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Acral melanoma, the most common melanoma subtype among non-Caucasian individuals, is associated with poor prognosis. However, its key molecular drivers remain obscure. Here, we performed integrative genomic and clinical profiling of acral melanomas from a cohort of 104 patients treated in North America or China. We found that recurrent, late-arising amplifications of cytoband chr22q11.21 are a leading determinant of inferior survival, strongly associated with metastasis, and linked to downregulation of immunomodulatory genes associated with response to immune checkpoint blockade. Unexpectedly, LZTR1 – a known tumor suppressor in other cancers – is a key candidate oncogene in this cytoband. Silencing of LZTR1 in melanoma cell lines caused apoptotic cell death independent of major hotspot mutations or melanoma subtypes. Conversely, overexpression of LZTR1 in normal human melanocytes initiated processes associated with metastasis, including anchorage-independent growth, formation of spheroids, and increased levels of MAPK and SRC activities. Our results provide new insights into the etiology of acral melanoma and implicate LZTR1 as a key tumor promoter and therapeutic target

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 (SHD) 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 (PS), but much weaker when either the background anionic lipids or PIP2 are removed. Through computational and experimental approaches, we show that this high affinity membrane interaction arises from concerted interaction at multiple sites on the C2A domain. In addition to a conserved PIP2-selective lysine cluster, there exists a larger cationic surface surrounding the cluster which contributes substantially to the affinity for physiologically relevant lipid compositions. While mutations at the PIP2-selective site decrease affinity for PIP2, multiple mutations are needed to decrease binding to physiologically relevant lipid compositions. Docking and molecular dynamics simulations indicate several conformationally flexible loops that contribute to the nonspecific cationic surface. We also identify and characterize a covalently modified variant in the bacterially expressed protein, which arises through reactivity of the PIP2-binding lysine cluster with endogenous bacterial compounds and has a low membrane affinity. 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.

MacDougall et al. review the structure and function of the calcium sensor synaptotagmin-7 in 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.

This study provides novel mechanistic insights into how Syt-1 and Syt-7 C2B domains regulate the later stages of exocytosis. We show that small structural differences within this region exert significant effects on fusion pore properties. We propose that these effects may arise from differences in C2B domain affinity for the plasma membrane., 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.

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.

Synaptotagmin-1 (Syt-1) and synaptotagmin-7 (Syt-7) contain analogous tandem C2 domains, C2A and C2B, which together serve as a Ca2+ sensor to bind membranes and promote the stabilization of exocytotic fusion pores. Functionally, Syt-1 triggers fast release of neurotransmitters, while Syt-7 is involved in lower-Ca2+ processes such as hormone secretion. Evidence suggests that Syt-1 C2 domains bind membranes cooperatively, penetrating farther into membranes as the C2AB tandem than as individual C2 domains. In contrast, we previously reported that the two C2 domains of Syt-7 bind membranes independently, based in part on measurements of their liposome dissociation kinetics. Here, we have investigated the effects of C2A-C2B interdomain cooperativity with Syt-1 and Syt-7 using directly comparable measurements. We report Ca2+ sensitivities, dissociation kinetics, and membrane insertion using liposomes approximating physiological lipid compositions. Equilibrium Ca2+ titrations confirm that the Syt-7 C2AB tandem has a greater Ca2+ sensitivity of membrane binding than either of its individual domains. Stopped-flow fluorescence kinetic measurements show that Syt-1 C2AB dissociates from liposome membranes much more slowly than either of its isolated C2 domains, suggesting that the two C2 domains of Syt-1 bind membranes cooperatively. In contrast, the dominant population of Syt-7 C2AB has a dissociation rate comparable to its C2A domain, indicating a lack of cooperativity, while only a small subpopulation dissociates at a slower rate. Measurements using an environment-sensitive fluorescent probe indicate that the Syt-7 C2B domain inserts more deeply into membranes as part of the C2AB tandem, similarly to Syt-1. Overall, these measurements are consistent with a model in which the structural linkage of C2A and C2B impacts the membrane-binding geometry of synaptotagmin C2B domains, but imparts little or no cooperativity to Syt-7 membrane binding and dissociation events that are dominated by its C2A domain.

Synaptotagmin-1 (Syt-1) and synaptotagmin-7 (Syt-7) contain analogous tandem C2 domains, C2A and C2B, which together sense Ca(2+) to bind membranes and promote the stabilization of exocytotic fusion pores. Syt-1 triggers fast release of neurotransmitters, whereas Syt-7 functions in processes that involve lower Ca(2+) concentrations such as hormone secretion. Syt-1 C2 domains are reported to bind membranes cooperatively, based on the observation that they penetrate farther into membranes as the C2AB tandem than as individual C2 domains. In contrast, we previously suggested that the two C2 domains of Syt-7 bind membranes independently, based in part on measurements of their liposome dissociation kinetics. Here, we investigated C2A-C2B interdomain cooperativity with Syt-1 and Syt-7 using directly comparable measurements. Equilibrium Ca(2+) titrations demonstrate that the Syt-7 C2AB tandem binds liposomes lacking phosphatidylinositol-4,5-bisphosphate (PIP(2)) with greater Ca(2+) sensitivity than either of its individual domains and binds to membranes containing PIP(2) even in the absence of Ca(2+). Stopped-flow kinetic measurements show differences in cooperativity between Syt-1 and Syt-7: Syt-1 C2AB dissociates from PIP(2)-free liposomes much more slowly than either of its individual C2 domains, indicating cooperativity, whereas the major population of Syt-7 C2AB has a dissociation rate comparable to its C2A domain, suggesting a lack of cooperativity. A minor subpopulation of Syt-7 C2AB dissociates at a slower rate, which could be due to a small cooperative component and/or liposome clustering. Measurements using an environment-sensitive fluorescent probe indicate that the Syt-7 C2B domain inserts deeply into membranes as part of the C2AB tandem, similar to the coinsertion previously reported for Syt-1. Overall, coinsertion of C2A and C2B domains is coupled to cooperative energetic effects in Syt-1 to a much greater extent than in Syt-7. The difference can be understood in terms of the relative contributions of C2A and C2B domains toward membrane binding in the two proteins.

