Multivalent lipid targeting by the calcium-independent C2A domain of synaptotagmin-like protein 4/granuphilin.

Synaptotagmin-like protein 4 (Slp-4), also known as granuphilin, is a Rab effector responsible for docking secretory vesicles to the plasma membrane before exocytosis. Slp-4 binds vesicular Rab proteins via an N-terminal Slp homology domain, interacts with plasma membrane SNARE complex proteins via a central linker region, and contains tandem C-terminal C2 domains (C2A and C2B) with affinity for phosphatidylinositol-(4,5)-bisphosphate (PIP2). The Slp-4 C2A domain binds with low nanomolar apparent affinity to PIP2 in lipid vesicles that also contain background anionic lipids such as phosphatidylserine but much weaker when either the background anionic lipids or PIP2 is removed. Through computational and experimental approaches, we show that this high-affinity membrane binding arises from concerted interaction at multiple sites on the C2A domain. In addition to a conserved PIP2-selective lysine cluster, a larger cationic surface surrounding the cluster contributes substantially to the affinity for physiologically relevant lipid compositions. Although the K398A mutation in the lysine cluster blocks PIP2 binding, this mutated protein domain retains the ability to bind physiological membranes in both a liposome-binding assay and MIN6 cells. Molecular dynamics simulations indicate several conformationally flexible loops that contribute to the nonspecific cationic surface. We also identify and characterize a covalently modified variant that arises through reactivity of the PIP2-binding lysine cluster with endogenous bacterial compounds and binds weakly to membranes. Overall, multivalent lipid binding by the Slp-4 C2A domain provides selective recognition and high-affinity docking of large dense core secretory vesicles to the plasma membrane.

Fluoride Ion Binding and Translocation in the CLCF Fluoride/Proton Antiporter: Molecular Insights from Combined Quantum-Mechanical/Molecular-Mechanical Modeling.

CLCF fluoride/proton antiporters move fluoride ions out of bacterial cells, leading to fluoride resistance in these bacteria. However, many details about their operating mechanisms remain unclear. Here, we report a combined quantum-mechanical/molecular-mechanical (QM/MM) study of a CLCF homologue from Enterococci casseliflavus (Eca), in accord with the previously proposed windmill mechanism. Our multiscale modeling sheds light on two critical steps in the transport cycle: (i) the external gating residue E118 pushing a fluoride in the external binding site into the extracellular vestibule and (ii) an incoming fluoride reconquering the external binding site by forcing out E118. Both steps feature competitions for the external binding site between the negatively charged carboxylate of E118 and the fluoride. Remarkably, the displaced E118 by fluoride accepts a proton from the nearby R117, initiating the next transport cycle. We also demonstrate the importance of accurate quantum descriptions of fluoride solvation. Our results provide clues to the mysterious E318 residue near the central binding site, suggesting that the transport activities are unlikely to be disrupted by the glutamate interacting with a well-solvated fluoride at the central binding site. This differs significantly from the structurally similar CLC chloride/proton antiporters, where a fluoride trapped deep in the hydrophobic pore causes the transporter to be locked down. A free-energy barrier of 10-15 kcal/mol was estimated via umbrella sampling for a fluoride ion traveling through the pore to repopulate the external binding site.

A conserved electrostatic membrane-binding surface in synaptotagmin-like proteins revealed using molecular phylogenetic analysis and homology modeling.

Protein structure prediction has emerged as a core technology for understanding biomolecules and their interactions. Here, we combine homology-based structure prediction with molecular phylogenetic analysis to study the evolution of electrostatic membrane binding among the vertebrate synaptotagmin-like protein (Slp) family. Slp family proteins play key roles in the membrane trafficking of large dense-core secretory vesicles. Our previous experimental and computational study found that the C2A domain of Slp-4 (also called granuphilin) binds with high affinity to anionic phospholipids in the cytoplasmic leaflet of the plasma membrane through a large positively charged protein surface centered on a cluster of phosphoinositide-binding lysine residues. Because this surface contributes greatly to Slp-4 C2A domain membrane binding, we hypothesized that the net charge on the surface might be evolutionarily conserved. To test this hypothesis, the known C2A sequences of Slp-4 among vertebrates were organized by class (from mammalia to pisces) using molecular phylogenetic analysis. Consensus sequences for each class were then identified and used to generate homology structures, from which Poisson-Boltzmann electrostatic potentials were calculated. For comparison, homology structures and electrostatic potentials were also calculated for the five human Slp protein family members. The results demonstrate that the charge on the membrane-binding surface is highly conserved throughout the evolution of Slp-4, and more highly conserved than many individual residues among the human Slp family paralogs. Such molecular phylogenetic-driven computational analysis can help to describe the evolution of electrostatic interactions between proteins and membranes which are crucial for their function.

Synaptotagmin 7 C2 domains induce membrane curvature stress via electrostatic interactions and the wedge mechanism.

