Structural and mutational analyses of residues within the hydrophobic pocket suggest that budding results in a switch between two conformations of the capsid hydrophobic pocket. This is the first description of a viral budding mechanism in molecular detail.

The crystal structure of tetrameric pyruvate decarboxylase from Zymomonas mobilis has been determined at 1.9 Å resolution and refined to a crystallographic R-factor of 16.2% and R free of 19.7%. The subunit consists of three domains, all of the ␣/␤ type. Two of the subunits form a tight dimer with an extensive interface area. The thiamin diphosphate binding site is located at the subunit-subunit interface, and the cofactor, bound in the V conformation, interacts with residues from the N-terminal domain of one subunit and the C-terminal domain of the second subunit. The 2-fold symmetry generates the second thiamin diphosphate binding site in the dimer. Two of the dimers form a tightly packed tetramer with pseudo 222 symmetry. The interface area between the dimers is much larger in pyruvate decarboxylase from Z. mobilis than in the yeast enzyme, and structural differences in these parts result in a completely different packing of the subunits in the two enzymes. In contrast to other pyruvate decarboxylases, the enzyme from Z. mobilis is not subject to allosteric activation by the substrate. The tight packing of the dimers in the tetramer prevents large rearrangements in the quaternary structure as seen in the yeast enzyme and locks the enzyme in an activated conformation. The architecture of the cofactor binding site and the active site is similar in the two enzymes. However, the x-ray analysis reveals subtle but significant structural differences in the active site that might be responsible for variations in the biochemical properties in these enzymes.

In order to facilitate the three-dimensional structure comparison of proteins, software for making comparisons and searching for similarities to protein structures in databases has been developed. The program identi®es the residues that share similar positions of both main-chain and side-chain atoms between two proteins. The unique functions of the software also include database processing via Internet-and Web-based servers for different types of users. The developed method and its friendly user interface copes with many of the problems that frequently occur in protein structure comparisons, such as detecting structurally equivalent residues, misalignment caused by coincident match of C atoms, circular sequence permutations, tedious repetition of access, maintenance of the most recent database, and inconvenience of user interface. The program is also designed to cooperate with other tools in structural bioinformatics, such as the 3DB Browser software [Prilusky (1998). Protein Data Bank Q. Newslett. 84, 3±4] and the SCOP database [Murzin, Brenner, Hubbard & Chothia (1995). J. Mol. Biol. 247, 536±540], for convenient molecular modelling and protein structure analysis. A similarity ranking score of`structure diversity' is proposed in order to estimate the evolutionary distance between proteins based on the comparisons of their three-dimensional structures. The function of the program has been utilized as a part of an automated program for multiple protein structure alignment. In this paper, the algorithm of the program and results of systematic tests are presented and discussed.

