicable. A new approach, ensemble optimization method (EOM), is proposed to quantitatively characterize flexible proteins in solution using small-angle X-ray scattering (SAXS). The flexibility is taken into account by allowing for the coexistence of different conformations of the protein contributing to the experimental scattering pattern. These conformers are selected using a genetic algorithm from a pool containing a large number of randomly generated models covering the protein configurational space. Quantitative criteria are developed to analyze the EOM selected models and to determine the optimum number of conformers in the ensemble. Simultaneous fitting of multiple scattering patterns from deletion mutants, if available, provides yet more detailed local information about the structure. The efficiency of EOM is demonstrated in model and practical examples on completely or partially unfolded proteins and on multidomain proteins interconnected by linkers. In the latter case, EOM is able to distinguish between rigid and flexible proteins and to directly assess the interdomain contacts.

We recently reported the discovery and preliminary characterization of Mimivirus, the largest known virus, with a 400-nanometer particle size comparable to mycoplasma. Mimivirus is a double-stranded DNA virus growing in amoebae. We now present its 1,181,404-base pair genome sequence, consisting of 1262 putative open reading frames, 10% of which exhibit a similarity to proteins of known functions. In addition to exceptional genome size, Mimivirus exhibits many features that distinguish it from other nucleocytoplasmic large DNA viruses. The most unexpected is the presence of numerous genes encoding central protein-translation components, including four amino-acyl transfer RNA synthetases, peptide release factor 1, translation elongation factor EF-TU, and translation initiation factor 1. The genome also exhibits six tRNAs. Other notable features include the presence of both type I and type II topoisomerases, components of all DNA repair pathways, many polysaccharide synthesis enzymes, and one intein-containing gene. The size and complexity of the Mimivirus genome challenge the established frontier between viruses and parasitic cellular organisms. This new sequence data might help shed a new light on the origin of DNA viruses and their role in the early evolution of eukaryotes.

Colicins are proteins produced by and toxic for some strains of Escherichia coli. They are produced by strains of E. coli carrying a colicinogenic plasmid that bears the genetic determinants for colicin synthesis, immunity, and release. Insights gained into each fundamental aspect of their biology are presented: their synthesis, which is under SOS regulation; their release into the extracellular medium, which involves the colicin lysis protein; and their uptake mechanisms and modes of action. Colicins are organized into three domains, each one involved in a different step of the process of killing sensitive bacteria. The structures of some colicins are known at the atomic level and are discussed. Colicins exert their lethal action by first binding to specific receptors, which are outer membrane proteins used for the entry of specific nutrients. They are then translocated through the outer membrane and transit through the periplasm by either the Tol or the TonB system. The components of each system are known, and their implication in the functioning of the system is described. Colicins then reach their lethal target and act either by forming a voltage-dependent channel into the inner membrane or by using their endonuclease activity on DNA, rRNA, or tRNA. The mechanisms of inhibition by specific and cognate immunity proteins are presented. Finally, the use of colicins as laboratory or biotechnological tools and their mode of evolution are discussed.

The structure of the protein-solvent interface is the subject of controversy in theoretical studies and requires direct experimental characterization. Three proteins with known atomic resolution crystal structure (lysozyme, Escherichia coli thioredoxin reductase, and protein R1 of E. coli ribonucleotide reductase) were investigated in parallel by x-ray and neutron scattering in H2O and D2O solutions. The analysis of the protein-solvent interface is based on the significantly different contrasts for the protein and for the hydration shell. The results point to the existence of a first hydration shell with an average density approximately 10% larger than that of the bulk solvent in the conditions studied. Comparisons with the results of other studies suggest that this may be a general property of aqueous interfaces.

Lipidic cubic phases provide a continuous three-dimensional bilayer matrix that facilitates nucleation and growth of bacteriorhodopsin microcrystals. The crystals diffract x-rays isotropically to 2.0 angstroms. The structure of this light-driven proton pump was solved at a resolution of 2.5 angstroms by molecular replacement, using previous results from electron crystallographic studies as a model. The earlier structure was generally confirmed, but several differences were found, including loop conformations and side chain residues. Eight water molecules are now identified experimentally in the proton pathway. These findings reveal the constituents of the proton translocation pathway in the ground state.

