Flexible pivoting of the pleckstrin domain domain in dynamin catalyzes fission by affecting membrane properties: Insights into the molecular degrees of freedom

Abstract

The neuronal dynamin1 functions in the release of synaptic vesicles by orchestrating the process of GTPase-dependent membrane fission. Dynamin1 associates with the plasma membrane-localized phosphatidylinositol-4,5-bisphosphate (PIP2) with its centrally-located pleckstrin homology domain (PHD). The PHD is dispensable as fission can be managed, albeit at much slower rates, even when the PHD-PIP2 interaction is replaced by a generic polyhistidine- or polylysine-lipid interaction. However, even when the PHD is present, the length of the dynamin scaffold and in turn the membrane remodeling and fission rates are severely restricted with mutations such as I533A on membrane-interacting variable loop 1 (VL1) of PHD. These observations suggest that PIP2-containing membrane interactions of PHD could have evolved to expedite fission to fulfill the requirement of rapid kinetics of synaptic vesicle recycling. Here, we use a suite of multiscale modeling approaches that combine atomistic molecular dynamics simulations, mixed resolution membrane mimetic models, coarse-grained molecular simulations and advanced free-energy sampling methods (metadynamics and umbrella sampling) to explore PHD-membrane interactions. Our results reveal that: (a) the binding of PHD to PIP2-containing membranes modulates the lipids towards fission-favoring conformations and softens the membrane, (b) that PHD engages another loop (VL4) for membrane association, which acts as an auxiliary pivot and modulates the orientation flexibility of PHD on the membrane - a mechanism we believe may be important for high fidelity dynamin collar assembly on the membrane. (c) Through analyses of our trajectories data and free-energy calculations on membrane-bound WT and mutant systems, we also identify key residues on multiple VLs that stabilizes PHD membrane association. And we suggest experiments to explore the ability of PHD to associate with membrane in orientations that favors faster fission. Together, these insights provide a molecular-level understanding of the "catalytic" role of the PHD in dynamin-mediated membrane fission. SIGNIFICANCEDynamin, a large multi-domain GTPase, remodels the membrane by self-assembling onto the neck of a budding vesicle and induces fission by its energy driven conformational changes. In this work, we use multi-scale molecular simulations to probe the role of dynamins pleckstrin-homology domain (PHD), which facilitates membrane interactions. Notably, PHD is dispensable for fission as is the case with extant bacterial and mitochondrial dynamins. However, reconstitution experiments suggest that the functional role of PHD in neuronal-membrane goes beyond that of an adaptor domain as it possibly expedites the fission reaction during synaptic vesicle recycling. We provide a molecular-dynamics picture of how PHDs make membranes more pliable for fission and suggest new insights into the molecular-level processes driving the expedited fission behavior.