Fusion and fission of cellular membranes involve dramatic, protein-mediated changes in membrane curvature. Many of the experimental methods useful for investigating curvature sensing or generation require specialized equipment. We have developed a system based on supported lipid bilayers (SLBs) in which lipid tubules are simple to produce and several types of membrane remodeling events can be readily imaged using widely available instrumentation (e.g., tubule fission and/or membrane budding). Briefly, high ionic strength during lipid bilayer deposition results in incorporation of excess lipids in the SLB. After sequentially washing with water and physiological ionic strength buffer solutions, lipid tubules form spontaneously. We find that tubule formation results from solution-dependent spreading of the SLB; washing from water into physiological ionic strength buffer solution leads to expansion of the bilayer and formation of tubules. Conversely, washing from physiological buffer into water results in contraction of the membrane and loss of tubules. We demonstrate the utility of these supported tubulated bilayers, termed "STuBs," with an investigation of Sar1B, a small Ras family G-protein known to influence membrane curvature. The addition of Sar1B to STuBs results in dramatic changes in tubule topology and eventual tubule fission. Overall, STuBs are a simple experimental system, useful for monitoring protein-mediated effects on membrane topology in real time, under physiologically relevant conditions.

The synaptotagmin (Syt) family of proteins plays an important role in vesicle docking and fusion during Ca(2+)-induced exocytosis in a wide variety of cell types. Its role as a Ca(2+) sensor derives primarily from its two C2 domains, C2A and C2B, which insert into anionic lipid membranes upon binding Ca(2+). Syt isoforms 1 and 7 differ significantly in their Ca(2+) sensitivity; the C2A domain from Syt7 binds Ca(2+) and membranes much more tightly than the C2A domain from Syt1, at least in part because of greater contributions from the hydrophobic effect. While the structure and membrane activity of Syt1 have been extensively studied, the structural origins of differences between Syt1 and Syt7 are unknown. This study used site-directed spin labeling and electron paramagnetic resonance spectroscopy to determine depth parameters for the Syt7 C2A domain, for comparison to analogous previous measurements with the Syt1 C2A domain. In a novel approach, the membrane docking geometry of both Syt1 and Syt7 C2A was modeled by mapping depth parameters onto multiple molecular dynamics-simulated structures of the Ca(2+)-bound protein. The models reveal membrane penetration of Ca(2+) binding loops 1 (CBL1) and 3 (CBL3), and membrane binding is more sensitive to mutations in CBL3. On average, Syt7 C2A inserts more deeply into the membrane than Syt1 C2A, although depths vary among the different structural models. This observation provides a partial structural explanation for the hydrophobically driven membrane docking of Syt7 C2A.

Synaptotagmin (Syt) family proteins contain tandem C2 domains, C2A and C2B, which insert into anionic membranes in response to increased cytosolic Ca2+ concentration and facilitate exocytosis in neuronal and endocrine cells. The C2A domain from Syt7 binds lipid membranes much more tightly than the corresponding domain from Syt1, but the implications of this difference for protein function are not yet clear. In particular, the ability of the isolated Syt7 C2A domain to initiate membrane apposition and/or aggregation has been previously unexplored. Here, we demonstrate that Syt7 C2A induces apposition and aggregation of liposomes using Forster resonance energy transfer (FRET) assays, dynamic light scattering, and spectroscopic techniques involving lipid-coated gold nanoparticles (LCAuNPs). Protein–membrane binding, membrane apposition, and macroscopic aggregation are three separate phenomena with distinct Ca2+ requirements: the threshold Ca2+ concentration for membrane binding is lowest, followed by apposit...

Maternal mRNAs are translationally regulated during early development. Zar1 and its closely related homolog, Zar2, are both crucial in early development. Xenopus laevis Zygote arrest 2 (Zar2) binds to the Translational Control Sequence (TCS) in maternal mRNAs and regulates translation. The molecular mechanism of Zar1 has not been described. Here we report similarities and differences between Xenopus Zar1 and Zar2. Analysis of Zar sequences in vertebrates revealed two Zar family members with conserved, characteristic amino acid differences in the C-terminal domain. The presence of only two vertebrate Zar proteins was supported by analyzing Zar1 synteny. We propose that the criteria for naming Zar sequences are based on the characteristic amino acids and the chromosomal context. We also propose reclassification of some Zar sequences. We found that Zar1 is expressed throughout oogenesis and is stable during oocyte maturation. The N-terminal domain of Zar1 repressed translation of a reporter construct in immature oocytes. Both Zar1 and Zar2 bound to the TCS in the Wee1 and Mos 3′ UTRs using a zinc finger in the C-terminal domain. However, Zar1 had much higher affinity for RNA than Zar2. To show the functional significance of the conserved amino acid substitutions, these residues in Zar2 were mutated to those found in Zar1. We show that these residues contributed to the different RNA binding characteristics of Zar1 compared to Zar2. Our study shows that Zar proteins have generally similar molecular functions in the translational regulation of maternal mRNAs, but they may have different roles in early development.