Cryptochromes are flavoprotein photoreceptors first identified in Arabidopsis thaliana, where they play key roles in growth and development. Subsequently identified in prokaryotes, archaea, and many eukaryotes, cryptochromes function in the animal circadian clock and are proposed as magnetoreceptors in migratory birds. Cryptochromes are closely structurally related to photolyases, evolutionarily ancient flavoproteins that catalyze light-dependent DNA repair. Here, we review the structural, photochemical, and molecular properties of cry-DASH, plant, and animal cryptochromes in relation to biological signaling mechanisms and uncover common features that may contribute to better understanding the function of cryptochromes in diverse systems including in man.

Cholinesterases are among the most efficient enzymes known. They are divided into two groups: acetylcholinesterase, involved in the hydrolysis of the neurotransmitter acetylcholine, and butyrylcholinesterase of unknown function. Several crystal structures of the former have shown that the active site is located at the bottom of a deep and narrow gorge, raising the question of how substrate and products enter and leave. Human butyrylcholinesterase (BChE) has attracted attention because it can hydrolyze toxic esters such as cocaine or scavenge organophosphorus pesticides and nerve agents. Here we report the crystal structures of several recombinant truncated human BChE complexes and conjugates and provide a description for mechanistically relevant non-productive substrate and product binding. As expected, the structure of BChE is similar to a previously published theoretical model of this enzyme and to the structure of Torpedo acetylcholinesterase. The main difference between the experimentally determined BChE structure and its model is found at the acyl binding pocket that is significantly bigger than expected. An electron density peak close to the catalytic Ser198 has been modeled as bound butyrate. Cholinesterases are among the most efficient enzymes known. They are divided into two groups: acetylcholinesterase, involved in the hydrolysis of the neurotransmitter acetylcholine, and butyrylcholinesterase of unknown function. Several crystal structures of the former have shown that the active site is located at the bottom of a deep and narrow gorge, raising the question of how substrate and products enter and leave. Human butyrylcholinesterase (BChE) has attracted attention because it can hydrolyze toxic esters such as cocaine or scavenge organophosphorus pesticides and nerve agents. Here we report the crystal structures of several recombinant truncated human BChE complexes and conjugates and provide a description for mechanistically relevant non-productive substrate and product binding. As expected, the structure of BChE is similar to a previously published theoretical model of this enzyme and to the structure of Torpedo acetylcholinesterase. The main difference between the experimentally determined BChE structure and its model is found at the acyl binding pocket that is significantly bigger than expected. An electron density peak close to the catalytic Ser198 has been modeled as bound butyrate. Cholinesterases are divided into two subfamilies according to their substrate and inhibitor specificities: acetylcholinesterase (AChE 1The abbreviations used are: AchE, acetylcholinesterase; BchE, butyrylcholinesterase; TcAChE, Torpedo californica acetylcholinesterase; DmAChE, Drosophila melanogaster acetylcholinesterase; BTC, butyrylthiocholine; Bicine, N,N-bis(2-hydroxyethyl)glycine; MES, 4-morpholineethanesulfonic acid.; EC 3.1.1.7) and butyrylcholinesterase (BChE; EC 3.1.1.8). Acetylcholinesterase is responsible for the hydrolysis of acetylcholine released at the synaptic cleft and the neuromuscular junction in response to nerve action potential (1Massoulie J. Sussman J. Bon S. Silman I. Prog. Brain Res. 1993; 98: 139-146Crossref PubMed Scopus (125) Google Scholar). In addition, both AChE and BChE seem to be involved in roles that are independent of their catalytic activities, such as cell differentiation and development (2Meshorer E. Erb C. Gazit R. Pavlovsky L. Kaufer D. Friedman A. Glick D. Ben-Arie N. Soreq H. Science. 2002; 295: 508-512Crossref PubMed Scopus (205) Google Scholar, 3Behra M. Cousin X. Bertrand C. Vonesch J.L. Biellmann D. Chatonnet A. Strahle U. Nat. Neurosci. 2002; 5: 111-118Crossref PubMed Scopus (307) Google Scholar). The catalytic mechanism of AChE is extremely efficient approaching diffusion-controlled rates (4Quinn D. Chem. Rev. 1987; 87: 955-979Crossref Scopus (946) Google Scholar). Unexpectedly, the crystal structure of the Torpedo californica enzyme (TcAChE) showed that the active site catalytic Ser-His-Glu triad is found at the bottom of a 20-Å deep gorge lined mostly with aromatic residues (5Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2426) Google Scholar). The structure also revealed the nature and the location of the previously described peripheral and “anionic” sites; the former, located at the outer rim of the gorge, has been postulated to be the initial substrate binding site (6Szegletes T. Mallender W.D. Thomas P.J. Rosenberry T.L. Biochemistry. 1999; 38: 122-133Crossref PubMed Scopus (152) Google Scholar). The binding of ligand to this site has been proposed to slow down the traffic of substrate and product at the acylation site (6Szegletes T. Mallender W.D. Thomas P.J. Rosenberry T.L. Biochemistry. 1999; 38: 122-133Crossref PubMed Scopus (152) Google Scholar, 7De Ferrari G.V. Mallender W.D. Inestrosa N.C. Rosenberry T.L. J. Biol. Chem. 2001; 276: 23282-23287Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Although a similar peripheral site has been described for human BChE, site-directed mutagenesis and photo-affinity labeling studies showed that its location and the response upon ligand binding differ significantly from those of AChE (8Nachon F. Ehret-Sabatier L. Loew D. Colas C. van Dorsselaer A. Goeldner M. Biochemistry. 