Dynamin stucture and function hinging on Ryngos
- Cell Signalling Unit, Children’s Medical Research Institute, University of Sydney, NSW, Australia.
- School of Environmental and Life Sciences, Faculty of Science, University of Newcastle, NSW, Australia.
Dynamins are GTPase enzymes responsible for performing the final scission of invaginated plasma membrane prior to completion of endocytosis. Pharmacological targeting in relevant mouse models has been shown to provide therapeutic relief for ailments as diverse as chronic kidney disease and epilepsy. We have generated a series of small molecule modulators (Ryngos) which ’lock’ dynamin into a ’ring’ oligomer that structurally differs from the ’helical’ state required for endocytosis. Ryngos exhibit different actions on enzyme activity in vitro (Ryngo-1, mixed-mode; Ryngo-3, stimulation). Due to their chemical similarity, it can be surmised that these compounds share a common binding pocket. This study aims to establish the binding site of Ryngos to allow for targeted drug design. Advanced computer modelling predicted lead compounds; Ryngo-1-23 and Ryngo-3-32, independently localised to, and differentially interacted with Hinge 1, located between middle domain and bundle-signalling element of dynamin. Partial overlap of residues between Ryngo-1-23 and Ryngo-3-32 suggests drug binding to different sub-regions of Hinge 1 may be capable of imparting different actions (inhibition/stimulation) on dynamin activity. To validate this, mutagenesis of Hinge 1 residues was undertaken and mutants characterised. Functional assays largely support these predictions (i.e. single mutations selectively lost drug action) whilst highlighting a broader role for Hinge 1 in dynamin characteristics (activity, oligomerisation). To account for allosteric effects of mutation, a chemically dissimilar dynamin inhibitor (Dynole-34-2) revealed loss of Ryngo action to be specific to Hinge 1. The data supports the model of these compounds differentially interacting with a flexible hinge within dynamin, an exceptionally rare binding site in pharmacology.