Protein complexes assembled on membrane surfaces regulate a wide array of signaling pathways and cell processes. Thus a molecular understanding of the membrane surface diffusion and regulatory events leading to the assembly of active membrane complexes is crucial to signaling biology and medicine. Here we present a novel single molecule diffusion analysis designed to detect complex formation on supported lipid bilayers. The usefulness of the method is illustrated by detection of an engineered, heterodimeric complex in which two membrane-bound pleckstrin homology (PH) domains associate stably, but reversibly, upon Ca2+-triggered binding of calmodulin (CaM) to a target peptide from myosin light chain kinase (MLCKp). Specifically, when a monomeric, fluorescent PH-CaM domain fusion protein diffusing on a supported bilayer binds a dark MLCKp-PH domain fusion protein, the heterodimeric complex is observed to diffuse nearly 2-fold more slowly than the monomer because both of its twin PH domains can simultaneously bind to the viscous bilayer. In a mixed population of monomers and heterodimers, the single molecule diffusion analysis resolves and quantitates the rapidly diffusing monomer and slowly diffusing heterodimer subpopulations. The affinity of the CaM-MLCKp interaction is measured by titrating dark MLCKp-PH construct into the system, while monitoring the changing average diffusion coefficient of the fluorescent PH-CaM population, yielding a saturating binding curve. Strikingly, the apparent affinity of the CaM-MLCKp complex is ∼102-fold greater in the membrane system than in solution, apparently due both to faster complex association and slower complex dissociation on the membrane surface. More broadly, the present findings suggest that single molecule diffusion measurements on supported bilayers will provide an important tool for analyzing the 2D diffusion and assembly reactions governing the formation of diverse membrane-bound complexes, including key complexes from critical signaling pathways. The approach may also prove useful in pharmaceutical screening for compounds that inhibit membrane complex assembly or stability.

Proteins containing membrane targeting domains play essential roles in many cellular signaling pathways. However, important features of the membrane-bound state are invisible to bulk methods, thereby hindering mechanistic analysis of membrane targeting reactions. Here we use total internal reflection fluorescence microscopy (TIRFM), combined with single particle tracking, to probe the membrane docking mechanism of a representative pleckstrin homology (PH) domain isolated from the general receptor for phosphoinositides, isoform 1 (GRP1). The findings show three previously undescribed features of GRP1 PH domain docking to membranes containing its rare target lipid, phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3]. First, analysis of surface diffusion kinetics on supported lipid bilayers shows that in the absence of other anionic lipids, the PI(3,4,5)P3-bound protein exhibits the same diffusion constant as a single lipid molecule. Second, the binding of the anionic lipid phosphatidylserine to a previously unidentified secondary binding site slows both diffusion and dissociation kinetics. Third, TIRFM enables direct observation of rare events in which dissociation from the membrane surface is followed by transient diffusion through solution and rapid rebinding to a nearby, membrane-associated target lipid. Overall, this study shows that in vitro single-molecule TIRFM provides a new window into the molecular mechanisms of membrane docking reactions.

Granuphilin, also known as synaptotagmin-like protein 4 (Slp4), is a Rab effector responsible for docking insulin secretory vesicles to the plasma membrane prior to exocytosis. Granuphilin binds vesicular Rab proteins via an N-terminal Slp homology (SHD) domain, binds 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). Granuphilin's C2A domain has previously been shown to bind PIP2 or its soluble analogues with low micromolar affinity; however, the domain docks with low nanomolar apparent affinity to PIP2 in lipid vesicles that also contain background anionic lipids such as phosphatidylserine (PS). Here we show using a combination of computational and experimental approaches that this high-affinity membrane interaction arises from concerted binding at two or more sites on the C2A domain. First, docking calculations predict at least two possible binding pockets for anionic ligands. Second, liposome binding measurements indicate that affinity depends on concentrations of PIP2 as well as PS and phosphatidic acid (PA) in the membrane. Third, fluorescence measurements indicate that different regions of the protein surface are responsible for binding membranes containing or lacking PIP2. Fourth, single-molecule lateral diffusion measurements indicate distinct membrane-bound states depending on the available target lipids. Mutational analysis is currently underway to confirm locations of these lipid binding sites. Overall, multivalent membrane binding by granuphilin likely contributes to selective recognition and high affinity docking of large dense-core secretory vesicles to the plasma membrane.

Synaptotagmin 1 (Syt1), a Ca2+ sensor involved in exocytosis in pre-synaptic neurons, contains a characteristic tandem C2 sequence (C2AB) which inserts into anionic membranes in the presence of Ca2+. Previous studies have shown that the C2AB fragment of Syt1 inserts into target membranes more deeply than either individual C2A or C2B domain, suggesting cooperative interaction between C2A and C2B in the membrane-docked state. This behavior stands in apparent contrast to that of another family member, Syt7, whose C2A and C2B domains have been shown to bind membranes independently. To compare the cooperative behaviors of the C2 domains in these two isoforms, the dissociation kinetics (off-rates) of protein-liposome complexes were measured upon addition of the Ca2+ chelator EDTA using stopped-flow fluorescence spectroscopy. Using liposomes composed of a 1:1 mixture of phosphatidylcholine and phosphatidylserine (PC/PS), the Syt1 C2AB tandem domain exhibits a much slower off-rate than either of the single domains. In contrast, dissociation kinetics for Syt7 C2AB were best fit to a two-step model in which each rate constant matches those from the individual domains. This preliminary result supports the presence of interdomain interactions in the membrane-docked state of Syt1 C2AB but not Syt7 C2AB. These experiments were performed with protein domains purified using affinity chromatography with high-salt washes and verified to be >95% free of nucleic acid contaminants; ongoing work aims to assess effects of polyanions on this apparent cooperative behavior, including ion-exchange steps in the protein purifications.