1998; 37: 10507-10513Crossref PubMed Scopus (52) Google Scholar, 9Masson P. Legrand P. Bartels C.F. Froment M.T. Schopfer L.M. Lockridge O. Biochemistry. 1997; 36: 2266-2277Crossref PubMed Scopus (133) Google Scholar). The site to which the positively charged quaternary ammonium of choline moiety productively binds is found half-way down the gorge, in between the peripheral and acylation sites. Originally, there was a great deal of controversy concerning the nature of the residues involved in this site. Both the crystal structure and labeling experiments showed that positively charged ligands form π-cation interactions with Phe 330 and Trp 84 (numbering in italics corresponds to that of torpedo AChE) (10Harel M. Schalk I. Ehret-Sabatier L. Bouet F. Goeldner M. Hirth C. Axelsen P.H. Silman I. Sussman J.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9031-9035Crossref PubMed Scopus (845) Google Scholar). The physiological role of BChE remains unclair (11Chatonnet A. Lockridge O. Biochem. J. 1989; 260: 625-634Crossref PubMed Scopus (472) Google Scholar, 12Mack A. Robitzki A. Prog. Neurobiol. 2000; 60: 607-628Crossref PubMed Scopus (122) Google Scholar). Although it is capable of hydrolyzing ACh and other acylcholines, so far no endogenous natural substrate has been described for this enzyme. Because BChE is relatively abundant in plasma (about 3 mg/liter), and can degrade a large number of ester-containing compounds, it plays important pharmacological and toxicological roles (13Lockridge O. Masson P. Neurotoxicology. 2000; 21: 113-126PubMed Google Scholar). For instance, BChE is a potential detoxifying enzyme to be used as a prophylactic scavenger against neurotoxic organophosphates such as the nerve gas soman (14Allon N. Raveh L. Gilat E. Cohen E. Grunwald J. Ashani Y. Toxicol. Sci. 1998; 43: 121-128PubMed Google Scholar, 15Broomfield C.A. Maxwell D.M. Solana R.P. Castro C.A. Finger A.V. Lenz D.E. J. Pharmacol. Exp. Ther. 1991; 259: 633-638PubMed Google Scholar, 16Raveh L. Grunwald J. Marcus D. Papier Y. Cohen E. Ashani Y. Biochem. Pharmacol. 1993; 45: 2465-2474Crossref PubMed Scopus (173) Google Scholar). We have recently published the engineering and crystallization of a monomeric and partially glycosylated recombinant human BchE (17Nachon F. Nicolet Y. Viguie N. Masson P. Fontecilla-Camps J.C. Lockridge O. Eur. J. Biochem. 2002; 269: 630-637Crossref PubMed Scopus (121) Google Scholar). Here we report several crystal structures of BChE complexed with a substrate, products, and conjugated to soman after aging. From these structures we propose alternative substrate and product binding that may be related to the high catalytic efficiency of the choline esterases. Crystallization of Recombinant BChE and Its Complexes—Recombinant human BChE suitable for crystallization was obtained, purified, and crystallized as described previously (17Nachon F. Nicolet Y. Viguie N. Masson P. Fontecilla-Camps J.C. Lockridge O. Eur. J. Biochem. 2002; 269: 630-637Crossref PubMed Scopus (121) Google Scholar). No butyrate was present in the culture medium and none was added during any step of the purification procedure. The 3-bromopropionate-BChE complex was obtained by soaking crystals for a few minutes in the mother liquor containing 100 mm bromopropionate (Sigma). The BChE-choline complex was obtained by soaking BChE crystals grown from a 2.1 m (NH4)2SO4 100 mm Bicine (Fluka), pH 9.0, crystallization solution, in the mother liquor containing 100 mm choline chloride. Crystallization of soman-aged BChE: racemic soman (pinacolyl methylphosphonofluoridate) was obtained from CEB Le Bouchet (Vert-le-Petit, France). The purified enzyme (6.6 mg/ml) was inhibited in the presence of 0.5 mm soman (∼5.5-fold molar excess) in 10 mm MES buffer, pH 6.5. The reaction mixture was further incubated for 3 days at 4 °C, allowing enough time for completion of the aging reaction and the disappearance of the remaining unreacted soman. The inhibited enzyme was crystallized under the same conditions as the uninhibited BChE except that the mother liquor was buffered at pH 8.0 using a 0.1 m Tris/HCl buffer solution. Soman-aged BChE and butyrylthiocholine (BTC) were cocrystallized at pH 6.5 (0.1 m MES buffer, 2.1 m (NH4)2SO4) with 10 mm BTC (Sigma). X-ray data were collected from a 4-day-old crystal to limit the loss of substrate by spontaneous hydrolysis. X-ray Data Collection and Structure Solution—All crystals were flash-cooled at 100 K in a nitrogen stream using 15–20% glycerol in the mother liquor as cryoprotectant. Data sets were collected at the following beamlines of the European Synchrotron Radiation Facility (Grenoble, France): ID14-eh1 for the soman-aged BChE, ID14-eh2 for the native BChE, BM30 for the choline-BChE complex and the somanaged BTC complex, and ID14-eh4 for the 3-bromopropionate-BChE complex (see Table I). Data sets for the native, the soman-aged BchE, and soman-aged BChE-BTC complex crystals were integrated, scaled, and reduced using MOSFLM, SCALA, and TRUNCATE from the CCP4 suite (18Collaborative Computational Project No. 