Protein-Cadmium Interactions in Crowded Biomolecular Environments Probed by In-cell and Lysate NMR Spectroscopy.

One of the mechanisms by which toxic metal ions interfere with cellular functions is ionic mimicry, where they bind to protein sites in lieu of native metals Ca 2+ and Zn 2+ . The influence of crowded intracellular environments on these interactions is not well understood. Here, we demonstrate the application of in-cell and lysate NMR spectroscopy to obtain atomic-level information on how a potent environmental toxin cadmium interacts with its protein targets. The experiments, conducted in intact E. coli cells and their lysates, revealed that Cd 2+ can profoundly affect the quinary interactions of its protein partners, and can replace Zn 2+ in both labile and non-labile protein structural sites without significant perturbation of the membrane binding function. Surprisingly, in crowded molecular environments Cd 2+ can effectively target not only all-sulfur and mixed sulfur/nitrogen but also all-oxygen coordination sites. The sulfur-rich coordination environments show significant promise for bioremedial applications, as demonstrated by the ability of the designed protein scaffold α 3 DIV to sequester intracellular cadmium. Our data suggests that in-cell NMR spectroscopy is a powerful tool for probing interactions of toxic metal ions with their potential protein targets, and for the assessment of potency of sequestering agents.

Mechanisms by which small molecules of diverse chemotypes arrest Sec14 lipid transfer activity.

Phosphatidylinositol (PtdIns) transfer proteins (PITPs) enhance the activities of PtdIns 4-OH kinases that generate signaling pools of PtdIns-4-phosphate. In that capacity, PITPs serve as key regulators of lipid signaling in eukaryotic cells. Although the PITP phospholipid exchange cycle is the engine that stimulates PtdIns 4-OH kinase activities, the underlying mechanism is not understood. Herein, we apply an integrative structural biology approach to investigate interactions of the yeast PITP Sec14 with small-molecule inhibitors (SMIs) of its phospholipid exchange cycle. Using a combination of X-ray crystallography, solution NMR spectroscopy, and atomistic MD simulations, we dissect how SMIs compete with native Sec14 phospholipid ligands and arrest phospholipid exchange. Moreover, as Sec14 PITPs represent new targets for the development of next-generation antifungal drugs, the structures of Sec14 bound to SMIs of diverse chemotypes reported in this study will provide critical information required for future structure-based design of next-generation lead compounds directed against Sec14 PITPs of virulent fungi.

Structural anatomy of Protein Kinase C C1 domain interactions with diacylglycerol and other agonists.

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.

Probing the Diacylglycerol Binding Site of Presynaptic Munc13-1.

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.

Structural insights into C1-ligand interactions: Filling the gaps by in silico methods.

Protein Kinase C isoenzymes (PKCs) are the key mediators of the phosphoinositide signaling pathway, which involves regulated hydrolysis of phosphatidylinositol (4,5)-bisphosphate to diacylglycerol (DAG) and inositol-1,4,5-trisphosphate. Dysregulation of PKCs is implicated in many human diseases making this class of enzymes an important therapeutic target. Specifically, the DAG-sensing cysteine-rich conserved homology-1 (C1) domains of PKCs have emerged as promising targets for pharmaceutical modulation. Despite significant progress, the rational design of the C1 modulators remains challenging due to difficulties associated with structure determination of the C1-ligand complexes. Given the dearth of experimental structural data, computationally derived models have been instrumental in providing atomistic insight into the interactions of the C1 domains with PKC agonists. In this review, we provide an overview of the in silico approaches for seven classes of C1 modulators and outline promising future directions.

Partial Metal Ion Saturation of C2 Domains Primes Synaptotagmin 1-Membrane Interactions.

Synaptotagmin 1 (Syt1) is an integral membrane protein whose phospholipid-binding tandem C2 domains, C2A and C2B, act as Ca2+ 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 Pb2+, a structural and functional surrogate of Ca2+, 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 Pb2+-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.

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

Pb2+ is a xenobiotic metal ion that competes for Ca2+-binding sites in proteins. Using the peripheral Ca2+-sensing domains of Syt1, we show that the chelating pH buffer Bis-Tris enables identification and functional characterization of high-affinity Pb2+ sites that are likely to be targeted by bioavailable Pb2+.

High affinity interactions of Pb2+ with synaptotagmin I.

Lead (Pb) is a potent neurotoxin that disrupts synaptic neurotransmission. We report that Synaptotagmin I (SytI), a key regulator of Ca2+-evoked neurotransmitter release, has two high-affinity Pb2+ binding sites that belong to its cytosolic C2A and C2B domains. The crystal structures of Pb2+-complexed C2 domains revealed that protein-bound Pb2+ ions have holodirected coordination geometries and all-oxygen coordination spheres. The on-rate constants of Pb2+ binding to the C2 domains of SytI are comparable to those of Ca2+ and are diffusion-limited. In contrast, the off-rate constants are at least two orders of magnitude smaller, indicating that Pb2+ can serve as both a thermodynamic and kinetic trap for the C2 domains. We demonstrate, using NMR spectroscopy, that population of these sites by Pb2+ ions inhibits further Ca2+ 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 Pb2+ ions can interfere with the Ca2+-dependent function of SytI in the cell.

Non-Native Metal Ion Reveals the Role of Electrostatics in Synaptotagmin 1-Membrane Interactions.

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, Förster resonance energy transfer, and vesicle cosedimentation 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, electron paramagnetic resonance 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 a 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.