Biomolecular interactions such as interfacial protein-membrane binding are often the cumulative effect of multivalent attractions. We have shown that the stoichiometry of peripheral protein-membrane interactions can be measured based on single-molecule diffusion using supported lipid bilayers. Here we apply this technique to granuphilin, a synaptotagmin-like protein containing tandem membrane-targeting C2 domains, C2A and C2B. Granuphilin C2A binds simple lipid membranes containing anionic lipids such as phosphatidylserine (PS), but C2B affinity for PS is undetectable using standard protein-membrane binding approaches. Here, we set out to determine the PS affinity of C2B based on a comparison of the diffusion rates of the C2A domain and the C2AB tandem on supported lipid bilayers composed of phosphatidylcholine (PC) and PS. Total internal reflection florescence (TIRF) microscopy with single particle tracking was used to identify diffusion constants of each individual or tandem C2 domain. Granuphilin C2A displays a lateral diffusion constant of ∼2 μm2/s when bound to 1:1 DOPC:DOPS bilayers, comparable to other C2 domains. However, the diffusion of the granuphilin C2AB tandem on the same membrane is significantly slower; suggesting substantial PS contacts for the C2B domain within the C2AB tandem. This effect represents a weak but potentially physiologically relevant interaction that influences the membrane-bound state of this strong membrane binding protein. In a separate experiment, we also show that the diffusion of the individual C2A domain decreases significantly in the presence of 2% phosphatidylinositol-(4,5)-bisphosphae (PIP2), suggesting separate binding sites for these two lipid ligands. Overall, single-molecule tracking can reveal membrane-binding states that are difficult to detect with traditional ensemble approaches.

Synaptotagmins are membrane trafficking proteins that contain two C-terminal C2 domains, C2A and C2B. In eight of the seventeen mammalian isoforms, the C2 domains serve as calcium-dependent membrane binding motifs that trigger exocytosis upon cellular calcium influx. C2 domains from the different isoforms display different kinetics and affinities for membranes, which reflect their functions in a variety of different cell types. For example, synaptotagmin-7 (Syt7) is mainly active in slow secretion events such as insulin release. Previous work in our lab has shown that the C2A domain of human Syt7 has membrane association and dissociation rates that are ∼2 and ∼60-fold slower than the corresponding domain of Syt1, an isoform involved in rapid neurotransmitter release [Brandt DS, et al., Biochemistry; 51(39):7654-7664.] The purpose of this study is to investigate the balance of forces governing the stronger membrane binding of Syt7 C2A. We have used kinetic measurements of association and dissociation to assess membrane affinity, while systematically varying buffer composition, liposome size, lipid composition, and mutant forms of the protein domain. The results indicate that this domain's unusually strong membrane binding strength is due to a combination of electrostatics and the hydrophobic effect. In particular, dissociation rates are governed mainly by the hydrophobic effect, as seen by slower dissociation in the presence of the kosmotropic agent trehalose. Furthermore, the F167 residue in the Syt7 C2A domain acts as a secondary hydrophobic anchor for the membrane-docked protein domain, as its mutation to methionine (as in Syt1) results in a reproducible 2-fold increase in off rate.

The C2A domain of synaptotagmin 7 (Syt7) is a Ca(2+) and membrane binding module that docks and inserts into cellular membranes in response to elevated intracellular Ca(2+) concentrations. Like other C2 domains, Syt7 C2A binds Ca(2+) and membranes primarily through three loop regions; however, it docks at Ca(2+) concentrations much lower than those required for other Syt C2A domains. To probe structural components of its unusually strong membrane docking, we conducted atomistic molecular dynamics simulations of Syt7 C2A under three conditions: in aqueous solution, in the proximity of a lipid bilayer membrane, and embedded in the membrane. The simulations of membrane-free protein indicate that Syt7 C2A likely binds three Ca(2+) ions in aqueous solution, consistent with prior experimental reports. Upon membrane docking, the outermost Ca(2+) ion interacts directly with lipid headgroups, while the other two Ca(2+) ions remain chelated by the protein. The membrane-bound domain was observed to exhibit large-amplitude swinging motions relative to the membrane surface, varying by up to 70° between a more parallel and a more perpendicular orientation, both during and after insertion of the Ca(2+) binding loops into the membrane. The computed orientation of the membrane-bound protein correlates well with experimental electron paramagnetic resonance measurements presented in the preceding paper ( DOI: 10.1021/acs.biochem.5b00421 ). In particular, the strictly conserved residue Phe229 inserted stably ∼4 A below the average depth of lipid phosphate groups, providing critical hydrophobic interactions anchoring the domain in the membrane. Overall, the position and orientation of Syt7 C2A with respect to the membrane are consistent with experiments.