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19770) Google Scholar). Data sets from the choline-BChE complex and the 3-bromopropionate-BChE species were processed using the program XDS (19Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3233) Google Scholar). Data collection from the 3-bromopropionate-BChE crystal was performed at a wavelength of 0.915 Å to measure the Br anomalous scattering signal. Subsequent data processing was performed without merging the Friedel mates to better evaluate the anomalous signal of the bromine atom. Molecular replacement was carried out with the native data set between 15- and 3.5-Å resolution using the program AMoRe (20Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5029) Google Scholar) and the TcAChE structure (Protein Data Bank ID code: 2ACE) as a search model. A well constrasted solution with R-factor = 42.4% and correlation coefficient = 46.7% was obtained for one monomer per asymmetric unit. This solution was used as a starting model for manual rebuilding and refinement of BChE against all data to 2.0-Å resolution with the programs TURBO (21Roussel A. Cambillaud C. TURBO computer program. Silicon Graphics, Mountain View, CA1989Google Scholar) and CNS (22Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gross P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. 1998; D54: 905-921Crossref Scopus (16967) Google Scholar), respectively. Observed structure factors were scaled anisotropically and a bulk solvent correction was applied. Several cycles of refinement, manual rebuilding, and solvent addition led to a model with good statistics (Table I). Residues 1–3, 378–379, and 455 did not have matching electron density and, consequently, were not included in the model. Carbohydrate chains corresponding to five of the six expected glycosylation sites (17Nachon F. Nicolet Y. Viguie N. Masson P. Fontecilla-Camps J.C. Lockridge O. Eur. J. Biochem. 2002; 269: 630-637Crossref PubMed Scopus (121) Google Scholar) have been included in the crystallographic model. They correspond to those connected to Asn57, Asn106, Asn241, Asn341, and Asn485. Although Asn256 was expected to be glycosylated, no electron density was observed for carbohydrate. Further examination of the electron density maps led to the inclusion of three molecules of glycerol, used as cryoprotectant, two sulfate ions, the precipitanting agent, one molecule of MES buffer, and two chloride ions. During refinement, an unexpected residual electron density was observed the of the catalytic the of refinement and after several at this density as corresponding to added during purification or a good was obtained butyrate was used as a model (see and refinement choline-BChE complex was to cell a = = of data processing was carried out without merging the Friedel mates to evaluate the anomalous signal from the bromine from The choline-BChE complex was to data processing was carried out without merging the Friedel mates to evaluate the anomalous signal from the bromine atom. in a The soman-aged BChE structure was using the refinement from the program The model statistics are shown in Table I. Residues 1–3, 378–379, and 455 and to Asn256 and no matching electron density and were not included in the model. The soman-aged BChE structure also the three glycerol the two chloride ions, and one of the two sulfate previously observed in the native The same refinement was to this and the choline-BChE structure (Table I). The structure of the 3-bromopropionate-BChE complex was using the native BChE structure as a starting set in which the modeled butyrate been difference and difference maps were with the program The starting Data Bank and for were obtained using R. M. R.W. J. PubMed Scopus Google Scholar). it was to the electron density for bromine and the other were obtained the to that the moiety was partially during the soaking of BChE crystal structure was by the replacement and to 2.0-Å resolution using CNS (see and Table I). As expected M. Sussman J.L. E. Bon S. P. J. Silman I. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, D. A. D. N. C. A. Biochemistry. 2001; PubMed Scopus Google Scholar, S. P. Biochemistry. 1993; PubMed Scopus Google Scholar, A. Jiang Lockridge O. Biochemistry. 1997; 36: PubMed Scopus Google Scholar, Y. S. P. Biochemistry. 1993; PubMed Scopus Google Scholar), the structure of BChE is similar to that of BChE not form the observed in structures of TcAChE (5Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2426) Google Scholar, M. Silman I. Sussman J.L. Structure Full Text Full Text PDF PubMed Scopus Google Scholar), AChE Y. P. P. Full Text PDF PubMed Scopus Google Scholar, Y. P. P. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), and human AChE Harel M. Toker L. A. C. D. N. A. Silman I. Sussman J.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; PubMed Scopus Google Scholar). In the involved in the are and the active site are located of the Although are found at the BChE and are not form a and the active site are located the same of the from the crystal be responsible for these between BChE and TcAChE are to the residues the gorge, the former enzyme has several of the aromatic of the by the with the of Phe and Phe of TcAChE by and these it for the binding of the butyrate substrate moiety in BChE and the of the acyl In addition, the catalytic is connected to a large electron density peak is in the We in that a previously modeled structure of BChE M. Sussman J.L. E. Bon S. P. J. Silman I. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) from the structure at these a the AChE starting As an the acyl pocket of the model is significantly than the one observed in the crystal A to the During the crystallographic refinement it that the catalytic was bound to an moiety that was modeled as butyrate with well matching electron density and this we native crystals with the butyrate in which the is by a bromine atom. we collected data at wavelength bromine to a anomalous signal. of the moiety by its with no in the of the corresponding electron In the of the enzyme the of the the acyl to the catalytic to with no residual in the difference electron density the other maps after crystallographic refinement of butyrate or the that one is a good model for the We are the of the using density Although the of the moiety is it to be for crystallization because were extremely to In the few crystals grown from these the butyrate bound to crystals were obtained or BTC, was added to the crystallization This may be related to the of the acyl pocket in BChE by butyrate. A we the native TcAChE, and the has the same (see residues to the acyl have in the three in BChE the acyl the at the catalytic by butyrate or soman The reaction of soman and other related esters with the catalytic of can be by such as in J. Appl. Toxicol. 