Purification and Crystallization of PBP2a from Methicilin-Resistant Staphylococcus aureus; poster

ROJAS-ALTUVE, A.; LLARRULL, L.I.; LASTOCHKIN, E.; HERMOSO, J.A.; MOBASHERY S.

Plymouth, Indiana

Jornada; 16th Annual Biochemistry Research Forum; 2011

Department of Chemistry and Biochemistry, University of Notre Dame

The Gram-positive bacterium Staphylococcus aureus is a leading cause of hospital- and community-associated infections. Of particular concern is the growing prevalence of methicillinresistant S. aureus (MRSA) in both hospital- and community-associated infections. Staphylococci have two primary mechanisms for resistance to beta-lactam antibiotics: the expression of PC1 beta-lactamase, an enzyme that hydrolyzes the beta-lactam ring, thus rendering the antibiotic inactive, and the acquisition of a gene encoding a modified penicillin-binding protein (PBP), known as PBP 2a, found in MRSA and coagulase-negative staphylococci. PBP 2a is intrinsically resistant to inhibition by beta-lactams. PBP 2a remains active in the presence of concentrations of beta-lactam antibiotics that inhibit most PBP enzymes, thus substituting for their functions in cell wall synthesis and allowing growth in the presence of the beta-lactam inhibitors. In this report, we describe a three-step purification of a soluble form of PBP 2a (PBP 2a?) to homogeneity using anion-exchange, cation-exchange, and size-exclusion chromatography. Purified PBP2a? was a 74-kDa monomeric protein, that retained the ability to be irreversibly acylated by beta-lactam antibiotics, as was shown using the reporter chromogenic beta-lactam nitrocefin. Purified PBP2a was crystallized, which diffracted at low resolution. Unexpectedly, mass spectrometry analysis of the protein in the crystals showed the presence of a truncated protein (amino acids 122-668). In order to improve the quality of the crystals for structure determination, and eventually co-crystallization with inhibitors, we constructed two truncated versions of PBP2a: one identical to the truncatedprotein observed in the crystals, and a smaller version in which a predicted mobile loop was removed (amino acids 138-668). The purification of these new truncated proteins is under way in the lab.