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Structural Characterization of the Rabphilin-3A–SNAP25 Interaction

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Structural Characterization of the Rabphilin-3A–SNAP25 Interaction Structural characterization of the Rabphilin- PNAS PLUS 3A–SNAP25 interaction Cristina Ferrer-Ortaa,1,2, María Dolores Pérez-Sánchezb,1, Teresa Coronado-Parrab, Cristina Silvaa, David López-Martínezb, Jesús Baltanás-Copadob, Juan Carmelo Gómez-Fernándezb, Senena Corbalán-Garcíab,2, and Núria Verdaguera,2 aStructural Biology Unit, Institut de Biologia Molecular de Barcelona, Consejo Superior de Investigaciones Cientificas, 08028 Barcelona, Spain; and bDepartamento de Bioquímica y Biología Molecular-A, Facultad de Veterinaria, Regional Campus of International Excellence “Campus Mare Nostrum,” Universidad de Murcia, Instituto Murciano de Investigación Biosanitaria Virgen de la Arrixaca, 30100 Murcia, Spain Edited by Axel T. Brunger, Stanford University, Stanford, CA, and approved May 30, 2017 (received for review February 14, 2017) Membrane fusion is essential in a myriad of eukaryotic cell biological the docking step of dense-core vesicles in PC12 cells (13, 14) and processes, including the synaptic transmission. Rabphilin-3A is a to limit the repriming of new vesicles during synaptic depression membrane trafficking protein involved in the calcium-dependent recovery in embryonic hippocampal neurons (15), suggesting regulation of secretory vesicle exocytosis in neurons and neuroendo- a fine-tuning role of Rph3A in the vesicle fusion machinery. crine cells, but the underlying mechanism remains poorly understood. Two distinct C2B–SNAP25 interfaces of interaction have been Here, we report the crystal structures and biochemical analyses of identified: the α-helices located at the bottom surface of the Rabphilin-3A C2B–SNAP25 and C2B–phosphatidylinositol 4,5-bisphos- domain (15) and the β3–β4 polybasic cluster (14). In contrast, phate (PIP2) complexes, revealing how Rabphilin-3A C2 domains oper- other works have demonstrated that this polybasic cluster is 2+ ate in cooperation with PIP2/Ca and SNAP25 to bind the plasma involved in direct contacts with the plasma membrane through membrane, adopting a conformation compatible to interact with the the phosphoinositide phosphatidylinositol 4,5-bisphosphate – complete SNARE complex. Comparisons with the synaptotagmin1 (PIP2) (11, 12, 16). However, the transient nature of these in- SNARE show that both proteins contact the same SNAP25 surface, teractions and the lack of high-resolution structures of Rph3A– but Rabphilin-3A uses a unique structural element. Data obtained here SNAP25–PIP complexes have prevented the detailed analysis of + 2 suggest a model to explain the Ca2 -dependent fusion process by residues involved and, consequently, hampered the efforts to membrane bending with a myriad of variations depending on the understand how these components orchestrate to bring about properties of the C2 domain-bearing protein, shedding light to under- vesicle fusion. stand the fine-tuning control of the different vesicle fusion events. In this study, we report the crystal structures of Rph3A C2B– SNAP25 and C2B–PIP2 complexes, revealing two different C2 domains | membrane fusion | Rabphilin-3A | SNAP-25 | contact interfaces: the former involves the bottom α-helices, and X-ray crystallography the latter, the polybasic cluster. Comparisons with the synapto- tagmin1–SNARE complex, solved by X-ray crystallography (17), eurons are able to communicate with others through the show that both proteins may contact the same SNAP25 surface Nliberation of neurotransmitters during synapses. Numerous but using distinct structural elements for the interaction. Lipid proteins are recruited to the presynaptic space to execute a highly controlled and precise mechanism, resulting in the liberation of Significance neurotransmitters to the synaptic cleft. Central components of the N process are the soluble -ethylmaleimide-sensitive factor attach- Membrane fusion is essential in multiple cell processes, including ment protein receptor (SNARE) proteins: syntaxin-1A (STX1A), neuronal communication. Numerous proteins are recruited to VAMP2 (synaptobrevin-2), and synaptosome-associated protein the presynaptic space to execute a highly controlled process, of 25 kDa (SNAP25), which fold into a stable four-helix bundle resulting in the liberation of neurotransmitters. Many of these that brings together vesicle and plasma membranes, driving proteins share C2 domains as common structural motifs, regu- – + membrane fusion (1 4). Other important players are complexin, lated by their ability to bind Ca2 , phospholipids, and other Munc18-1, Munc13, and a collection of proteins that share a proteins, endowing them with properties to fine-tune a wide common structural