Structural Determinants for Ca2+ and Phosphatidylinositol 4,5-Bisphosphate Binding by the C2A Domain of Rabphilin-3A

Abstract

Rabphilin-3A is a neuronal C2 domain tandem containing protein involved in vesicle trafficking. Both its C2 domains (C2A and C2B) are able to bind phosphatidylinositol 4,5-bisphosphate, a key player in the neurotransmitter release process. The rabphilin-3A C2A domain has previously been shown to bind inositol-1,4,5-trisphosphate (IP3; phosphatidylinositol 4,5-bisphosphate headgroup) in a Ca 2؉ -dependent manner with a relatively high affinity (50 M 2؉ binding enables the interaction with IP3 and vice versa, in line with a target-activated messenger affinity mechanism. Our data provide detailed structural insight into the functional properties of the rabphilin-3A C2A domain and reveal for the first time the structural determinants of a target-activated messenger affinity mechanism.)C2 domains are ubiquitous protein modules initially identified in protein kinase C (1). They all consist of ϳ130 residues (2) and share a common fold composed of two four-stranded ␤-sheets arranged in a compact ␤-sandwich and surrounded by variable loops and helices. C2 domain-containing proteins such as phospholipase C, cytosolic phospholipase A2, or protein kinase C are generally involved in signal transduction or membrane trafficking. The main function of the C2 domains in these proteins appears to be the targeting to biological membranes via their Ca 2ϩ -dependent phospholipid binding properties (3).Primary lipid targets are phosphatidylcholine (for cytosolic phospholipase A2 (4)) and phosphatidylserine (for phospholipase C␦ (5), synaptotagmins (6), and protein kinase C␣ (7)), the most abundant phospholipids in the inner membrane leaflet. Although not known to be classical phosphoinositide binding modules, some C2 domains have been shown to interact rather specifically with phosphoinositides (phospholipase C, protein kinase C␣), especially phosphatidylinositol 4,5-bisphosphate (PIP2) 3 (8). PIP2 represents less than 1% of the lipids in the plasma membrane and forms microdomains to regulate several key signaling events (9, 10). The interaction between the C2 domain of protein kinase C␣ and PIP2 is essential for the proper subcellular localization of protein kinase C␣ (11, 12). Several C2 domains of proteins involved in synaptic vesicle fusion have been described to bind PIP2, e.g. C2 domains of some synaptotagmins (6), CAPS-1 (13), and rabphilin-3A (14). The binding of the C2B domain of synaptotagmin 1 to PIP2 prelocalizes the protein to PIP2-enriched membranes (15). Recently a role for the CAPS-1 PIP2 interaction has been shown in the prefusion step of dense core vesicle fusion (13).PIP2 is a key player in the different stages of the neurotransmitter release process. It is required for clathrin-mediated endocytosis (16) or for specific stages of exocytosis and synaptic vesicle trafficking (10). PIP2 recruits proteins to specific locations in the plasma membrane to modulate spatially localized events involved in membrane trafficking (17).Rabphilin-3A was originally identified as a Rab3 effector in neuronal cells (18). It was found t...

Structural insights into the Ca 2+ and PI(4,5)P 2 binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1

“…However, this movement does not explain the large conformational change needed to open the CBR, as seen in the two Ca 2+ -bound structure (SI Appendix, Fig. S1 C and F) (27).…”

“…Strikingly, this ion was located close to the conventional position of Ca2 described for most of the Ca 2+ -dependent C2 domains (2,5,32), indicating the existence of an intermediate step in the calcium-binding process. Thus, we compared both the 3D structures and electrostatic potentials of the Ca 2+ -free (Protein Data Bank, PDB ID code 2CHD) (29) and the 2Ca 2+ -bound structures (PDB ID code 2K3H) (27) with the structure determined in this work.…”

“…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 Ca 2+ and phosphoinositides has hindered the understanding of the molecular mechanism driving the PI(4,5)P 2 -C2 domain interaction.…”

“…This site is also conserved in a wide variety of C2 domains of topology I, for example synaptotagmins, rabphilin 3A, DOC2, and PI3KC2α (10,(16)(17)(18)(19). Given the importance of PI(4,5)P 2 for bringing the vesicle and plasma membranes together before exocytosis to ensure rapid and efficient fusion upon calcium influx (20-23), 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 Ca 2+ and PI(4,5)P 2 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,(24)(25)(26)(27)(28). 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).…”

“…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 Ca 2+ and PI(4,5)P 2 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,(24)(25)(26)(27)(28). 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).…”