1994; PubMed Scopus Google with of an the a J. Science. PubMed Scopus Google Scholar). for its the is a of both the we have in the BChE crystals and of the postulated The a with one of the a was also observed in the crystal structures of and TcAChE A. Harel M. Y. D. A. Silman I. Sussman J.L. Biochemistry. 1999; 38: PubMed Scopus Google Scholar). A of the acyl has also been for the crystal structure of these in that the acyl is and are with that the AChE active site the gorge, this or an alternative located it J. Chem. 1998; Scopus Google Scholar). As of the acyl by butyrate to be for at the an of the Ser198 in BChE, we crystals in a 100 mm choline chloride solution, pH maps with data collected from a crystal no electron density corresponding to the In this structure the from Ser198 two a one it a with the catalytic and a expected it has from the and a with the main from at the a and is that this is to the of at pH 9.0, because crystals grown at pH 6.5 similar Ser198 with or and a native structure at pH has the butyrate bound to the active site in this structure is the of the bound as expected, its quaternary ammonium binds close to a π-cation the the gorge and a with a molecule in with the main from Although choline binding may not be relevant the high choline in the soaking solution and the it that this reaction product may at two at the active the as of bound to the active site and an alternative as observed a and The of an alternative choline binding to an crystals obtained from previously inhibited with soman were in a solution containing the substrate In the complex between soman-aged BChE and BTC the binds with its ammonium close to and its to a molecule to the one described for the BChE-choline complex This the that substrate binding π-cation interactions is and that a bound to the catalytic is with site substrate binding as by the soman-aged BChE-BTC data for all the structures are in Table I. the in with data from the concerning the catalytic mechanism of enzymes to an step in the reaction is by the and extremely efficient is of the substrate binding sites. sites as for substrate at the outer rim of the gorge by a not From one or several of these substrate can into an to the one observed in the soman-aged BChE-BTC as by Masson P. Legrand P. Bartels C.F. Froment M.T. Schopfer L.M. Lockridge O. Biochemistry. 1997; 36: 2266-2277Crossref PubMed Scopus (133) Google Scholar), a of the molecule its quaternary ammonium bound at the π-cation can the substrate to binding. of the of BTC to a than to may this has been proposed that the of the catalytic triad in at the bottom of a gorge the of the in a M. D.M. Silman I. Sussman J.L. J. Chem. Scopus Google Scholar). The provide an for the location of the catalytic triad as the presence of peripheral and sites at the gorge for a of substrate it to the active site in a most efficient The presence of a butyrate bound to Ser198 be related to the of the catalytic This is by the by Y. P. P. J. PubMed Scopus Google Scholar) described a or molecule found close to the catalytic of AChE in complex with a peripheral site other structures of AChE also electron close to the catalytic M. Rosenberry T.L. Mallender W.D. T. R.J. Silman I. Sussman J.L. Sci. 2000; PubMed Scopus Google Scholar, M. S. Harel M. P. Silman I. J. Sussman J.L. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). Although the between of Ser198 and of of Å is with the between and is a that is for that of In this were to the of the choline moiety be have been For the A structure at resolution T. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. PubMed Scopus Google Scholar) a of Å and the is far from that the is or to the of of the is to propose that the of to to Ser198 this is not this to be well and be in all the structures the other we have observed that in several BChE complexes the of Ser198 is and no to to for a report by Masson P. F. Bartels C.F. Froment M.T. F. C. Lockridge O. Eur. J. Biochem. PubMed Scopus Google Scholar) that in the presence of substrate and BChE is under these previously data T. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. PubMed Scopus Google Scholar, P. F. Bartels C.F. Froment M.T. F. C. Lockridge O. Eur. J. Biochem. PubMed Scopus Google Scholar), the of the catalytic by during alternative to of the catalytic by during be a product binding at the catalytic The presence of a butyrate moiety in the purified BChE at this As upon substrate binding at the active site the its with the a the and an the the of the of Ser198 the partially atom. This the step of the reaction by the the of the A similar mechanism a molecule the X-ray structures of both and soman-aged BChE have a glycerol molecule bound to the π-cation site. As glycerol was used as (see the its presence in the active site gorge the of the π-cation site and that the substrate is to residues and the acyl The presence of a moiety to the catalytic is Several of that in it is most butyrate as it can be by it is by and the presence of butyrate is for experiments be to its and structure factors have been with the Data Bank with with and with soman and and be released upon We are to the of and beamlines from the European Synchrotron Radiation is for with data collection from the 3-bromopropionate-BChE

