Glutamate optimizes enzymatic activity under high hydrostatic pressure in Desulfovibrio species: effects on the ubiquitous thioredoxin system.

In piezophilic microorganisms, enzymes are optimized to perform under high hydrostatic pressure. The two major reported mechanisms responsible for such adaptation in bacterial species are changes in amino acids in the protein structure, favoring their activity and stability under high-pressure conditions, and the possible accumulation of micromolecular co-solutes in the cytoplasm. Recently, the accumulation of glutamate in the cytoplasm of piezophilic Desulfovibrio species has been reported under high-pressure growth conditions. In this study, analysis of the effect of glutamate on the enzymatic activity of the thioredoxin reductase/thioredoxin enzymatic complex of either a piezosensitive or a piezophilic microorganism confirms its role as a protective co-solute. Analysis of the thioredoxin structures suggests an adaptation both to the presence of glutamate and to high hydrostatic pressure in the enzyme from the piezophilic strain. Indeed, the presence of large surface pockets could counterbalance the overall compression that occurs at high hydrostatic pressure to maintain enzymatic activity. A lower isoelectric point and a greater dipolar moment than that of thioredoxin from the piezosensitive strain would allow the protein from the piezophilic strain to compensate for the presence of the charged amino acid glutamate to interact with its partner.

Solution structure of the module X2 1 of unknown function of the cellulosomal scaffolding protein CipC of Clostridium cellulolyticum.

Multidimensional, homo- and heteronuclear magnetic resonance spectroscopy combined with dynamical annealing has been used to determine the structure of a 94 residue module (X2 1) of the scaffolding protein CipC from the anaerobic bacterium Clostridium cellulolyticum. An experimental data set comprising 1647 nuclear Overhauser effect-derived restraints, 105 hydrogen bond restraints and 66 phi torsion angle restraints was used to calculate 20 converging final solutions. The calculated structures have an average rmsd about the mean structure of 0.55(+/-0.11) A for backbone atoms and 1.40(+/-0.11) A for all heavy atoms when fitted over the secondary structural elements. The X2 1 module has an immunoglobulin-like fold with two beta-sheets packed against each other. One sheet contains three strands, the second contains four strands. An additional strand is intercalated between the beta-sandwich, as well as two turns of a 3(.10) helix. X2 1 has a surprising conformational stability and may act as a conformational linker and solubility enhancer within the scaffolding protein. The fold of X2 1 is very similar to that of telokin, titin Ig domain, hemolin D2 domain, twitchin immunoglobulin domain and the first four domains of the IgSF portion of transmembrane cell adhesion molecule. As a consequence, the X2 1 module is the first prokaryotic member assigned to the I set of the immunoglobulin superfamily even though no sequence similarity with any member of this superfamily could be detected.

Interactions of bile salt micelles and colipase studied through intermolecular nOes.

Colipase is a small protein (10 kDa), which acts as a protein cofactor for the pancreatic lipase. Various models of the activated ternary complex (lipase-colipase-bile salt micelles) have been proposed using detergent micelles, but no structural information has been established with bile salt micelles. We have investigated the organization of sodium taurodeoxycholate (NaTDC) micelles and their interactions with pig and horse colipases by homonuclear nuclear magnetic resonance (NMR) spectroscopy. The NMR data supply evidence that the folding of horse colipase is similar to that already described for pig colipase. Intermolecular nuclear Overhauser effects have shown that two conserved aromatic residues interact with NaTDC micelles.

Evaluation of slaved pulses to study protein hydration.

The new concept of slaved pulses is evaluated in the context of the study of protein hydration. The inversion properties of these pulses are shown to be superior in quality to the previously published schemes. High-quality water selective homonuclear 2D 1H NOESY-NOESY and NOESY-TOCSY experiments were recorded on horse heart ferrocytochrome c.

Solution structure of a selectively 13C-labeled intramolecular DNA triplex.

The three-dimensional structure of an intramolecular triple helix whose three strands have been linked by a hexaethylene glycol chain, and selectively 13C-enriched in position C1' on the third strand was investigated by NMR spectroscopy and constrained molecular mechanics calculations. Starting from different initial conformations, we show that the NOE constraints determined by the complete relaxation matrix calculation and iterative back-calculations allowed us to reach the same final restrained triple helix, taking into account implicitly the solvent effect. We conclude that this triplex adopted a B-type conformation rather than a A-type. The sugar pucker was found predominantly in the S-type conformation, in the range of C2'-endo geometry.

