CAREER: Molecular evolution of antibiotic export by the SMR family of membrane proteins

One way bacteria gain resistance to toxins (such as antibiotics or antiseptics) is through the evolution of transporters that eject toxic compounds from the cell. This adaptation mechanism utilizes protein assemblies in the membrane to "pump" the toxins out, yet maintain the integrity of the impermeable membrane barrier that defines the cell. Such proteins undermine antibiotic treatment because they actively remove these drugs from the cell. This project will investigate the evolution of antibiotic and antiseptic resistance in bacteria, focusing on a particular class of membrane proteins known as the "small multidrug resistance transporters." This project will reveal fundamental knowledge that will inform the fight against drug-resistant bacteria. In addition, this research will elucidate how transport of select compounds across cell membranes evolved in nature. This knowledge will impact bioengineering strategies, with potential applications in biofuel production, bioremediation, and nutritional fortification of foods. This project will also provide training opportunities for undergraduates to learn cutting-edge scientific techniques. It will also include development of an augmented reality app that will engage undergraduate students and the general public in learning how molecular machines function and can be exploited in biotechnology.<br/><br/>The overarching scientific objective of this project is to gain insight into two key evolutionary events for membrane proteins: a) duplication of a progenitor single-domain protein to generate the two-domain architecture found in a plurality of transporters; and b) evolution of novel substrate specificity of such transporters. The PI has recently identified and functionally surveyed a family of microbial membrane proteins that is an excellent model for both of these processes, the SMR (small, multidrug resistance) family, which comprises two subtypes: exporters of guanidinium ion, and exporters of bulky hydrophobic antimicrobials. Objective 1 will characterize, in atomic detail, the structure, mechanism and mutational robustness of representative SMR proteins that, because of their position in the phylogenetic tree, will be particularly informative for evolutionary analyses. Objective 2 will test hypotheses about the evolutionary mechanism by which drug export function emerged in the SMR family. To do this, we will use a multidisciplinary approach combining evolutionary techniques such as phylogenetics, ancestral reconstruction, and direct coupling analysis with membrane protein biochemistry, electrophysiology, and crystallographic approaches.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.