Hydrogen is an important metabolite for many microrganisms. H2 is either produced or consumed by hydrogenase enzymes, according to the reaction: H2⇌2 H+ + 2 e−. The catalytic sites of the two major families of hydrogenases include a dinuclear center, either NiFe (see picture) or FeFe, along with proteic and nonproteic ligands. The structure and activity of these sites are currently being studied, with the aim of designing cheap biological or chemical catalysts able to produce hydrogen, the ideal clean fuel.

Acinetobacter baumannii is a species of nonfermentative gram-negative bacteria commonly found in water and soil. This organism was susceptible to most antibiotics in the 1970s. It has now become a major cause of hospital-acquired infections worldwide due to its remarkable propensity to rapidly acquire resistance determinants to a wide range of antibacterial agents. Here we use a comparative genomic approach to identify the complete repertoire of resistance genes exhibited by the multidrug-resistant A. baumannii strain AYE, which is epidemic in France, as well as to investigate the mechanisms of their acquisition by comparison with the fully susceptible A. baumannii strain SDF, which is associated with human body lice. The assembly of the whole shotgun genome sequences of the strains AYE and SDF gave an estimated size of 3.9 and 3.2 Mb, respectively. A. baumannii strain AYE exhibits an 86-kb genomic region termed a resistance island--the largest identified to date--in which 45 resistance genes are clustered. At the homologous location, the SDF strain exhibits a 20 kb-genomic island flanked by transposases but devoid of resistance markers. Such a switching genomic structure might be a hotspot that could explain the rapid acquisition of resistance markers under antimicrobial pressure. Sequence similarity and phylogenetic analyses confirm that most of the resistance genes found in the A. baumannii strain AYE have been recently acquired from bacteria of the genera Pseudomonas, Salmonella, or Escherichia. This study also resulted in the discovery of 19 new putative resistance genes. Whole-genome sequencing appears to be a fast and efficient approach to the exhaustive identification of resistance genes in epidemic infectious agents of clinical significance.

