Structural characterization of the Rabphilin-3A-SNAP25 interaction

Membrane fusion is essential in a myriad of eukaryotic cell biological processes, including the synaptic transmission. Rabphilin-3A is a membrane trafficking protein involved in the calcium-dependent regulation of secretory vesicle exocytosis in neurons and neuroendocrine cells, but the underlying mechanism remains poorly understood. Here, we report the crystal structures and biochemical analyses of Rabphilin-3A C2B–SNAP25 and C2B–phosphatidylinositol 4,5-bisphosphate (PIP2) complexes, revealing how Rabphilin-3A C2 domains operate in cooperation with PIP2/Ca2+ and SNAP25 to bind the plasma membrane, adopting a conformation compatible to interact with the complete SNARE complex. Comparisons with the synaptotagmin1–SNARE show that both proteins contact the same SNAP25 surface, but Rabphilin-3A uses a unique structural element. Data obtained here suggest a model to explain the Ca2+-dependent fusion process by membrane bending with a myriad of variations depending on the properties of the C2 domain-bearing protein, shedding light to understand the fine-tuning control of the different vesicle fusion events.
Work in Barcelona was supported by Grants BIO2014-54588-P [Ministry of Economy and Competitiveness (MINECO), Spain–European Fund for Economic and Regional Development (FEDER)] and Maria de Maeztu Unit of Excellence MDM-2014-0435. Work in Murcia was supported by Grants BFU2014-52269-P (MINECO, Spain–FEDER) and Fundación Séneca Region de Murcia 19409/PI/14. X-ray data were collected at ALBA-CELLS (beamline XALOC) (Cerdanyola del Valles, Barcelona, Spain) with the collaboration of ALBA staff and at ESRF beamline ID23.2 (Grenoble, France). Financial support was also provided by ALBA and ESRF.