The conversion of soluble protein into beta-sheet-rich amyloid fibers is the hallmark of a number of serious diseases. Precursors for many of these systems (e.g., Abeta from Alzheimer's disease) reside in close association with a biological membrane. Membrane bilayers are reported to accelerate the rate of amyloid assembly. Furthermore, membrane permeabilization by amyloidogenic peptides can lead to toxicity. Given the beta-sheet-rich nature of mature amyloid, it is seemingly paradoxical that many precursors are either intrinsically alpha-helical or transiently adopt an alpha-helical state upon association with membrane. In this work, we investigate these phenomena in islet amyloid polypeptide (IAPP). IAPP is a 37-residue peptide hormone which forms amyloid fibers in individuals with type II diabetes. Fiber formation by human IAPP (hIAPP) is markedly accelerated by lipid bilayers despite adopting an alpha-helical state on the membrane. We further show that IAPP partitions into monomeric and oligomeric helical assemblies. Importantly, it is this latter state which most strongly correlates to both membrane leakage and accelerated fiber formation. A sequence variant of IAPP from rodents (rIAPP) does not form fibers and is reputed not to permeabilize membranes. Here, we report that rIAPP is capable of permeabilizing membranes under conditions that permit rIAPP membrane binding. Sequence and spectroscopic comparisons of rIAPP and hIAPP enable us to propose a general mechanism for the helical acceleration of amyloid formation in vitro. As rIAPP cannot form amyloid fibers, our results show that fiber formation need not be directly coupled to toxicity.

Synaptotagmin (Syt) acting as a calcium sensor promotes SNARE-mediated membrane fusion by docking to target membrane. Here we study the C2A domain of Syt7, which triggers Ca2+-dependent release of large dense-core vesicles in several cell types, by doing atomistic molecular dynamics simulations and Poisson-Boltzmann calculations. The association of the Syt7 C2A with membrane (POPC:POPS=3:1) was found accompanied by seesaw-like movements of the protein, primarily due to two significant interactions: (1) the electrostatic attractions between the negatively-charged lipid headgroups and the positively-charged residues in the loops L1-L3 and (2) the hydrophobic interactions between the lipid tails and a critical phenylalanine residue F167. Good linear correlation was found between the EPR measured penetration depth parameters and the theoretically calculated average penetration depths for a large number of residues.

The synaptotagmin (Syt) family of proteins contains tandem C2 domains, C2A and C2B, which bind membranes in the presence of Ca(2+) to trigger vesicle fusion during exocytosis. Despite recent progress, the role and extent of interdomain interactions between C2A and C2B in membrane binding remain unclear. To test whether the two domains interact on a planar lipid bilayer (i.e., experience thermodynamic interdomain contacts), diffusion of fluorescent-tagged C2A, C2B, and C2AB domains from human Syt7 was measured using total internal reflection fluorescence microscopy with single-particle tracking. The C2AB tandem exhibits a lateral diffusion constant approximately half the value of the isolated single domains and does not change when additional residues are engineered into the C2A-C2B linker. This is the expected result if C2A and C2B are separated when membrane-bound; theory predicts that C2AB diffusion would be faster if the two domains were close enough together to have interdomain contact. Stopped-flow measurements of membrane dissociation kinetics further support an absence of interdomain interactions, as dissociation kinetics of the C2AB tandem remain unchanged when rigid or flexible linker extensions are included. Together, the results suggest that the two C2 domains of Syt7 bind independently to planar membranes, in contrast to reported interdomain cooperativity in Syt1.

Protein kinase C-α (PKCα) is a member of the conventional family of protein kinase C isoforms (cPKCs) that regulate diverse cellular signaling pathways, share a common activation mechanism, and are linked to multiple pathologies. The cPKC domain structure is modular, consisting of an N-terminal pseudosubstrate peptide, two inhibitory domains (C1A and C1B), a targeting domain (C2), and a kinase domain. Mature, cytoplasmic cPKCs are inactive until they are switched on by a multistep activation reaction that occurs largely on the plasma membrane surface. Often, this activation begins with a cytoplasmic Ca(2+) signal that triggers C2 domain targeting to the plasma membrane where it binds phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP2). Subsequently, the appearance of the signaling lipid diacylglycerol (DAG) activates the membrane-bound enzyme by recruiting the inhibitory pseudosubstrate and one or both C1 domains away from the kinase domain. To further investigate this mechanism, this study has utilized single-molecule total internal reflection fluorescence microscopy (TIRFM) to quantitate the binding and lateral diffusion of full-length PKCα and fragments missing specific domain(s) on supported lipid bilayers. Lipid binding events, and events during which additional protein is inserted into the bilayer, were detected by their effects on the equilibrium bound particle density and the two-dimensional diffusion rate. In addition to the previously proposed activation steps, the findings reveal a major, undescribed, kinase-inactive intermediate. On bilayers containing PS or PS and PIP2, full-length PKCα first docks to the membrane via its C2 domain, and then its C1A domain embeds itself in the bilayer even before DAG appears. The resulting pre-DAG intermediate with membrane-bound C1A and C2 domains is the predominant state of PKCα while it awaits the DAG signal. The newly detected, membrane-embedded C1A domain of this pre-DAG intermediate confers multiple useful features, including enhanced membrane affinity and longer bound state lifetime. The findings also identify the key molecular step in kinase activation: because C1A is already membrane-embedded in the kinase off state, recruitment of C1B to the bilayer by DAG or phorbol ester is the key regulatory event that stabilizes the kinase on state. More broadly, this study illustrates the power of single-molecule methods in elucidating the activation mechanisms and hidden regulatory states of membrane-bound signaling proteins.

