Linking Protein Sequence to Structure and Function in Bacterial DHFR Enzymes

Abstract

Evolution has led to significant sequence variation between homologous proteins present in different organisms. Natural variation is a part of the theoretical sequence space that can fold into a specific protein structure, which in turn plays an important role in determining the functionality of the protein. Understanding how sequence variation affects protein structure and function will enable the development of effective therapeutic strategies to curb infections by bacteria. In this study, the impact of interspecific and intraspecific variation on protein structure and function in bacterial Dihydrofolate Reductase (DHFR) enzymes is investigated. Trimethoprim resistance in Escherichia coli was used as a phenotypic read-out to investigate the impact of single and combinatorial mutations in DHFR on protein function. The results indicate that natural sequence variation in DHFR has the potential to influence intrinsic and mutationally acquired trimethoprim resistance. The results show that the ability of intraspecific variants in E. coli DHFR to confer trimethoprim resistance depends on the physiochemical properties of the residues. Moreover, the results also show that a combination of mutations has drastically different phenotypic effects than their corresponding single mutations. The results display the impact of intraspecific variations on the phenotypic effects of other resistance-conferring mutations. At least some of these findings can be explained by how sequence variation alters the stability of DHFR, as well as its tolerance to mutation. This study, thus, demonstrates the potential of standing sequence variation in proteins to significantly impact the evolution of organismal traits with biomedical significance, such as antimicrobial resistance.