Fe-only hydrogenases, as well as their NiFe counterparts, display unusual intrinsic high-frequency IR bands that have been assigned to CO and CN(-) ligation to iron in their active sites. FTIR experiments performed on the Fe-only hydrogenase from Desulfovibrio desulfuricans indicate that upon reduction of the active oxidized form, there is a major shift of one of these bands that is provoked, most likely, by the change of a CO ligand from a bridging position to a terminal one. Indeed, the crystal structure of the reduced active site of this enzyme shows that the previously bridging CO is now terminally bound to the iron ion that most likely corresponds to the primary hydrogen binding site (Fe2). The CO binding change may result from changes in the coordination sphere of Fe2 or its reduction. Superposition of this reduced active site with the equivalent region of a NiFe hydrogenase shows a remarkable coincidence between the coordination of Fe2 and that of the Fe ion in the NiFe cluster. Both stereochemical and mechanistic considerations suggest that the small organic molecule found at the Fe-only hydrogenase active site and previously modeled as 1,3-propanedithiolate may, in fact, be di-(thiomethyl)-amine.

An effective environmental force constant is introduced to quantify the molecular resilience (or its opposite, "softness") of a protein structure and relate it to biological function and activity. Specific resilience-function relations were found in neutron-scattering experiments on purple membranes containing bacteriorhodopsin, the light-activated proton pump of halobacteria; the connection between resilience and stability is illustrated by a study of myoglobin in different environments. Important advantages of the neutron method are that it can characterize the dynamics of any type of biological sample-which need not be crystalline or monodisperse-and that it enables researchers to focus on the dynamics of specific parts of a complex structure with deuterium labeling.

The identification of dynamical domains in proteins and the description of the low-frequency domain motions are one of the important applications of numerical simulation techniques. The application of these techniques to large proteins requires a substantial computational effort and therefore cannot be performed routinely, if at all. This article shows how physically motivated approximations permit the calculation of low-frequency normal modes in a few minutes on standard desktop computers. The technique is based on the observation that the low-frequency modes, which describe domain motions, are independent of force field details and can be obtained with simplified mechanical models. These models also provide a useful measure for rigidity in proteins, allowing the identification of quasi-rigid domains. The methods are validated by application to three well-studied proteins, crambin, lysozyme, and ATCase. In addition to being useful techniques for studying domain motions, the success of the approximations provides new insight into the relevance of normal mode calculations and the nature of the potential energy surface of proteins.

The reactive oxygen species (ROS)-producing enzyme NADPH oxidase (NOX) was first identified in the membrane of phagocytic cells. For many years, its only known role was in immune defense, where its ROS production leads to the destruction of pathogens by the immune cells. NOX from phagocytes catalyzes, via one-electron trans-membrane transfer to molecular oxygen, the production of the superoxide anion. Over the years, six human homologs of the catalytic subunit of the phagocyte NADPH oxidase were found: NOX1, NOX3, NOX4, NOX5, DUOX1, and DUOX2. Together with the NOX2/gp91phox component present in the phagocyte NADPH oxidase assembly itself, the homologs are now referred to as the NOX family of NADPH oxidases. NOX are complex multidomain proteins with varying requirements for assembly with combinations of other proteins for activity. The recent structural insights acquired on both prokaryotic and eukaryotic NOX open new perspectives for the understanding of the molecular mechanisms inherent to NOX regulation and ROS production (superoxide or hydrogen peroxide). This new structural information will certainly inform new investigations of human disease. As specialized ROS producers, NOX enzymes participate in numerous crucial physiological processes, including host defense, the post-translational processing of proteins, cellular signaling, regulation of gene expression, and cell differentiation. These diversities of physiological context will be discussed in this review. We also discuss NOX misregulation, which can contribute to a wide range of severe pathologies, such as atherosclerosis, hypertension, diabetic nephropathy, lung fibrosis, cancer, or neurodegenerative diseases, giving this family of membrane proteins a strong therapeutic interest.

