Partial metal ion saturation of C2 domains primes Syt1-membrane interactions

Abstract

Synaptotagmin 1 (Syt1) is an integral membrane protein that acts as a Ca 2+ sensor of neurotransmitter release. How the Ca 2+ -sensing function of Syt1 is coupled to its interactions with anionic membranes and synaptic fusion machinery is not well understood. Here, we investigated the dynamics and membrane-binding properties of Syt1 under conditions where its highest affinity Ca 2+ sites, which are thought to drive the initial membrane recruitment, are selectively populated by divalent metal ions. To create such protein states for the Ca 2+ -sensing C2 domains of Syt1, we exploited the unique chemistry of Pb 2+ , a xenobiotic metal ion that is isostructural and isofunctional to Ca 2+ . NMR experiments revealed that binding of a single metal ion results in the loss of conformational plasticity of the C2 domain loop regions that are involved in both coordinating metal ions and membrane interactions. In the C2A domain, a single metal ion is sufficient to drive its weak association with PtdSer-containing membranes; in C2B, it enhances the interactions with the signaling lipid PtdIns(4,5)P 2 . In fulllength Syt1, both C2 domains associate with PtdSer-containing membranes, with the depth of insertion modulated by the occupancy of the metal ion sites. Our data suggest that Syt1 adopts a shallow membrane-bound state upon initial recruitment of its C2 domains to the membranes. The properties of this state, such as conformationally restricted loop regions and positioning of C2 domains in close proximity to anionic lipid headgroups, "prime" Syt1 for binding a full complement of metal ions required for activation of protein function.

Interference of pH buffer with Pb2+-peripheral domain interactions: obstacle or opportunity?

“…The attenuation was quantified as the ratio of the N-H N cross-peak intensities in the absence (I 0 ) and presence (I) of DPS. Consistent with previous data obtained using PtdSer containing bicelles, 16 residues that belong to the loop regions showed significant decrease in I/I 0 values, along with several residues in the polylysine region between loops 1 and 2 (Fig. 4A).…”

“…In MES, the C2A•Pb1 complex prepared by mixing stoichiometric amounts of C2A and Pb 2+ associates weakly with PtdSer-containing bicelles. 16 The chemical exchange process between the C2A•Pb1-PtdSer ternary complex and the C2A•Pb1 binary complexes occurs on the "fast-to-intermediate" NMR chemical shift timescale. This produces a specific pattern of resonance intensity decrease in the NMR spectra that predominantly affects the loop regions of the C2A domain.…”

“…C2 domains with partially occupied metal-ion binding sites, through weak association with anionic membranes, are brought close to the polar membrane region. 16,20 It is believed that the resulting proximity to anionic lipid moieties elicits cooperative metal-ion binding to the remaining weaker sites by enhancing their affinities. This mutual cooperativity enables the domain to perform its function at physiological concentrations of Ca 2+ .…”

“…Site 1 of each C2 domain. Upon population of Site 1 by Pb 2+ , C2B domain will interact with anionic lipids of the presynaptic membranes, PtdIns(4,5)P 2 and PtdSer, 16 via the polylysine motif; due to the positive cooperativity, the affinity of C2B to divalent metal ions will be enhanced. As we demonstrated previously, 8,11 Ca 2+ is unable to share ligands with Pb1 and occupy the remaining vacant metal ion sites.…”

Interference of pH buffer with Pb2+-peripheral domain interactions: obstacle or opportunity?

“…In MES, the C2A·Pb1 complex prepared by mixing stoichiometric amounts of C2A and Pb 2+ associates weakly with PtdSer-containing bicelles. 16 The chemical exchange process between the C2A·Pb1-PtdSer ternary complex and the C2A·Pb1 16 residues that belong to the loop regions showed significant decrease in I/I 0 values, along with several residues in the polylysine region between loops 1 and 2 (Fig. 4A).…”