High affinity interactions of Pb²⁺with Synaptotagmin I

Abstract

words]Lead (Pb) is a potent neurotoxin that disrupts synaptic neurotransmission. We report that Synaptotagmin I (SytI), a key regulator of Ca 2+ -evoked neurotransmitter release, has two highaffinity Pb 2+ binding sites that belong to its cytosolic C2A and C2B domains. The crystal structures of Pb 2+ -complexed C2 domains revealed that protein-bound Pb 2+ ions have holodirected coordination geometries and all-oxygen coordination spheres. The on-rate constants of Pb 2+ binding to the C2 domains of SytI are comparable to those of Ca 2+ and are diffusion-limited. In contrast, the off-rate constants are at least two orders of magnitude smaller, indicating that Pb 2+ can serve as both thermodynamic and kinetic trap for the C2 domains. We demonstrate, using NMR spectroscopy, that population of these sites by Pb 2+ ions inhibits further Ca 2+ binding despite the existing coordination vacancies. Our work offers a unique insight into the bioinorganic chemistry of Pb(II) and suggests a mechanism by which low concentrations of Pb 2+ ions can interfere with the Ca 2+ -dependent function of SytI in the cell.

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

“…[ 15 N-1 H] TROSY HSQC spectra of [U-15 N, ~80%-2 H] C2AB at 1:1 and 1:2 protein-to-Pb 2+ ratios (Fig. S7) revealed the same chemical exchange regime, saturation behavior, and chemical shift perturbation patterns as for the isolated C2 domains (27). These data indicate that Pb 2+ selectively populates Site 1 of the C2A and C2B domains in the context of the C2AB fragment and that the linker region is not involved in metalion binding.…”

“…What sets Pb 2+ apart from other metal ions is two features. First, it supports the membrane interactions of C2 domains and is therefore isofunctional to Ca 2+ (24,26,27). Second, although Pb 2+ populates the exact same C2 domain sites as Ca 2+ in solution, there are significant differences in its affinities to Site 1 and 2-3: the affinity of Pb 2+ to Site 1 is ~450-fold higher than that to Site 2 ( Fig.…”

“…We previously obtained high-resolution crystal structures of individual Pb 2+ -complexed Syt1 C2 domains, where Pb 2+ ions bind to Site 1 with two-orders of magnitude higher affinity than Ca 2+ (27) (Figs. 1A,B).…”

“…The gene segments encoding Syt1 C2A (residues 137-265), C2B (residues 271-421), and C2AB (residues 137-421) were cloned into pET-SUMO vector (Novagen, Madison, WI) and expressed as 6xHis-tagged SUMO fusion proteins in the E. coli BL21(DE3) (C2A, C2AB) and Rosetta(DE3) (C2B) strains as described previously (22,27). The full length-Syt1 (FL-Syt1) (residues 1-421) with the native cysteines mutated as: C73A, C74A, C76A, C78A, C82S, and C277S was cloned into a pET-28a vector.…”

“…Second, although Pb 2+ populates the exact same C2 domain sites as Ca 2+ in solution, there are significant differences in its affinities to Site 1 and 2-3: the affinity of Pb 2+ to Site 1 is ~450-fold higher than that to Site 2 ( Fig. 1B) (27). These unique properties of Pb 2+ with respect to its interactions with C2 domains enabled us to simultaneously achieve selective population of specific metal binding site, Site 1, with minimum perturbation of the membrane binding function of the proteins.…”