Ca2+ binding to synaptotagmin: how many Ca2+ ions bind to the tip of a C2-domain?

Abstract

C 2 -domains are widespread protein modules with diverse Ca 2⍣ -regulatory functions. Although multiple Ca 2⍣ ions are known to bind at the tip of several C 2 -domains, the exact number of Ca 2⍣ -binding sites and their functional relevance are unknown. The first C 2 -domain of synaptotagmin I is believed to play a key role in neurotransmitter release via its Ca 2⍣ -dependent interactions with syntaxin and phospholipids. We have studied the Ca 2⍣ -binding mode of this C 2 -domain as a prototypical C 2 -domain using NMR spectroscopy and site-directed mutagenesis. The C 2 -domain is an elliptical module composed of a β-sandwich with a long axis of 50 Å. Our results reveal that the C 2 -domain binds three Ca 2⍣ ions in a tight cluster spanning only 6 Å at the tip of the module. The Ca 2⍣ -binding region is formed by two loops whose conformation is stabilized by Ca 2ϩ binding. Binding involves one serine and five aspartate residues that are conserved in numerous C 2 -domains. All three Ca 2⍣ ions are required for the interactions of the C 2 -domain with syntaxin and phospholipids. These results support an electrostatic switch model for C 2 -domain function whereby the β-sheets of the domain provide a fixed scaffold for the Ca 2⍣ -binding loops, and whereby interactions with target molecules are triggered by a Ca 2⍣ -induced switch in electrostatic potential.

The C2b Domain of Synaptotagmin Is a Ca2+–Sensing Module Essential for Exocytosis

“…The C2A domain exhibits a similar conformational change in limited proteolysis experiments (Davletov and Südhof 1994). These changes may reflect the movement of charged residues within or near the Ca 2+ -binding loops (Ubach et al 1998b; Chae et al 1998), as indicated by structural studies of the C2 domain of phospholipase C-δ1 (Grobler et al 1996). Given the diffusion-limited kinetics of the C2 domains of synaptotagmin, these conformational changes are unlikely to involve large-scale structural rearrangements (Davis et al 1999).…”

“…(A) Schematic diagram of putative Ca 2+ ligands within the C2B domain of synaptotagmin I. This model is based on structural studies of a variety of C2 domains (Sutton and Sprang 1995; Sutton et al 1995; Grobler et al 1996; Perisic et al 1998; Ubach et al 1998a,Ubach et al 1998b) and alignments between the C2A and C2B domains of synaptotagmin. (B) Wild-type (WT) and the indicated synaptotagmin Ib point mutants were analyzed for Ca 2+ -triggered oligomerization activity as described in Fig.…”

“…Three flexible loops protrude from the tip of the domain. In some cases, two of these loops form a pocket that mediates the binding of Ca 2 + and other divalent cations (Sutton and Sprang 1995; Sutton et al 1995, Sutton et al 1999; Grobler et al 1996; Perisic et al 1998; Ubach et al 1998a,Ubach et al 1998b; Pratt et al 1999; Macedo-Ribeiro et al 1999). Ca 2 + binding regulates the interaction of some C2 domains with target molecules including lipids and proteins, while other C2 domains do not appear to bind Ca 2 +.…”

“…The C2A domain of synaptotagmin I binds multiple Ca 2+ ions (Sutton et al 1995; Ubach et al 1998b), and becomes partially inserted into membranes that contain anionic phospholipids (Chapman and Davis 1998; Davis et al 1999; Bai et al 2000). Equilibrium and kinetic Ca 2+ binding properties of C2A are consistent with the Ca 2+ requirement and speed of secretion (Davis et al 1999).…”

Ca2+ Activation of the cPLA2 C2 Domain: Ordered Binding of Two Ca2+ Ions with Positive Cooperativity

“…Together they provide Ca 2+ coordination for up to four potential Ca 2+ -binding sites labeled sites I-IV; however, in a given C2 domain only a subset of the four sites may actually bind Ca 2+ in solution, since not all the sites provide adequate Ca 2+ coordination. Thus, the Ca 2+ -loaded C2 domains characterized to date possess from one to three Ca 2+ bound in different combinations of sites I-IV (12,15,(17)(18)(19).…”