motif: the C2 domain (5, 6). These domains + variety of vesicle release modes. Here, by solving the structures are regulated by their ability to bind Ca2 , phospholipids, and of Rabphilin-3A (Rph3A) C2B–SNAP25 and Rph3A C2B–PIP2 protein interactions, endowing them with properties to fine-tune complexes, we revealed a membrane-binding mode in which the the wide variety of vesicle release modes (7). Nowadays, we have 2+ Rph3A-C2 domains operate in cooperation with PIP2/Ca and detailed information on many of the steps contributing to SNAP25, adopting a conformation able to promote membrane docking, priming, and fusion of these vesicles, but a clear picture bending, suggesting a model to explain how Rph3A regulates on how these regulators contribute in each particular state still various steps of the vesicle fusion process. remains under debate, mainly due to the lack of high-resolution structural data. Author contributions: C.F.-O., S.C.-G., and N.V. designed research; C.F.-O., M.D.P.-S., and Rabphilin-3A (Rph3A) is a membrane trafficking protein in- T.C.-P. performed research; C.S., D.L.-M., and J.B.-C. contributed new reagents/analytic + volved in the Ca2 -dependent regulation of secretory vesicle tools; C.F.-O., M.D.P.-S., T.C.-P., J.C.G.-F., S.C.-G., and N.V. analyzed data; and C.F.-O., exocytosis in neurons and neuroendocrine cells; however, its exact S.C.-G., and N.V. wrote the paper. role in the process still remains under debate. The protein is The authors declare no conflict of interest. targeted to synaptic or secretory vesicles through the interaction This article is a PNAS Direct Submission. with the small G-proteins Rab3 or Rab27, via its N-terminal Rab- Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 5LO8, 5LOW, and 5LOB). binding domain (8–10). Its C-terminal domain consists of tandem + 1C.F.-O. and M.D.P.-S. contributed equally to this work. C2 domains that are responsible for the Ca2 - and phospholipids- 2To whom correspondence may be addressed. Email: [email protected], [email protected], dependent membrane specificity of the protein (11, 12) and also or [email protected]. – BIOPHYSICS AND participate in other protein protein interactions. In particular, the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. C2B domain has been shown to interact with SNAP25 to regulate 1073/pnas.1702542114/-/DCSupplemental. COMPUTATIONAL BIOLOGY www.pnas.org/cgi/doi/10.1073/pnas.1702542114 PNAS | Published online June 20, 2017 | E5343–E5351 Downloaded by guest on September 26, 2021 sedimentation assays also indicate that the bottom α-helices are faces were relatively small (<350 Å2) and only partially con- crucial to interact with SNAP25, whereas the polybasic region is served between crystals (Fig. S4). essential for the phosphoinositide-dependent interaction with the In both crystal forms, C2B-C participated in contacts with two plasma membrane. Altogether, these data suggest that Rph3A might distinct SNAP25-N molecules using two different interfaces: the α adopt a specific PIP2-dependent conformation at the membrane first involved the bottom -helices mentioned above that in this that facilitates the interaction not only with SNAP25 molecules environment contacted the most N-terminal amino acids: M7, R8, but also with the SNARE complex, providing a plausible model to E10, M14, and Q15 of SNAP25 (Nt-region III) (contact area, explain how Rph3A C2B might regulate different steps in the ∼530 Å2;Fig.2andTable S2), and the second was contributed vesicle fusion process. mainly by the β3–β4 polybasic region through crystal packing in- teractions (contact area, ∼358 Å2; Fig. S4). Results and Discussion In all complexes, the calcium binding loops (CBRs) of the 2+ Structure of the Rph3A C2B–SNAP25 Complex. Cocrystals of Rph3A three bound C2B molecules appear coordinating two Ca ions, C2B domain (residues 536–680) in complex with the SNAP25 as previously described (16). In addition, the C2 crystals show the helical domains (SNAP25-N, residues 7–82; SNAP25-C, residues CBRs of molecules C2B-A and C2B-B free and exposed to the 2+ 141–203) were obtained in two different space groups, C2 and P21, solvent with a sulfate molecule tightly bound to the Ca ions, and the structures were solved at 3.3- and 2.8-Å resolution, re- probably mimicking the membrane phospholipids. spectively (Materials and Methods and Table S1). Structure de- Structural comparisons between the six independent SNAP25 termination was performed by molecular replacement using first molecules determined here with the equivalent helices in the – the X-ray data of the C2 crystals and the coordinates of the C2B crystallographic SNARE Syt1 complex (PDB ID 5CCG; ref. 17) domain [Protein Data Bank (PDB) ID 2CM5] as a search model. showed rmsd ranging from 2.27 to 2.67 Å for the superimposition
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