Solution structure of BmKTX, a K+ blocker toxin from the Chinese scorpion Buthus Martensi.

BmKTX is a toxin recently purified from the venom of Buthus Martensi, which belongs to the kaliotoxin family. We have determined its solution structure by use of conventional two-dimensional NMR techniques followed by distance-geometry and energy minimization. The calculated structure is composed of a short alpha-helix (residues 14 to 20) connected by a tight turn to a two-stranded antiparallel beta-sheet (sequences 25-27 and 32-34). The beta-turn connecting these strands belongs to type I. The N-terminal segment (sequence 1 to 8) runs parallel to the beta-sheet although it cannot be considered as a third strand. Comparison of the conformation of BmKTX and toxins of the kaliotoxin family clearly demonstrates that they are highly related. Therefore, analysis of the residues belonging to the interacting surface of those toxins allows us to propose a functional map of BmKTX slightly different from the one of KTX and AgTX2, which may explain the variations in affinities of these toxins towards the Kv1.3 channels.

Selectively 13C-enriched DNA: 13C and 1H assignments of a triple helix by two-dimensional relayed HMQC experiments.

We present NMR studies of an intramolecular triple helix, the three strands of which have been linked by a hexaethylene glycol chain. To overcome the generally encountered difficulties of assignment in the homo-pyrimidine strands, the carbon Cl' of the pyrimidines were selectively 13C-enriched. Assignments of the aromatic and sugar protons were obtained from NOESY-HMQC and TOCSY-HMQC spectra. We show that the recognition of a DNA duplex by a third strand via triplex formation is easily carried out in solution by observing the changes of the 1Hl'-13Cl' connectivities as a function of pH. Furthermore, the conformation of the sugars has been found to be C2'-endo, on the basis of the coupling constant values directly measured in an HSQC spectrum.

Solution structure of two new toxins from the venom of the Chinese scorpion Buthus martensi Karsch blockers of potassium channels.

The solution structure of BmTX2 purified from the venom of the Chinese Buthid Buthus martensi has been determined by 2D NMR spectroscopy techniques which led to the description of its 3D conformation. The structure consists of a triple-stranded beta-sheet connected to a helical structure. This helix encompasses 10 residues, from 11 to 20, begins with a turn of 310 helix, and ends with an alpha helix. The three strands of beta sheet comprise residues 2-6, with a bulge covering residues 4 and 5, 26-29, and 32-35, with a type I' beta turn centered on residues 30-31. We also characterized the solution structure of BmTX1. The two toxins which are potent blockers of both large-conductance calcium-activated potassium channels (BKCa channels) and voltage-gated potassium channels (Kv1. 3) are highly superimposable and possess the same structural characteristics. Analysis of these structures allows us to hypothesize that, besides the main surface of interaction described by the functional map of charybdotoxin, one can expect that the binding of scorpion toxins on BKCa channels may involve residues on the edge of this surface.

Solution structure of oligonucleotides covalently linked to a psoralen derivative.

Psoralen (pso) was attached via its C-5 position to the 5'-phosphate group of an oligodeoxynucleotide d(TAAGCCG) by a hexamethylene linker (m6). Complex formation between pso-m6-d(TAAGCCG) and the complementary strands d(CGGCTTA)[7-7mer] or d(CGGCTTAT)[7-8mer] was investigated by nuclear magnetic resonance in aqueous solution. Structural informations derived from DQF-COSY and NOESY maps, revealed that the mini double helix adopts a B-form conformation and that the deoxyriboses preferentially adopt a C2'-endo conformation. The nOe connectivities observed between the protons of the bases or the sugars in each duplex, and the protons of the psoralen and the hexamethylene chain, led us to propose a model involving an equilibrium between two conformations due to different locations of the psoralen. Upon UV-irradiation, the psoralen moiety cross-linked the two DNA strands at the level of 5'TpA3' sequences. NMR studies of the single major photo-cross-linked duplex pso-m6-d(TAAGCCG) and d(CGGCTTA) were performed. The stereochemistry of the diadduct is indeed cis-syn at both cyclobutane rings. In addition, the effects of this diadduct on the helical structure are analyzed in detail.

Chemosensory protein from the moth Mamestra brassicae. Expression and secondary structure from 1H and 15N NMR.