Membrane-targeting proteins are crucial components of many cell signaling pathways, including the secretion of insulin. Granuphilin, also known as synaptotagmin-like protein 4, functions in tethering secretory vesicles to the plasma membrane prior to exocytosis. Granuphilin docks to insulin secretory vesicles through interaction of its N-terminal domain with vesicular Rab proteins; however, the mechanisms of granuphilin plasma membrane targeting and release are less clear. Granuphilin contains two C2 domains, C2A and C2B, that interact with the plasma membrane lipid phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. The goal of this study was to determine membrane-binding mechanisms, affinities, and kinetics of both granuphilin C2 domains using fluorescence spectroscopic techniques. Results indicate that both C2A and C2B bind anionic lipids in a Ca(2+)-independent manner. The C2A domain binds liposomes containing a physiological mixture of lipids including 2% PI(4,5)P2 or PI(3,4,5)P3 with high affinity (apparent K(d, PIPx) of 2-5 nM), and binds nonspecifically with moderate affinity to anionic liposomes lacking phosphatidylinositol phosphate (PIPx) lipids. The C2B domain binds with sub-micromolar affinity to liposomes containing PI(4,5)P2 but does not have a measurable affinity for background anionic lipids. Both domains can be competed away from their target lipids by the soluble PIPx analog inositol-(1,2,3,4,5,6)-hexakisphosphate (IP6), which is a positive regulator of insulin secretion. Potential roles of these interactions in the docking and release of granuphilin from the plasma membrane are discussed.

Synaptotagmin (Syt) triggers Ca(2+)-dependent membrane fusion via its tandem C2 domains, C2A and C2B. The 17 known human isoforms are active in different secretory cell types, including neurons (Syt1 and others) and pancreatic β cells (Syt7 and others). Here, quantitative fluorescence measurements reveal notable differences in the membrane docking mechanisms of Syt1 C2A and Syt7 C2A to vesicles comprised of physiological lipid mixtures. In agreement with previous studies, the Ca(2+) sensitivity of membrane binding is much higher for Syt7 C2A. We report here for the first time that this increased sensitivity is due to the slower target membrane dissociation of Syt7 C2A. Association and dissociation rate constants for Syt7 C2A are found to be ~2-fold and ~60-fold slower than Syt1 C2A, respectively. Furthermore, the membrane dissociation of Syt7 C2A but not Syt1 C2A is slowed by Na(2)SO(4) and trehalose, solutes that enhance the hydrophobic effect. Overall, the simplest model consistent with these findings proposes that Syt7 C2A first docks electrostatically to the target membrane surface and then inserts into the bilayer via a slow hydrophobic mechanism. In contrast, the membrane docking of Syt1 C2A is known to be predominantly electrostatic. Thus, these two highly homologous domains exhibit distinct mechanisms of membrane binding correlated with their known differences in function.

Many signaling pathways are controlled by protein complexes assembled on membrane surfaces via collisions between diffusing membrane components. Subsequently, the active, membrane-bound complex often collides with other effector proteins or lipids on the membrane surface to transmit its essential downstream signal. Thus, 2D-diffusion of membrane-bound proteins, complexes and effectors plays a central role in signaling biology. We have proposed that single molecule analysis of membrane protein diffusion on supported lipid bilayers could provide a powerful approach for studies of complex formation between membrane-associated proteins (Knight, Lerner, Marcano-Velazquez, Pastor & Falke (2010) Biophys J 99:2879-87). In principle the approach could yield information about complex stoichiometry, stability, and kinetics, and about specific lipid requirements for complex formation. Here we test these ideas using a simple model system in which membrane-bound pleckstrin homology (PH) domains are forced to dimerize by the calcium-triggered association of calmodulin (CaM) and its target peptide from skeletal muscle light chain kinase (MLCKp). Two fusion protein constructs (CaM-PH) and (MLCKp-PH) have been generated and bind normally to the PH domain target lipid, PIP3. Single molecule TIRF analysis of CaM-PH diffusion reveals that in the presence of calcium and MLCKp-PH a stable CaM-PH/MLCKp-PH heterodimer is formed on the membrane surface that diffuses approximately 2-fold more slowly than the single CaM-PH molecule. Addition of excess EDTA to chelate calcium reverses the dimer back to monomers. Substoichiometric levels of MLCKp-PH yield a mixed population of monomers and dimers, and the two populations can be resolved by single molecule analysis. Overall, the findings indicate that single molecule diffusion provides a useful new window into many critical, molecular features of protein complex formation on membrane surfaces. [Supported by NIH grant GM063235.]