The crystal structure of the complex of the thiamine diphosphate dependent tetrameric enzyme pyruvate decarboxylase (PDC) from brewer's yeast strain with the activator pyruvamide has been determined to 2.4 A Ê resolution. The asymmetric unit of the crystal contains two subunits, and the tetrameric molecule is generated by crystallographic symmetry. Structure analysis revealed conformational nonequivalence of the active sites. One of the two active sites in the asymmetric unit was found in an open conformation, with two active site loop regions (residues 104±113 and 290±304) disordered. In the other subunit, these loop regions are well-ordered and shield the active site from the bulk solution. In the closed enzyme subunit, one molecule of pyruvamide is bound in the active site channel, and is located in the vicinity of the thiazolium ring of the cofactor. A second pyruvamide binding site was found at the interface between the Pyr and the R domains of the subunit in the closed conformation, about 10 A Ê away from residue C221. This second pyruvamide molecule might function in stabilizing the unique orientation of the R domain in this subunit which in turn is important for dimer±dimer interactions in the activated tetramer. No difference electron density in the close vicinity of the side chain of residue C221 was found, indicating that this residue does not form a covalent adduct with an activator molecule. Kinetic experiments showed that substrate activation was not affected by oxidation of cysteine residues and therefore does not seem to be dependent on intact thiol groups in the enzyme. The results suggest that a disorder± order transition of two active-site loop regions is a key event in the activation process triggered by the activator pyruvamide and that covalent modification of C221 is not required for this transition to occur. Based on these findings, a possible mechanism for the activation of PDC by its substrate, pyruvate, is proposed.Keywords: enzyme mechanism; thiamine diphosphate; protein crystallography; conformational change.Thiamine diphosphate (ThDP) is an essential cofactor for a number of enzyme-catalysed reactions, especially in carbohydrate metabolism. Among ThDP dependent enzymes, pyruvate decarboxylase (PDC; EC 4.1.1.1.) has been widely studied to understand the mechanism of thiamine catalysis [1]. PDC catalyses the penultimate step in the alcoholic fermentation process, the nonoxidative decarboxylation of pyruvate to acetaldehyde. In yeast and bacteria, the catalytically active enzyme is composed of four identical or almost identical subunits. In the yeast Saccharomyces cerevisiae the major structural gene PDC1 codes for 563 amino acids [2]. PDCs from plants form higher oligomeric complexes with subunit sizes in the range of 59±67 kDa [3].All PDCs studied so far, except the enzyme from the bacterium Zymomonas mobilis [4,5], are subject to substrate activation. This process, characterized by a sigmoidal deviation from the hyperbolic v/S plot, was observed as early as 1967 in the enzyme specie...

Alphaviruses are enveloped, insect-borne viruses, which contains a positive-sense RNA genome. The protein capsid is surrounded by a lipid membrane, which is penetrated by glycoprotein spikes. The structure of the Sindbis virus (SINV) (the type virus) core protein (SCP) was previously determined and found to have a chymotrypsin-like structure. SCP is a serine proteinase which cleaves itself from a polyprotein. Semliki Forest virus (SFV) is among the most distantly related alphaviruses to SINV. Similar to SCP, autocatalysis is inhibited in SFCP after cleavage of the polyprotein by leaving the carboxy-terminal tryptophan in the specificity pocket. The structures of two different crystal forms (I and II) of SFV core protein (SFCP) have been determined to 3.0 A and 3.3 A resolution, respectively. The SFCP monomer backbone structure is very similar to that of SCP. The dimeric association between monomers, A and B, found in two different crystal forms of SCP is also present in both crystal forms of SFCP. However, a third monomer, C, occurs in SFCP crystal form I. While monomers A and B make a tail-to-tail dimer contact, monomers B and C make a head-to-head dimer contact. A hydrophobic pocket on the surface of the capsid protein, the proposed site of binding of the E2 glycoprotein, has large conformational differences with respect to SCP and, in contrast to SCP, is found devoid of bound peptide. In particular, Tyr184 is pointing out of the hydrophobic pocket in SFCP, whereas the equivalent tyrosine in SCP is pointing into the pocket. The conformation of Tyr184, found in SFCP, is consistent with its availability for iodination, as observed in the homologous SINV cores. This suggests, by comparison with SCP, that E2 binding to cores causes major conformational changes, including the burial of Tyr184, which would stabilize the intact virus on budding from an infected cell. The head-to-tail contacts found in the pentameric and hexameric associations within the virion utilize in the same monomer surface regions as found in the crystalline dimer interfaces.

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A new crystal form of thiamine diphosphate dependent pyruvate decarboxylase from Sacchavomyces cerevisiae has been obtained in the presence of the activator pyruvamide. The crystallographic structure analysis reveals differences in the domain packing in the enzyme subunit and a novel assembly of the subunits in the tetramer, when compared to the structure of native PDC. The orientation of the ß domains in the subunit differs by a 6.3° and 8.3° rotation, respectively, whereas the subunit-subunit interface in the dimer, formed by the a and y domains, is essentially maintained. In the tetramer, one of the dimers rotates relative to the second dimer by approximately 30° creating a new dimer-dimer interface.