UC Riverside

Structure and dynamics are essential elements of protein function. Protein structure is constantly fluctuating and undergoing conformational transitions, which are typically captured by molecular dynamics (MD) simulations. Conformational state transitions in a protein involve shifts in its equilibrium conformations that occur either independently or as a response to external perturbations. In this work, we describe the effect of ligand binding and post-translational modifications (PTMs) to proteins as an external perturbation responsible for conformational changes in chemokine receptor 7 (CCR7) and the KU70-KU80 protein complex, respectively. In both systems, we isolate specific side chain rearrangements that act as molecular switches, and mediate the allosteric communication between distant functional sites in a protein, as a mechanism to regulate conformational state transitions and sampling. Specifically, in CCR7, we focus on the role of allostery in regulating the information transduced from the ligand-binding site to the intracellular region of the receptor to allow discrimination in binding intracellular effectors. This phenomenon is known as biased activation and is critical to G protein-coupled receptor function. In our work, we detect a series of molecular switches in CCR7 that are coupled to various ligand-induced allosteric events. Although these molecular switches mediate the transitioning between different states, the receptor remains inactive (absence of the canonical TM6 outward movement), illustrating loose coupling between the extracellular ligand-binding site and the intracellular effector-binding site. This finding might justify the existence of a novel hybrid model in CCR7, consisting of a “rhodopsin-like” sequential network of allosteric events (mediated by molecular switches) and a “β2-adrenergic-like” loose coupling between the extracellular and intracellular regions of the receptor. Furthermore, MD simulations of the ligand-free receptor highlight the importance of the ligand in coordinating the receptor’s side-chain fluctuations. We also focus on developing new methods to systematically detect coupled molecular switches and large domain motions in membrane proteins. Finally, we used MD simulations and electrostatic calculations to identify the role of PTMs, such as acetylation and methylation, on KU70-KU80’s dynamics. Such PTMs are shown to regulate conformational changes within several of KU70’s functional domains through acetylation-dependent alteration of the electrostatic profile of the DNA-binding and linker-SAP domains, and methylation-dependent molecular switching that is responsible for regulating a “pendulum-like” motion in linker-SAP domain.