Synaptotagmin (Syt) triggers Ca2+-dependent membrane fusion during secretion via its tandem C2 domains, termed C2A and C2B. The seventeen known human isoforms are active in different secretory cell types, including neurons (SytI and others) and pancreatic β cells (SytVII and others). Here, quantitative fluorescence measurements reveal notable differences in the membrane docking affinities, kinetics, and molecular driving forces for C2A and C2B domains from SytI and SytVII, using vesicles comprised of physiological target lipid mixtures. In agreement with previous studies, the Ca2+ sensitivity of membrane binding is greater for both domains from SytVII than for their counterparts in SytI. We demonstrate that for C2A, this increased sensitivity is due to a stronger SytVIIC2A membrane interaction, which involves substantial contribution from the hydrophobic effect. Association and dissociation rate constants for both SytVII domains are found to be significantly slower than their counterparts in SytI. For SytVIIC2A, the dissociation rate constant is ∼50-fold slower than SytIC2A and is reminiscent of the cPLA2C2 domain that is known to insert deeply into membranes. Addition of sodium sulfate decreases the dissociation rate of SytVIIC2A but not SytIC2A, further indicating that hydrophobic contacts play a major role in SytVIIC2A membrane docking. Thus, SytVIIC2A docks to membranes via both hydrophobic and electrostatic interactions, while the membrane docking interaction of SytIC2A is predominantly electrostatic. The inclusion of phosphatidylinositol-4,5-bisphosphate (PIP2) in membrane mixtures leads to increased affinity and slower dissociation for both C2B domains, but has minimal effects on C2A domains. Overall, highly homologous domains from these two proteins exhibit distinct mechanisms of membrane binding that may reflect their functions in different cell types.

Granuphilin is a membrane binding protein responsible, in part, for docking insulin secretory vesicles to the plasma membrane. Granuphilin, also known as synaptotagmin like protein 4 (Slp4), contains tandem C2 domains. Unlike synaptotagmin, however, the granuphilin C2A domain binds strongly to plasma membrane lipids independent of intracellular Ca2+. Despite the strong binding, neither the lipid targets for this protein nor the residues responsible for binding are well characterized. Our study attempts to identify the respective roles of (a) phosphoinositides such as phosphatidylinositol (4,5)-bisphosphate (PIP2) and (b) anionic backgroundlipids such as phosphatidylserine (PS) in binding of the granuphilin C2A domain to liposomes. Affinities are measured using protein-to-membrane FRET and titration with the competitive inhibitor inositol (1,2,3,4,5,6)-hexakisphosphate (IP6). Granuphilin C2A binds to liposomes containing phosphatidylcholine, dansyl-phosphatidylethanolamine, and PS and/or PIP2. Decreased FRET is measured as the protein is displaced from lipid upon titration with IP6. Granuphilin C2A has similar affinity for liposomes containing either 24% PS or 2% PIP2, but affinity increases ∼100-fold in the presence of both target lipids. Binding site(s) for the two lipids are being probed using site-directed mutagenesis and NMR. Overall, granuphilin demonstrates the capability to bind to background anionic lipids and phosphoinositides independently, but the presence of both lipids greatly increases the binding affinity of this protein. This suggests that granuphilin may serve as a coincidence detector to target vesicles to sites of secretion on the plasma membrane.

Islet amyloid polypeptide (IAPP, also known as amylin) is the major protein component of pancreatic amyloid fibers in type II diabetes and is normally cosecreted with insulin from the beta-cells of the pancreas. IAPP forms amyloid fibrils rapidly at concentrations well below those found in vivo, yet progression of type II diabetes occurs over many years. Insulin, a known inhibitor of IAPP fibrillogenesis, exists as a dense crystalline or near-crystalline core in the secretory vesicle, while IAPP localizes to the region between the crystal and the secretory vesicle membrane. In vitro, IAPP fibrillogenesis is both accelerated by lipid membranes and inhibited by monomeric insulin. In this work, we investigate insulin-IAPP-lipid interactions in vitro under conditions chosen to approximate native secretory vesicle physiology and the amyloid disease state. The effect of insulin on IAPP fibrillogenesis is investigated using fluorescence spectrometry. Additionally, interactions of IAPP and lipids with crystalline insulin are studied using fluorescence microscopy. We find that, while soluble states of insulin and IAPP do not interact significantly, large assemblies of either insulin (crystals) or IAPP (fibers) can lead to stable IAPP-insulin interactions. The results raise the possibility of multiple physiological interactions between these two beta-cell hormones.

The proper execution of Ca2+-triggered exocytosis requires extreme changes in bilayer shape to be precisely regulated. Because membranes alone are unlikely to undergo the required changes, proteins are necessary to mediate events. However, the identity of these proteins and the conditions under which they act are not well understood. Within this context, our studies focus on membrane-targeting C2 domains from synaptotagmin-7 (Syt-7) - an isoform of the Syt protein family that is important for secretion in neuroendocrine cells. To define how Syt-7 drives changes in membrane morphology, we have used recombinant C2AB protein fragments, supported lipid bilayers (SLBs), and total internal reflection fluorescence microscopy (TIRFM). We have developed conditions for forming SLBs under which the membrane spontaneously forms long tubule-like structures extending away from the support surface. Upon addition of purified Syt-7 C2AB in the presence of Ca2+, these long tubules disappear and are replaced by shorter tubules or vesicles. The effect is reversible upon removal of Ca2+. These studies demonstrate that Syt-7 can alter membrane morphology, ostensibly by driving changes in membrane curvature. They also demonstrate the utility of a novel experimental system in which protein-mediated changes in membrane topology can be studied in aqueous media and in real-time.

