T. Alwyn Jones

Phi/psi-chology: Ramachandran revisited.

The errors that can be introduced into a protein model during model building and refinement vary tremendously in their importance and severity [1,2]. At one extreme, the mainchain may be totally incorrectly traced in the experimental map, or the molecular replacement solution may be wrong. Minor errors may include an incorrect peptide orientation, or misplaced or excessive water molecules. The reasons why errors creep into models are many, but for a structure built into an experimental map, the main ones are limited resolution and poorly phased diffraction data. Other things being equal, the resolution of the diffraction data should be the ultimate variable that determines the accuracy of a structural investigation. Inevitably, life is more complicated, and a successful structural investigation is often a learning experience for the people involved.

The Uppsala Electron-Density Server

The Uppsala Electron Density Server (EDS; http://eds.bmc.uu.se/) is a web-based facility that provides access to electron-density maps and statistics concerning the fit of crystal structures and their maps. Maps are available for approximately 87% of the crystallographic Protein Data Bank (PDB) entries for which structure factors have been deposited and for which straightforward map calculations succeed in reproducing the published R value to within five percentage points. Here, an account is provided of the methods that are used to generate the information contained in the server. Some of the problems that are encountered in the map-generation process as well as some spin-offs of the project are also discussed.

Where freedom is given, liberties are taken

We thank the Editors for allowing us to publish this diatribe, and Dr Alex Cameron for providing us with the coordinates of his unpublished glutathione transferase structure. We are preparing a considerably longer and more technical paper concerning rebuilding and refinement for a forthcoming volume of Methods in Enzymology, a preprint of which will be made available via the World Wide Web (http://onyx.bmc.uu.se/).

Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Ce16A from Trichoderma reesei

BACKGROUND Cel6A is one of the two cellobiohydrolases produced by Trichoderma reesei. The catalytic core has a structure that is a variation of the classic TIM barrel. The active site is located inside a tunnel, the roof of which is formed mainly by a pair of loops. RESULTS We describe three new ligand complexes. One is the structure of the wild-type enzyme in complex with a nonhydrolysable cello-oligosaccharide, methyl 4-S-beta-cellobiosyl-4-thio-beta-cellobioside (Glc)(2)-S-(Glc)(2), which differs from a cellotetraose in the nature of the central glycosidic linkage where a sulphur atom replaces an oxygen atom. The second structure is a mutant, Y169F, in complex with the same ligand, and the third is the wild-type enzyme in complex with m-iodobenzyl beta-D-glucopyranosyl-beta(1,4)-D-xylopyranoside (IBXG). CONCLUSIONS The (Glc)(2)-S-(Glc)(2) ligand binds in the -2 to +2 sites in both the wild-type and mutant enzymes. The glucosyl unit in the -1 site is distorted from the usual chair conformation in both structures. The IBXG ligand binds in the -2 to +1 sites, with the xylosyl unit in the -1 site where it adopts the energetically favourable chair conformation. The -1 site glucosyl of the (Glc)(2)-S-(Glc)(2) ligand is unable to take on this conformation because of steric clashes with the protein. The crystallographic results show that one of the tunnel-forming loops in Cel6A is sensitive to modifications at the active site, and is able to take on a number of different conformations. One of the conformational changes disrupts a set of interactions at the active site that we propose is an integral part of the reaction mechanism.

Crystal Structures of a Poplar Xyloglucan Endotransglycosylase Reveal Details of Transglycosylation Acceptor Binding

Xyloglucan endotransglycosylases (XETs) cleave and religate xyloglucan polymers in plant cell walls via a transglycosylation mechanism. Thus, XET is a key enzyme in all plant processes that require cell wall remodeling. To provide a basis for detailed structure–function studies, the crystal structure of Populus tremula x tremuloides XET16A (PttXET16A), heterologously expressed in Pichia pastoris, has been determined at 1.8-Å resolution. Even though the overall structure of PttXET16A is a curved β-sandwich similar to other enzymes in the glycoside hydrolase family GH16, parts of its substrate binding cleft are more reminiscent of the distantly related family GH7. In addition, XET has a C-terminal extension that packs against the conserved core, providing an additional β-strand and a short α-helix. The structure of XET in complex with a xyloglucan nonasaccharide, XLLG, reveals a very favorable acceptor binding site, which is a necessary but not sufficient prerequisite for transglycosylation. Biochemical data imply that the enzyme requires sugar residues in both acceptor and donor sites to properly orient the glycosidic bond relative to the catalytic residues.

Structure of Satellite tobacco necrosis virus at 3.0 Å resolution

Abstract The structure of Satellite tobacco necrosis virus (STNV) has been determined to 3.0 A resolution by X-ray crystallography. Electron density maps were obtained with phases based on one heavy-atom derivative and several cycles of phase refinement using the 60-fold non-crystallographic symmetry in the particle. A model for one protein subunit was built using a computer graphics display. The subunit is constructed mainly of a β-roll structure forming two β-sheets, each of four antiparallel strands. The N-termini of the subunits form bundles of three α-helices extending into the RNA region of the virus at the 3-fold axis. The topology of the polypeptide chain is the same as, and the conformation clearly similar to, that of the shell domains of the Tomato bushy stunt virus (TBSV) and Southern bean mosaic virus (SBMV) protein subunits. The subunit packing in the T = 1 STNV structure is, however, significantly different from the packing of these T = 3 viruses: parts of some of the structural elements facing the RNA in TBSV and SBMV are utilized for subunit-subunit contacts in STNV. No RNA structure is obvious in the present icosahedrally averaged electron density maps. The protein surface facing the RNA contains mainly hydrophilic residues, especially lysine and arginine.

