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Structural insights into the Ca2+ and PI(4,5)P2 binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1
Authors:Jaime Guillén  Cristina Ferrer-Orta  Mònica Buxaderas  Dolores Pérez-Sánchez  Marta Guerrero-Valero  Ginés Luengo-Gil  Joan Pous  Pablo Guerra  Juan C. Gómez-Fernández  Nuria Verdaguer  Senena Corbalán-García
Affiliation:aDepartamento de Bioquímica y Biología Molecular A, Facultad de Veterinaria, Regional Campus of International Excellence “Campus Mare Nostrum,” Universidad de Murcia, 30100 Murcia, Spain;;bInstituto de Biología Molecular de Barcelona, Consejo Superior de Investigaciones Cientificas, 08028 Barcelona, Spain; and;cInstitute for Research in Biomedicine, Parc Científic de Barcelona, 08028 Barcelona, Spain
Abstract:Proteins containing C2 domains are the sensors for Ca2+ and PI(4,5)P2 in a myriad of secretory pathways. Here, the use of a free-mounting system has enabled us to capture an intermediate state of Ca2+ binding to the C2A domain of rabphilin 3A that suggests a different mechanism of ion interaction. We have also determined the structure of this domain in complex with PI(4,5)P2 and IP3 at resolutions of 1.75 and 1.9 Å, respectively, unveiling that the polybasic cluster formed by strands β3–β4 is involved in the interaction with the phosphoinositides. A comparative study demonstrates that the C2A domain is highly specific for PI(4,5)P2/PI(3,4,5)P3, whereas the C2B domain cannot discriminate among any of the diphosphorylated forms. Structural comparisons between C2A domains of rabphilin 3A and synaptotagmin 1 indicated the presence of a key glutamic residue in the polybasic cluster of synaptotagmin 1 that abolishes the interaction with PI(4,5)P2. Together, these results provide a structural explanation for the ability of different C2 domains to pull plasma and vesicle membranes close together in a Ca2+-dependent manner and reveal how this family of proteins can use subtle structural changes to modulate their sensitivity and specificity to various cellular signals.C2 modules are most commonly found in enzymes involved in lipid modifications and signal transduction and in proteins involved in membrane trafficking. They consist of 130 residues and share a common fold composed of two four-stranded β-sheets arranged in a compact β-sandwich connected by surface loops and helices (14). Many of these C2 domains have been demonstrated to function in a Ca2+-dependent membrane-binding manner and hence act as cellular Ca2+ sensors. Calcium ions bind in a cup-shaped invagination formed by three loops at one tip of the β-sandwich where the coordination spheres for the Ca2+ ions are incomplete (57). This incomplete coordination sphere can be occupied by neutral and anionic (79) phospholipids, enabling the C2 domain to dock at the membrane.Previous work in our laboratory has shed light on the 3D structure of the C2 domain of PKCα in complex with both PS and PI(4,5)P2 simultaneously (10). This revealed an additional lipid-binding site located in the polybasic region formed by β3–β4 strands that preferentially binds to PI(4,5)P2 (1115). This site is also conserved in a wide variety of C2 domains of topology I, for example synaptotagmins, rabphilin 3A, DOC2, and PI3KC2α (10, 1619). Given the importance of PI(4,5)P2 for bringing the vesicle and plasma membranes together before exocytosis to ensure rapid and efficient fusion upon calcium influx (2023), it is crucial to understand the molecular mechanisms beneath this event.Many studies have reported different and contradictory results about the membrane binding properties of C2A and C2B domains of synaptotagmin 1 and rabphilin 3A providing an unclear picture about how Ca2+ and PI(4,5)P2 combine to orchestrate the vesicle fusion and repriming processes by acting through the two C2 domains existing in each of these proteins (16, 20, 22, 2428). A myriad of works have explored the 3D structure of the individual C2 domains of both synaptotagmins and rabphilin 3A (5, 26, 27, 29, 30). However, the impossibility of obtaining crystal structures of these domains in complex with Ca2+ and phosphoinositides has hindered the understanding of the molecular mechanism driving the PI(4,5)P2–C2 domain interaction. Here, we sought to unravel the molecular mechanism of Ca2+ and PI(4,5)P2 binding to the C2A domain of rabphilin 3A by X-ray crystallography. A combination of site-directed mutagenesis together with isothermal titration calorimetry (ITC), fluorescence resonance of energy transfer (FRET), and aggregation experiments has enabled us to propose a molecular mechanism of Ca2+/PI(4,5)P2-dependent membrane interaction through two different motifs that could bend the membrane and accelerate the vesicle fusion process. A comparative analysis revealed the structural basis for the different phosphoinositide affinities of C2A and -B domains. Furthermore, the C2A domain of synaptotagmin 1 lacks one of the key residues responsible for the PI(4,5)P2 interaction, confirming it is a non-PI(4,5)P2 responder.
Keywords:PIP2   calcium   vesicle fusion
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