Synaptotagmin 7 (Syt-7) is part of the synaptotagmin protein family that regulates exocytotic lipid membrane fusion. Among the family, Syt-7 stands out by its membrane binding strength and stabilization of long-lived membrane fusion pores. Given that Syt-7 vesicles form long-lived fusion pores, we hypothesize that its interactions with the membrane stabilize the specific curvatures, thicknesses, and lipid compositions that support a metastable fusion pore. Using all-atom molecular dynamics simulations and FRET-based assays of Syt-7's membrane-binding C2 domains (C2A and C2B), we found that Syt-7 C2 domains sequester anionic lipids, are sensitive to cholesterol, thin membranes, and generate lipid membrane curvature by two competing, but related mechanisms. First, Syt-7 forms strong electrostatic contacts with the membrane, generating negative curvature stress. Second, Syt-7's calcium binding loops embed in the membrane surface, acting as a wedge to thin the membrane and induce positive curvature stress. These curvature mechanisms are linked by the protein insertion depth as well as the resulting protein tilt. Simplified quantitative models of the curvature-generating mechanisms link simulation observables to their membrane-reshaping effectiveness.

A Conserved Electrostatic Membrane-Binding Surface in Synaptotagmin-Like Proteins Revealed Using Molecular Phylogenetic Analysis and Homology Modeling.

Protein structure prediction has emerged as a core technology for understanding biomolecules and their interactions. Here, we combine homology-based structure prediction with molecular phylogenetic analysis to study the evolution of electrostatic membrane binding among vertebrate synaptotagmin-like proteins (Slps). Slp family proteins play key roles in the membrane trafficking of large dense-core secretory vesicles. Our previous experimental and computational study found that the C2A domain of Slp-4 (also called granuphilin) binds with high affinity to anionic phospholipids in the cytoplasmic leaflet of the plasma membrane through a large positively charged protein surface centered on a cluster of phosphoinositide-binding lysine residues. Because this surface contributes greatly to Slp-4 C2A domain membrane binding, we hypothesized that the net charge on the surface might be evolutionarily conserved. To test this hypothesis, the known C2A sequences of Slp-4 among vertebrates were organized by class (from mammalia to pisces) using molecular phylogenetic analysis. Consensus sequences for each class were then identified and used to generate homology structures, from which Poisson-Boltzmann electrostatic potentials were calculated. For comparison, homology structures and electrostatic potentials were also calculated for the five human Slp protein family members. The results demonstrate that the charge on the membrane-binding surface is highly conserved throughout the evolution of Slp-4, and more highly conserved than many individual residues among the human Slp family paralogs. Such molecular phylogenetic-driven computational analysis can help to describe the evolution of electrostatic interactions between proteins and membranes which are crucial for their function.

The high-affinity calcium sensor synaptotagmin-7 serves multiple roles in regulated exocytosis.

Synaptotagmin (Syt) proteins comprise a 17-member family, many of which trigger exocytosis in response to calcium. Historically, most studies have focused on the isoform Syt-1, which serves as the primary calcium sensor in synchronous neurotransmitter release. Recently, Syt-7 has become a topic of broad interest because of its extreme calcium sensitivity and diversity of roles in a wide range of cell types. Here, we review the known and emerging roles of Syt-7 in various contexts and stress the importance of its actions. Unique functions of Syt-7 are discussed in light of recent imaging, electrophysiological, and computational studies. Particular emphasis is placed on Syt-7-dependent regulation of synaptic transmission and neuroendocrine cell secretion. Finally, based on biochemical and structural data, we propose a mechanism to link Syt-7's role in membrane fusion with its role in subsequent fusion pore expansion via strong calcium-dependent phospholipid binding.

The synaptotagmin C2B domain calcium-binding loops modulate the rate of fusion pore expansion.

In chromaffin cells, the kinetics of fusion pore expansion vary depending on which synaptotagmin isoform (Syt-1 or Syt-7) drives release. Our recent studies have shown that fusion pores of granules harboring Syt-1 expand more rapidly than those harboring Syt-7. Here we sought to define the structural specificity of synaptotagmin action at the fusion pore by manipulating the Ca2+-binding C2B module. We generated a chimeric Syt-1 in which its C2B Ca2+-binding loops had been exchanged for those of Syt-7. Fusion pores of granules harboring a Syt-1 C2B chimera with all three Ca2+-binding loops of Syt-7 (Syt-1:7C2B123) exhibited slower rates of fusion pore expansion and neuropeptide cargo release relative to WT Syt-1. After fusion, this chimera also dispersed more slowly from fusion sites than WT protein. We speculate that the Syt-1:7 C2B123 and WT Syt-1 are likely to differ in their interactions with Ca2+ and membranes. Subsequent in vitro and in silico data demonstrated that the chimera exhibits a higher affinity for phospholipids than WT Syt-1. We conclude that the affinity of synaptotagmin for the plasma membrane, and the rate at which it releases the membrane, contribute in important ways to the rate of fusion pore expansion.