Elucidating beta-lactamase functional mechanisms via evolutionary conservation

DESCRIPTION (provided by applicant): Penicillins and chemically related molecules are our most abundant and common used class of antibiotics, which are characterized by a conserved 4-atom ¿-lactam ring. Historically, they have been an effective treatment to gram-positive bacterial infections; however, the cell wall of gram-negative bacteria poses an effective barrier to antibiotic penetration. Conversely, second generation cephalosporins are also effective against gram-negative bacteria because they are able to penetrate the cell wall. Nevertheless, an increasing number of bacteria are resistant due to the enzyme ?-lactamase (BL). BL, which is expressed in the cell wall, hydrolyzes the ? -lactam ring, thus rendering the antibiotic ineffective. Due to decades of antibiotic overuse, BL enzymes have alarmingly evolved additional resistances that are now breaking down our last lines of defense. For example, extended spectrum ? -lactamases (ESBL) also hydrolyze the ? -lactam ring of cephalosporins, which have generally been resistant to BL activity. As such, a better understanding of BL resistance mechanisms is imperative so that new and more effective antibiotics can be developed quickly. There are four common classes of BL enzymes, which reflect specific sequence, structure and antibiotic resistance patterns. The Class A, C and D enzymes share a serine-based hydrolysis, whereas the catalytic mechanism of Class B enzymes is based on a zinc ion. However, little is known about how dynamics and stability vary across the superfamily. Are stability and/or dynamical mechanisms conserved across the superfamily? Are certain mechanisms critical to function? Can mechanistic differences help explain antibiotic resistance patterns? Is allostery conserved across the superfamily? These are the types of unanswered questions this proposal seeks to answer. To that end, we will employ a powerful and fast computational distance constraint model (DCM) to characterize the serine-based classes. While broad characterization across the BL superfamily has not yet been done, a small number of Class A structures have been studied by NMR and molecular dynamics simulation. Interestingly, these structures appear extraordinarily rigid, punctuated by flexible loop regions that may or may not be related to function. Our preliminary DCM characterizations across Class A enzymes reproduce these results. Even so, there is significant diversity within dynamical quantities across the family, which reflects evolutionary out-groups and, in many cases, parallels the onset of extended-spectrum activities. Taken together, these preliminary results highlight how the synthesis of biophysical descriptions with the paradigm of comparative bioinformatics synergistically improves the importance and accuracy of our characterizations. As such, we propose a series of additional studies along these lines to expand our understanding of BL structure and function, potentially paving the way to new therapeutic opportunities.