We demonstrate for different protein samples that 2D 1H-15N correlation NMR spectra can be recorded in a few seconds of acquisition time using a new band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence experiment. This has enabled us to measure fast hydrogen-deuterium exchange rate constants along the backbone of a small globular protein fragment by real-time 2D NMR.

A number of ways and means have evolved to provide resistance to eubacteria challenged by beta-lactams. This review is focused on pathogens that resist by expressing low-affinity targets for these antibiotics, the penicillin-binding proteins (PBPs). Even within this narrow focus, a great variety of strategies have been uncovered such as the acquisition of an additional low-affinity PBP, the overexpression of an endogenous low-affinity PBP, the alteration of endogenous PBPs by point mutations or homologous recombination or a combination of the above.

Visibly fluorescent proteins (FPs) from jellyfish and corals have revolutionized many areas of molecular and cell biology, but the use of FPs in intact animals, such as mice, has been handicapped by poor penetration of excitation light. We now show that a bacteriophytochrome from Deinococcus radiodurans, incorporating biliverdin as the chromophore, can be engineered into monomeric, infrared-fluorescent proteins (IFPs), with excitation and emission maxima of 684 and 708 nm, respectively; extinction coefficient >90,000 M(-1) cm(-1); and quantum yield of 0.07. IFPs express well in mammalian cells and mice and spontaneously incorporate biliverdin, which is ubiquitous as the initial intermediate in heme catabolism but has negligible fluorescence by itself. Because their wavelengths penetrate tissue well, IFPs are suitable for whole-body imaging. The IFPs developed here provide a scaffold for further engineering.

Crystallographic data on the [NiFe] hydrogenase from Desulfovibrio gigas are presented that provide new information on the structure and mode of action of its dihydrogen activating metal center. Recently we found this center to contain, besides Ni, a second metal ion which was tentatively assigned to Fe (Volbeda, A.; Charon, M. H.; Piras, C.; Hatchikian, E. C.; Frey, M.; Fontecilla-Camps, J. C. Nature 1995, 373, 580−587). This assignment is now unambiguously confirmed by a crystallographic analysis using 3 Å resolution X-ray data collected at wavelengths close to either side of the Fe absorption edge. Moreover, we report the structure of another crystal form of the as-purified D. gigas hydrogenase refined at 2.54 Å resolution, showing that the active site Fe binds three diatomic ligands. The electron density map shows an additional small peak at a position bridging the two active site metal ions, which may be assigned to some form of oxygen. This bridging oxygen species is proposed to be the signature of the inactive form of the enzyme. An infrared analysis similar to the one reported for Chromatium vinosum hydrogenase (Bagley, K. A.; Duin, E. C.; Roseboom, W.; Albracht, S. P. J.; Woodruff, W. H. Biochemistry 1995, 34, 5527−5535) shows the existence of three bands at exceptionally high frequencies, that shift their position in a concerted fashion depending on the redox state of the enzyme. Based on these high frequencies, the diatomic Fe ligands may be assigned to nonexchangeable triply bonded molecules, possible candidates being CO, CN- and NO. The frequency shifts of the infrared bands suggest a redox role for the Fe center during catalysis. Based on the new crystal structure and a number of spectroscopic results, possible modes of hydrogen binding to the active site are discussed.

The biological activity of macromolecules is accompanied by rapid structural changes. The photosensitivity of the carbon monoxide complex of myoglobin was used at the European Synchrotron Radiation Facility to obtain pulsed, Laue x-ray diffraction data with nanosecond time resolution during the process of heme and protein relaxation after carbon monoxide photodissociation and during rebinding. These time-resolved experiments reveal the structures of myoglobin photoproducts, provide a structural foundation to spectroscopic results and molecular dynamics calculations, and demonstrate that time-resolved macromolecular crystallography can elucidate the structural bases of biochemical mechanisms on the nanosecond time scale.