Crystal structure of PBP2x from a highly penicillin-resistant Streptococcus pneumoniae clinical isolate - A mosaic framework containing 83 mutations

Penicillin-binding proteins (PBPs) are the main targets for beta -lactam an tibiotics, such as penicillins and cephalosporins, in a wide range of bacte rial species. In some Gram-positive strains, the surge of resistance to tre atment with beta -lactams is primarily the result of the proliferation of m osaic PBP-encoding genes, which encode novel proteins by recombination. PBP 2x is a primary resistance determinant in Streptococcus pneumoniae, and its modification is an essential step in the development of high level beta -l actam resistance. To understand such a resistance mechanism at an atomic le vel, we have solved the x-ray crystal structure of PBP2x from a highly peni cillin-resistant clinical isolate of S. pneumoniae, Sp328, which harbors 83 mutations in the soluble region. In the proximity of the Sp328 PBP2x* acti ve site, the Thr(338)--> Ala mutation weakens the local hydrogen bonding ne twork, thus abrogating the stabilization of a crucial buried water molecule . In addition, the Ser(389)--> Leu and Asn(514)--> His mutations produce a destabilizing effect that generates an "open" active site. It has been sugg ested that peptidoglycan substrates for beta -lactam-resistant PBPs contain a large amount of abnormal, branched peptides, whereas sensitive strains t end to catalyze cross-linking of linear forms. Thus, in vivo, an "open" act ive site could facilitate the recognition of distinct, branched physiologic al substrates.