STRUCTURAL STUDIES OF PENICILLIN BINDING PROTEINS

Elspeth Gordon, Nicolas Mouz, Emile Duee and Otto Dideberg

Laboratoire de Cristallographie Macromoléculaire, Institut de Biologie Structurale, 41, avnue des Martyrs, 38027 Grenoble Cedex 1, FRANCE
gordon@godot.ibs.fr

Penicillin binding proteins are the primary targets of ß-lactam antibiotics. Inhibition of these enzymes arrest the critical step of transpeptidation/cross-linking of the cell wall and result in cell death. In pathogenic strains of Streptococcus pneumoniae, drug resistance is due to modification of several of the essential PBPs. In particular, PBP2x has been shown to be involved in both low level and in high level drug resistance.

The aim of the study is to understand how antibiotics are recognised and react with PBP2x. And how aquisition of resistance changes these interactions.

Naturally occuring mutants from clinical isolated drug resistant strains have up to 100 changes in their amino acid sequence. The two most frequent substitutions, T338 and E552, are probably the most important in terms of aquisition of resistance. We have used crystallographic and site ditrected mutagenisis techniques to demonstrate the structural importance of these substitutions.

Thr338 adjacent to the active site serine points away from the active site but binds to a buried water molecule. Site directed mutation of the water ligands result in an enzyme with a reduced acetylation rate by antibiotics and modified kinetic parameters : a result demonstrating the structural importance of this water molecule.

Glu552 points into the active site and interacts directly with the bound antibiotic, a mutation at this point alters the charged nature of the active site.

Structural and biochemical data will be presented.