A group of ubiquitous small proteins (average 13 kDa) has been isolated from several sensory organs of a wide range of insect species. They are believed to be involved in chemical communication and perception (olfaction or taste) and have therefore been called chemo-sensory proteins (CSPs). Several CSPs have been identified in the antennae and proboscis of the moth Mamestra brassicae. We have expressed one of the antennal proteins (CSPMbraA6) in large quantities as a soluble recombinant protein in Escherichia coli periplasm. This 112-residue protein is a highly soluble monomer of 13 072 Da with a pI of 5.5. NMR data (1H and 15N) indicate that CSPMbraA6 is well folded and contains seven alpha helices (59 amino acids) and two short extended structures (12 amino acids) from positions 5 to 10 and from 107 to 112. Thirty-seven amino acids are involved in beta turns and coiled segments and four amino acids are not assigned in the NMR spectra (the N-terminus and the residue 52 in the loop 48-53), probably due to their mobility. This is the first report on the expression and structural characterization of a recombinant CSP.

Solution structure of potassium channel-inhibiting scorpion toxin Lq2.

Lq2 is a unique scorpion toxin. Acting from the extracellular side, Lq2 blocks the ion conduction pore in not only the voltage- and Ca2+ -activated channels, but also the inward-rectifier K+ channels. This finding argues that the three-dimensional structures of the pores in these K+ channels are similar. However, the amino acid sequences that form the external part of the pore are minimally conserved among the various classes of K+ channels. Because Lq2 can bind to all the three classes of K+ channels, we can use Lq2 as a structural probe to examine how the non-conserved pore-forming sequences are arranged in space to form similar pore structures. In the present study, we determined the three-dimensional structure of Lq2 using nuclear magnetic resonance (NMR) techniques. Lq2 consists of an alpha-helix (residues S10 to L20) and a beta-sheet, connected by an alphabeta3 loop (residues N22 to N24). The beta-sheet has two well-defined anti-parallel strands (residues G26 to M29 and residues K32 to C35), which are connected by a type I' beta-turn centered between residues N30 and K31. The N-terminal segment (residues Z1 to T8) appears to form a quasi-third strand of the beta-sheet.

Structural model of the Fe-hydrogenase/cytochrome c553 complex combining transverse relaxation-optimized spectroscopy experiments and soft docking calculations.

Fe-hydrogenase is a 54-kDa iron-sulfur enzyme essential for hydrogen cycling in sulfate-reducing bacteria. The x-ray structure of Desulfovibrio desulfuricans Fe-hydrogenase has recently been solved, but structural information on the recognition of its redox partners is essential to understand the structure-function relationships of the enzyme. In the present work, we have obtained a structural model of the complex of Fe-hydrogenase with its redox partner, the cytochrome c(553), combining docking calculations and NMR experiments. The putative models of the complex demonstrate that the small subunit of the hydrogenase has an important role in the complex formation with the redox partner; 50% of the interacting site on the hydrogenase involves the small subunit. The closest contact between the redox centers is observed between Cys-38, a ligand of the distal cluster of the hydrogenase and Cys-10, a ligand of the heme in the cytochrome. The electron pathway from the distal cluster of the Fe-hydrogenase to the heme of cytochrome c(553) was investigated using the software Greenpath and indicates that the observed cysteine/cysteine contact has an essential role. The spatial arrangement of the residues on the interface of the complex is very similar to that already described in the ferredoxin-cytochrome c(553) complex, which therefore, is a very good model for the interacting domain of the Fe-hydrogenase-cytochrome c(553).

The location of the ligand-binding site of carbohydrate-binding modules that have evolved from a common sequence is not conserved.

Polysaccharide-degrading enzymes are generally modular proteins that contain non-catalytic carbohydrate-binding modules (CBMs), which potentiate the activity of the catalytic module. CBMs have been grouped into sequence-based families, and three-dimensional structural data are available for half of these families. Clostridium thermocellum xylanase 11A is a modular enzyme that contains a CBM from family 6 (CBM6), for which no structural data are available. We have determined the crystal structure of this module to a resolution of 2.1 A. The protein is a beta-sandwich that contains two potential ligand-binding clefts designated cleft A and B. The CBM interacts primarily with xylan, and NMR spectroscopy coupled with site-directed mutagenesis identified cleft A, containing Trp-92, Tyr-34, and Asn-120, as the ligand-binding site. The overall fold of CBM6 is similar to proteins in CBM families 4 and 22, although surprisingly the ligand-binding site in CBM4 and CBM22 is equivalent to cleft B in CBM6. These structural data define a superfamily of CBMs, comprising CBM4, CBM6, and CBM22, and demonstrate that, although CBMs have evolved from a relatively small number of ancestors, the structural elements involved in ligand recognition have been assembled at different locations on the ancestral scaffold.