Kinetic and Structural Analyses of a Dynamin Mechanoenzyme

Abstract

Proteins in the dynamin superfamily are large, multi-domain mechanoenzymes that harness the energy of GTP hydrolysis to remodel intracellular membranes. Defects in these proteins result in severe human diseases, including cardiomyopathy, neurological abnormalities and neonatal death. While previous enzymological studies on dynamin proteins suggest that they exhibit assembly-stimulated GTP hydrolysis, the mechanism by which self-assembly stimulates GTPase activity is poorly understood. Here, we present the first quantitative kinetic model for GTP hydrolysis by a dynamin superfamily member that accounts for the complex equilibria between multiple self-assembling species of enzyme, all of which are catalytically active. We found that assembly-stimulated hydrolysis was not well described by a monomer-n-mer model, but could be fit equally well to dimer-n-mer and tetramer-n-mer models. Discrimination between these and other models required the design and use of enzyme variants trapped in different assembly states. Iterative global analysis of these data coupled with statistical analyses suggests a kinetic model containing a dimer-tetramer-n-mer equilibrium. Together, these studies allow for a quantitative understanding of how self-assembly of dynamin proteins stimulates their hydrolytic activities and lay the foundation for discerning the effect of multiple factors including interacting proteins, small molecular inhibitors and activators, post-translational modifications and membrane interaction on dynamin self-assembly and GTP hydrolysis.