Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

Dynamin structure and function hinging on Ryngos (#108)

David A. Cardoso 1 , Mohammed K. Abdel-Hamid 2 , Adam McCluskey 2 , Phil J. Robinson 1
  1. Cell Signalling Unit , Children's Medical Research Institute, University of Sydney, Westmead, NSW, Australia
  2. Faculty of Science, University of Newcastle, Newcastle, NSW, Australia

Dynamins are multi-domain GTPase enzymes capable of performing the final scission of invaginated plasma membrane prior to the completion of endocytosis. Pharmacological targeting of dynamin in relevant mouse models has been shown to provide therapeutic relief for diseases such as chronic kidney disease and epilepsy. We have generated a series of small molecule dynamin modulators (Ryngos) which ‘lock’ dynamin into a ‘ring’ oligomeric state that structurally differs from the ‘helical’ state required for endocytosis. These compounds exhibit different actions on dynamin enzyme activity in vitro (Ryngo-1: mixed-mode / Ryngo-3: stimulation). Due to their chemical similarity, it can be surmised that these pharmacological agents share a common binding pocket. To establish their binding site, advanced computer modelling techniques were employed. The model 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. A partial overlap of implicated 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 (stimulation/inhibition) on dynamin activity in vitro. To validate this model, mutagenesis of implicated Hinge 1 residues was carried out and resultant mutants characterised. Functional assays largely support these predictions (i.e. single mutations specifically lost drug action) as well as highlight a broader role for Hinge 1 in dynamin characteristics (e.g. activity, oligomerisation). To account for allosteric effects of mutation, a chemically dissimilar dynamin-targeting compound (Dynole-34-2) was utilised and revealed loss of Ryngo action to be specific to Hinge 1. The data supports the proposed model of these compounds differentially interacting with a flexible hinge within dynamin.