Cloning and X-Ray Structure of the Pyrrolidone Carboxyl Peptidase from the Archaeon Thermococcus litoralis
Jennifer LITTLECHILD*, Martin SINGLETON, and Michail ISUPOV
Protein Structure Group,Departments of Chemistry and Biological Sciences, University of Exeter, Exeter, U.K.
Pyrrolidone carboxyl peptidases (Pcps) (EC 3.4.19.3) catalyse the hydrolysis of amino terminal pyroglutamate residues from peptides and proteins. Pcps have been divided into two classes; the type I enzyme, found in both prokaryotes and eukaryotes is a soluble cysteine protease, while the type II enzyme, isolated from mammalian brain tissue, is a membrane-bound metallopeptidase. The thermostable type I enzyme from the hyperthermophilic archaeon Thermococcus litoralis has been cloned and overexpressed in Escherichia coli. The structure of the enzyme has been solved by multiple isomorphous replacement with averaging and solvent flattening and refined at 1.76Å resolution to a crystallographic R factor of 18.7%. The enzyme, of total o molecular weight 96 kDa is a homotetramer of dimensions 70 x 75 x 53 Å. Each monomer of the enzyme is folded into a single α/β domain, consisting of seven parallel and antiparallel strands surrounded by helices on both sides. The pairs of subunits are linked by two disulphide bridges located in a short helix at the subunit interface. The residue Cys 143, proposed to be the nucleophilic group in substrate hydrolysis appears to form a catalytic triad with His 167 and Glu80. These residues line a prominent cleft in the structure, formed by the two sets of helices and the edge of the β-sheet. A striking feature of the structure is a long loop which contains an extremely hydrophobic sequence of residues, unique to the archaeal enzyme. This forms a tightly packed core in the centre of the tetramer, and is an important determinant of the enzymes thermostability. The overall fold of the enzyme is similar to that of several zinc-containing metallopeptidases such as carboxypeptidase A, but shows no similarity to any eukaryotic cysteine proteases.
