UCSF

Abstract Exploiting conformational dynamics is a powerful method for inhibiting enzymes and can produce new allosteric inhibitors for managing disease. In this work, we outline the discovery and characterization of an antibody fragment called Fab5 which inhibits the human Cytomegalovirus protease (HCMV Pr). Using ensemble biophysical techniques, we describe the dynamic relationship between Fab5 and HCMV Pr, from which we propose a mechanism of inhibition along with potential insights into the broader conformational network that controls all human Herpesvirus Protease (HHV Pr) activation. Mutagenesis and biochemical characterization allow us to validate our hypotheses in vitro. We provide novel tools and strategies for HHV Pr inhibition that we believe can be leveraged in future therapeutic development. In Chapter 1.1, we use cryogenic electron microscopy (cryo-EM) to reveal the Fab5 binding site on HCMV Pr which we call the Latch Loop. We propose that Fab5 inhibits HCMV Pr by preventing dimerization through an allosteric mechanism that is both mechanistic and dynamic, ultimately preventing the protease from activating. Mutagenic studies demonstrate the previously unrecognized importance of the Latch Loop in the conformational network that links dimerization to HHV Pr activation. In Chapter 1.2, we use X-ray radiolytic Footprinting to survey solvent accessibility changes on HCMV Pr after Fab5 binding. These studies reveal a residue network near the HCMV Pr active site that influences dimerization and, thus, activity despite being buried in the center of the protease.