Staphylococcus aureus is among the most prevalent and antibiotic-resistant of pathogenic bacteria. However, beta-lactam antibiotics, the most effective therapy for treating staphylococcal infections, are becoming less effective as resistance to them among staphylococci increases. Resistance is mediated by a beta-lactamase (encoded by blaZ) that hydrolyzes penicillins and an alternate target, penicillin binding protein (PBP2a, encoded by mecA), to which beta-lactam antibiotics bind poorly. Transcription of mecA and blaZ is activated by signal transduction through either MecRl or BlaRl when the bacterial cell is exposed to an inducing beta-lactam. The mec (MecRI and Mecl) and bla (BlaRl and Blal) regulators seem to have a signal transduction mechanism of action that is unique in prokaryotic biology but its mechanism is unclear, due in part to lack of structural information. Answers to this question not only solve an interesting biological question but may also uncover an induction cascade involving other molecules that could link regulation of P-lactam resistance to essential cellular systems. The major goal of this application is to determine the crystal structures of the multidomain integral membrane MecRl and/or BlaRl proteins that will provide a unique insight into the relationship between the parts of the molecules that are responsible for signal reception and signal transduction. An important long-term objective of this application is to provide a structural framework for understanding the molecular mechanisms involved in antibiotic resistance and the development of novel therapeutics. Moreover, the studies will also provide a wealth of other information, including membrane protein production, solubilization and crystallization techniques that are crucial to the continued understanding of integral membrane proteins.
The specific aims are: (1) Structure determination of the intact multidomain BlaRl/MecRl proteins, (2) structure determination of rationally chosen fragments of the BlaRl/MecRl protein, and (3) structure determination of complex between BlaRl/MecRl and their homologous repressers Blal/MecI. The clones and subclones of BlaRl and MecRl will be expressed in E. coli and purified. Crystallization experiments to obtain diffraction-quality crystals using the detergent-micelle, cubic-phase, vesicle-fusion, and bicelle techniques will be undertaken. Subsequently, the structures of the proteins will be determined using X-ray crystallography. ? ?
