X-RAY STRUCTURE OF A PRIMARY PENICILLIN TARGET ENZYME: PBP2X FROM S. PNEUMONIAE O. Dideberg, E. Gordon, S. Pares & N. Mouz, L.C.M., Institut de Biologie Structurale Jean-Pierre Ebel 41, avenue des Martyrs, 38027 GRENOBLE CEDEX 1, FRANCE.PP

All [[beta]]-lactam antibiotics exert their biological effects by interacting with a unique class of proteins, the penicillin-binding proteins (PBPs). These membrane proteins are involved in the biosynthesis of the murein or peptidoglycan, a mesh-like structure which completely surrounds the bacterial cell. Sequence similarities indicate that one domain of these proteins belongs to a large family of [[beta]]-lactam-recognizing proteins, which includes the active-site serine [[beta]]-lactamases. Since all PBPs are membrane bound, molecular genetic techniques were used to delete the hydrophobic N-terminal membrane-anchoring domain of PBP2x from S. pneumoniae and to overexpress the resulting protein (PBP2x*) in an E. coli system. The water-soluble PBP2x* has been crystallised. Its structure was solved to 3.5 Å resolution using the multiple isomorphous replacement method with density modification and phase combination using X-ray data collected on the PBP2x* protein and heavy atom derivative crystals. The present model has a crystallographic R-factor of 22.5% and r.m.s deviations of 0.012 Å and 2.0deg. from the ideal bond lengths and bond angles, respectively.

PBP2x* is a three-domain protein with dimensions of 97 Å x 60 Å x 49 Å. The domain folds bear no resemblance to any known structure, except for the central domain which shows similarities to the active-site serine [[beta]]-lactamases. The N-terminal domain (up to residue 265) is sugar-tongs shaped and shows a hole with a diameter of about 10 Å . This hole is 65 Å away from serine 337, which catalyses the transpeptidase activity and is located in the central domain. No biological function has been reported so far for the N-terminal domain of PBP2x. The central domain (266-616) contains the active site serine 337 and can therefore be called the transpeptidase domain. The structure of the Carboxy-terminal domain (635-750), found in some, but not all, hmm PBPs , suggests a gene duplication of a small [[alpha]][[beta]][[beta]][[beta]] domain. Its function is unknown.