Using Structure to Study the Function of Proteins Involved in Antibiotic Resistance and Viral Infection

Abstract

Understanding the mechanisms that underpin antibiotic resistance is an important first step towards the development of therapeutics for the treatment of resistant bacterial infections. Bacteria have evolved diverse mechanisms to evade antibiotic induced cell death, one example being antibiotic target modification, such as the expression of low affinity class B penicillin binding proteins. X-ray crystallography is the primary approach for studying this family of proteins. Mutations that affect antibiotic adduct formation result in minimal structural changes; however, failing to explain how sequence variations lead to resistance. Our initial experiments on penicillin binding protein 5 (PBP5) identified residues that inhibit β-lactam adduct formation, yet the structural implications of these mutations were minimal, similar to what has been observed with other PBPs. In response, we developed a method for studying PBP5, a large (70 kDa), multi-domain protein, in solution, using NMR spectroscopy. We demonstrated that a serine insertion at position 466 works synergistically with an alanine mutation at position 485 to increase resistance to penicillin G (0.73-fold relative to wild type PBP5) and that this causes minimal structural changes (RMSDs of 0.1 and 0.2 Å when main chain atoms were aligned with apo- and PenG-acylated PBP5 WT, respectively). We then refolded PBP5 and characterized the quality of the protein using NMR, which showed an increased number of peaks and uniform peak intensity, and by using X-ray crystallography, which demonstrated that the refolded protein has the same structure as the protein before refolding (0.2 Å RMSD when main chain atoms were aligned with PBP5 before refolding). The second project described in this dissertation examines mechanisms of SARS-CoV-2 spike protein receptor binding domain (RBD) neutralization by antibodies. Many antibody-RBD structures fail to crystalize, necessitating the use of an alternative technique to characterize these interactions. NMR spectroscopy allows for the rapid determination of antibody epitopes, thus eliminating the need for the protein to be crystallized. We demonstrate that 39 residues experience chemical shift perturbation upon titration with the CR3022 fab. When mapped onto the structure, the most significantly perturbed residues correspond well with residues known to be located at the CR3022-RBD interface.

Taken together, these findings demonstrate that NMR spectroscopy can be used to gain atomic-resolution structural data on drug targets that require complicated protein refolding protocols. The refolding methods used to study PBP5 will allow for the characterization of interactions between PBP5 and variants with inhibitors and peptide substrates in solution. Finally, selective labeling, refolding, and purification of the SARS-CoV-2 RBD has allowed for the completion of the sequence specific backbone assignment, which can be used to rapidly determine antibody epitopes for RBD-antibody complexes that fail to crystalize.