C2 domains are independently folded modules that often target their host proteins to anionic membranes in a Ca2+-dependent manner. In these cases, membrane association is triggered by Ca2+ binding to the negatively charged loop region of the C2 domain. Here, we used a non-native metal ion, Cd2+, in lieu of Ca2+ to gain insight into the contributions made by long-range Coulombic interactions and direct metal ion-lipid bridging to membrane binding. Using X-ray crystallography, NMR, FRET, and vesicle co-sedimentation assays, we demonstrate that, although Cd2+ binds to the loop region of C2A/B domains of Synaptotagmin 1 with high affinity, long-range Coulombic interactions are too weak to support membrane binding of individual domains. We attribute this behavior to two factors: the stoichiometry of Cd2+ binding to the loop regions of the C2A and C2B domains and the impaired ability of Cd2+ to directly coordinate the lipids. In contrast, EPR experiments revealed that Cd2+ does support membrane binding of the C2 domains in full-length Synaptotagmin 1, where the high local lipid concentrations that result from membrane tethering can partially compensate for lack of full complement of divalent metal ions and specific lipid coordination in Cd2+-complexed C2A/B domains. Our data suggest that long-range Coulombic interactions alone can drive the initial association of C2A/B with anionic membranes, and that Ca2+ further augments membrane binding by the formation of metal ion-lipid coordination bonds and additional Ca2+ ion binding to the C2 domain loop regions.

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.

Diacylglycerol (DAG) is a versatile lipid whose 1,2-sn-stereoisomer serves both as second messenger in signal transduction pathways that control vital cellular processes, and as metabolic precursor for downstream signaling lipids such as phosphatidic acid. Effector proteins translocate to available DAG pools in the membranes by using conserved homology 1 (C1) domains as DAG-sensing modules. Yet, how C1 domains recognize and capture DAG in the complex environment of a biological membrane has remained unresolved for the 40 years since the discovery of Protein Kinase C (PKC) as the first member of the DAG effector cohort. Herein, we report the high-resolution crystal structures of a C1 domain (C1B from PKCδ) complexed to DAG and to each of four potent PKC agonists that produce different biological readouts and that command intense therapeutic interest. This structural information details the mechanisms of stereospecific recognition of DAG by the C1 domains, the functional properties of the lipid-binding site, and the identities of the key residues required for the recognition and capture of DAG and exogenous agonists. Moreover, the structures of the five C1 domain complexes provide the high-resolution guides for the design of agents that modulate the activities of DAG effector proteins.

Many pheromones have very low water solubility, posing experimental difficulties for quantitative binding measurements. A new method is presented for determining thermodynamically valid dissociation constants for ligands binding to pheromone-binding proteins (OBPs), using β-cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila OBP that binds the pheromone 11-cis vaccenyl acetate (cVA). Refolding of LUSH expressed in E. coli was assessed by measuring N-phenyl-1-naphthylamine (NPN) binding and Förster resonance energy transfer between LUSH tryptophan 123 (W123) and NPN. Binding of cVA was measured from quenching of W123 fluorescence as a function of cVA concentration. The equilibrium constant for transfer of cVA between β-cyclodextrin and LUSH was determined from a linked equilibria model. This constant, multiplied by the β-cyclodextrin-cVA dissociation constant, gives the LUSH-cVA dissociation constant: ~100 nM. It was also found that other ligands quench W123 fluorescence. The LUSH-ligand dissociation constants were determined to be ~200 nM for the silk moth pheromone bombykol and ~90 nM for methyl oleate. The results indicate that the ligand-binding cavity of LUSH can accommodate a variety ligands with strong binding interactions. Implications of this for the pheromone receptor model proposed by Laughlin et al. (Cell 133: 1255–65, 2008) are discussed.

Munc13-1 is a presynaptic active zone protein that acts as a master regulator of synaptic vesicle priming and neurotransmitter release in the brain. It has been implicated in the pathophysiology of several neurodegenerative diseases. Diacylglycerol and phorbol ester activate Munc13-1 by binding to its C1 domain. The objective of this study is to identify the structural determinants of ligand binding activity of the Munc13-1 C1 domain. Molecular docking suggested that residues Trp-588, Ile-590, and Arg-592 of Munc13-1 are involved in ligand interactions. To elucidate the role of these three residues in ligand binding, we generated W588A, I590A, and R592A mutants in full-length Munc13-1, expressed them as GFP-tagged proteins in HT22 cells, and measured their ligand-induced membrane translocation by confocal microscopy and immunoblotting. The extent of 1,2-dioctanoyl-sn-glycerol (DOG)-and phorbol ester-induced membrane translocation decreased in the following order: wild type > I590A > W588A > R592A and wild type > W588A > I590A > R592A, respectively. To understand the effect of the mutations on ligand binding, we also measured the DOG binding affinity of the isolated wild-type C1 domain and its mutants in membrane-mimicking micelles using nuclear magnetic resonance methods. The DOG binding affinity decreased in the following order: wild type > I590A > R592A. No binding was detected for W588A with DOG in micelles. This study shows that Trp-588, Ile-590, and Arg-592 are essential determinants for the activity of Munc13-1 and the effects of the three residues on the activity are ligand-dependent. This study bears significance for the development of selective modulators of Munc13-1.

Synaptotagmin 1 (Syt1) is an integral membrane protein whose phospholipid-binding tandem C2 domains, C2A and C2B, act as Ca 2þ sensors of neurotransmitter release. Our objective was to understand the role of individual metal-ion binding sites of these domains in the membrane association process. We used Pb 2þ , a structural and functional surrogate of Ca 2þ , to generate the protein states with well-defined protein-metal ion stoichiometry. NMR experiments revealed that binding of one divalent metal ion per C2 domain results in loss of conformational plasticity of the loop regions, potentially pre-organizing them for additional metal-ion and membrane-binding events. In C2A, a divalent metal ion in site 1 is sufficient to drive its weak association with phosphatidylserine-containing membranes, whereas in C2B, it enhances the interactions with the signaling lipid phosphatidylinositol-4,5-bisphosphate. In full-length Syt1, both Pb 2þ -complexed C2 domains associate with phosphatidylserine-containing membranes. Electron paramagnetic resonance experiments show that the extent of membrane insertion correlates with the occupancy of the C2 metal ion sites. Together, our results indicate that upon partial metal ion saturation of the intra-loop region, Syt1 adopts a dynamic, partially membrane-bound state. 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 cooperative binding of a full complement of metal ions and deeper membrane insertion.

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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.