Kinetic and structural adaptation in P-loop type NTPases: The role of nucleotide and exchange factor interactions in GTPase and myosin function

Nucleotide triphosphatases (NTPases) containing phosphate-binding loop (P-loop) comprise a family of structurally related enzymes that utilize the free energy of nucleotide triphosphate binding and hydrolysis to perform useful biological work. To couple these processes efficiently, these NTPases are tightly controlled in vivo, and their biochemical cycles are highly adapted to perform their specific biological functions. A fundamental aspect of the NTPase cycle that governs NTPase activity are the linked equilibria for binding nucleotide and nucleotide exchange factors. Careful and quantitative analysis of the kinetics and thermodynamics of these NTPases with nucleotides and exchange factors, provides an understanding of how their interactions control the NTPase system in a manner that is specifically adapted to achieve the biological role(s) of these proteins.;The primary focus of this dissertation is to understand how the extent of coupling between nucleotide and exchange factor binding dictates physiological function in myosins and GTPases. Members of different myosin classes exhibit varying degrees of coupling between ADP and actin, and weak coupling in myosins V, VI, and VII results in ternary complex formation that allows these myosins to function as tension sensors and processive transporters. I address several consequences of weak coupling in these myosins in relation to their biological functions. In addition, I extend the principles and consequences of weak coupling into the GTPase systems, where it previously had not been identified. I show that the Rab GTPase Ypt1p functions with weak coupling between binding GDP/GTP and its guanine nucleotide exchange factor, TRAPP. Just as in the myosin superfamily, weak coupling in this GTPase is utilized to generate a stable ternary TRAPP-Ypt1p-GTP complex that presumably is important for the biological function of this GTPase in regulation of membrane tethering and vesicle fusion. Our exploration into coupling between nucleotide and exchange factor binding in NTPases has contributed to the understanding of how kinetic and thermodynamic adaptations in NTPase biochemical properties contribute to biological function in energy transducing systems.