X-ray Structure and Ligand Binding Study of a Moth Chemosensory Protein

Chemosensory proteins (CSPs) are believed to be involved in chemical communication and perception. Such proteins, ofM r 13,000, have been isolated from several sensory organs of a wide range of insect species. Several CSPs have been identified in the antennae and proboscis of the mothMamestra brassicae. One of them, CSPMbraA6, a 112-amino acid antennal protein, has been expressed in large quantities and is soluble in the Escherichia coli periplasm. X-ray structure determination has been performed in parallel with ligand binding assays using tryptophan fluorescence quenching. The protein has overall dimensions of 25 × 30 × 32 Å and exhibits a novel type of α-helical fold with six helices connected by α−α loops. A narrow channel extends within the protein hydrophobic core. Fluorescence quenching with brominated alkyl alcohols or fatty acids and modeling studies indicates that CSPMbraA6 is able to bind such compounds with C12–18 alkyl chains. These ubiquitous proteins might have the role of extracting hydrophobic linear compounds (pheromones, odors, or fatty acids) dispersed in the phospholipid membrane and transporting them to their receptor.

Structure of Rhodococcus erythropolis limonene-1,2-epoxide hydrolase reveals a novel active site

Epoxide hydrolases are essential for the processing of epoxide‐containing compounds in detoxification or metabolism. The classic epoxide hydrolases have an α/β hydrolase fold and act via a two‐step reaction mechanism including an enzyme–substrate intermediate. We report here the structure of the limonene‐1,2‐epoxide hydrolase from Rhodococcus erythropolis, solved using single‐wavelength anomalous dispersion from a selenomethionine‐substituted protein and refined at 1.2 Å resolution. This enzyme represents a completely different structure and a novel one‐step mechanism. The fold features a highly curved six‐stranded mixed β‐sheet, with four α‐helices packed onto it to create a deep pocket. Although most residues lining this pocket are hydrophobic, a cluster of polar groups, including an Asp–Arg–Asp triad, interact at its deepest point. Site‐directed mutagenesis supports the conclusion that this is the active site. Further, a 1.7 Å resolution structure shows the inhibitor valpromide bound at this position, with its polar atoms interacting directly with the residues of the triad. We suggest that several bacterial proteins of currently unknown function will share this structure and, in some cases, catalytic properties.

Tryptophan 272: an essential determinant of crystalline cellulose degradation by Trichoderma reesei cellobiohydrolase Cel6A

Trichoderma reesei cellobiohydrolase Cel6A (formerly CBHII) has a tunnel shaped active site with four internal subsites for the glucose units. We have predicted an additional ring stacking interaction for a sixth glucose moiety with a tryptophan residue (W272) found on the domain surface. Mutagenesis of this residue selectively impairs the enzyme function on crystalline cellulose but not on soluble or amorphous substrates. Our data shows that W272 forms an additional subsite at the entrance of the active site tunnel and suggests it has a specialised role in crystalline cellulose degradation, possibly in guiding a glucan chain into the tunnel.

The missing piece of the type II fatty acid synthase system from Mycobacterium tuberculosis

The Mycobacterium tuberculosis fatty acid synthase type II (FAS-II) system has the unique property of producing unusually long-chain fatty acids involved in the biosynthesis of mycolic acids, key molecules of the tubercle bacillus. The enzyme(s) responsible for dehydration of (3R)-hydroxyacyl-ACP during the elongation cycles of the mycobacterial FAS-II remained unknown. This step is classically catalyzed by FabZ- and FabA-type enzymes in bacteria, but no such proteins are present in mycobacteria. Bioinformatic analyses and an essentiality study allowed the identification of a candidate protein cluster, Rv0635-Rv0636-Rv0637. Its expression in recombinant Escherichia coli strains leads to the formation of two heterodimers, Rv0635-Rv0636 (HadAB) and Rv0636-Rv0637 (HadBC), which also occurs in Mycobacterium smegmatis, as shown by split-Trp assays. Both heterodimers exhibit the enzymatic properties expected for mycobacterial FAS-II dehydratases: a marked specificity for both long-chain (≥C12) and ACP-linked substrates. Furthermore, they function as 3-hydroxyacyl dehydratases when coupled with MabA and InhA enzymes from the M. tuberculosis FAS-II system. HadAB and HadBC are the long-sought (3R)-hydroxyacyl-ACP dehydratases. The correlation between the substrate specificities of these enzymes, the organization of the orthologous gene cluster in different Corynebacterineae, and the structure of their mycolic acids suggests distinct roles for both heterodimers during the elongation process. This work describes bacterial monofunctional (3R)-hydroxyacyl-ACP dehydratases belonging to the hydratase 2 family. Their original structure and the fact that they are essential for M. tuberculosis survival make these enzymes very good candidates for the development of antimycobacterial drugs.