Islet amyloid polypeptide (IAPP) is a 37-residue hormone that forms cytotoxic amyloid fibers in the endocrine pancreas of patients with type II diabetes (NIDDM). A potential origin for cytotoxicity is disruption of lipid membranes by IAPP as has been observed in vitro. The cause of amyloid formation during NIDDM is not known, nor is the mechanism by which membrane disruption occurs in vitro. Here, we use kinetic studies in conjunction with assessments of lipid binding and electron microscopy to investigate the interactions of IAPP with phospholipid bilayers and the morphological effects of membranes on IAPP fibers. Fibrillogenesis of IAPP is catalyzed by synthetic and human tissue-derived phospholipids, leading to >tenfold increases in the rate of fibrillogenesis. The molecular basis of this phenomenon includes a strong dependence on the concentration and charge density of the membrane. IAPP binds to lipid membranes of mixed anionic (DOPG) and zwitterionic (DOPC) content. The transition for binding occurs over a physiologically relevant range of anionic content. Membrane binding by IAPP occurs on timescales that are short compared to fibrillogenesis and results in assembly into preamyloid states via ordered interactions at the N but not C terminus of the protein. These assemblies lead both to gross morphological changes in liposomes and to alterations in the appearance of early fibers when compared to liposome-free fibril formation. Intact bilayer surfaces are regenerated upon dissociation of fibers from the membrane surface. These findings offer a structural mechanism of membrane destabilization and suggest that changes in lipid metabolism could induce IAPP fiber formation in NIDDM.

A novel method for protection of DNA from high shear induced damage is presented. This method uses simple divalent cations and the lyophilizable alcohol, tert-butanol, to self-assemble DNA into condensed, shear-resistant forms. The DNA used in these studies was a 5600 BP plasmid DNA encoding a therapeutic gene. Various solvents and salts were used to identify optimal conditions to condense plasmid DNA. A stable formulation was identified with plasmid DNA condensed in a cosolvent solution containing 20% (v/v) tert-butanol and 1 mM calcium chloride. The DNA was formulated at 100 μg/ml and condensed into rod and toroidal shapes that were approximately 50–300 nm in diameter. The rods were found to be kinetically stable for greater than 24 h following their preparation. Condensation of the plasmid DNA in this manner results in nearly 100% of the plasmid DNA remaining intact after 1 min of high shear stress applied by a 50 W probe sonicator. Uncondensed control plasmid DNA is completely fragmented following 30 s of identical sonication. It is believed that condensation of DNA in this manner will permit utilization of high shear-stress inducing processing techniques, such as lyophilization or spray-drying without resulting in damage to the DNA.

A debilitating complication of long-term hemodialysis is the deposition of ‚-2-microglobulin (‚2m) as amyloid plaques in the joint space. We have recently shown that Cu 2+ can be a contributing, if not causal, factor at concentrations encountered during dialysis therapy. The basis for this effect is destabilization and incorporation of ‚2m into amyloid fibers upon binding of Cu 2+ . In this work, we demonstrate that while ‚2m binds Cu 2+ specifically in the native state, it is binding of Cu 2+ by non- native states of ‚2m which is responsible for destabilization. Mutagenesis of potential coordinating groups for Cu 2+ shows that native state binding of Cu 2+ is mediated by residues and structures that are different than those which bind in non-native states. An increased affinity for copper by non-native states compared to that of the native state gives rise to overall destabilization. Using mass spectrometry, NMR, and fluorescence techniques, we show that native state binding is localized to H31 and W60 and is highly specific for Cu 2+ over Zn 2+ and Ni 2+ . Binding of Cu 2+ in non-native states of ‚2m is mediated by residues H13, H51, and H84, but not H31. Although denatured ‚2m has characteristics of a globally unfolded state, it nevertheless demonstrates the following strong specificity of binding: Cu 2+ > Zn 2+ . Ni 2+ . This requires the existence of a well-defined structure in the unfolded state of this protein. As Cu 2+ effects are reported in many other amyloidoses, e.g., PrP, R-synuclein, and A‚, our results may be extended to the emerging field of divalent ion-associated amyloidosis. The conversion of normally soluble proteins into amyloid fiber is a common feature of a number of clinical disorders, including Alzheimer's, type II diabetes, and spongiform encephalopathies (1). Furthermore, many proteins which do not form pathogenic fibers, e.g., myoglobin, have neverthe- less been shown to convert to amyloid under laboratory conditions (2). Amyloid fibers formed from different proteins have many features in common. They are unbranched, resist proteolytic digestion, and display green birefringence upon staining with the histological dye Congo Red. The tertiary and quaternary structure of amyloid fibers has been deter- mined from X-ray diffraction studies to be cross-‚, with the ‚-strands arranged orthogonal to the fiber axis and hydrogen bonding parallel to the fiber axis (3). The kinetics of amyloid formation include a lag phase suggesting nucleation-depend- ent kinetics akin to crystallization ( 4). The similarity of fiber formation kinetics to crystallization includes the ability to bypass the lag phase by providing exogenous seed. Interest- ingly, while amyloids from different proteins share common histological and ultrastructural features, seeded reaction kinetics are dependent on using the seed of the same or

Synaptotagmin (Syt) proteins serve as Ca2+ sensitive triggers in many exocytotic pathways, with the seventeen different human isoforms active in various cell types. Syt proteins contain two C2 domains, C2A and C2B, which bind membranes in response to Ca2+ to drive vesicle fusion. Much is known about the biophysical mechanism of function for Syt1, which triggers fusion for rapid neurotransmitter secretion, but less mechanistic information is available for other isoforms. Syt7 typically operates in slower pathways requiring smaller peak Ca2+ concentrations, and its C2 domains are known to bind membranes with a much higher Ca2+ sensitivity compared to Syt1. using kinetic and equilibrium fluorescence measurements of C2A domain docking to synthetic liposomes approximating the lipid composition of physiological membranes, we report that the differences between the two isoforms include kinetic and solute effects consistent with much greater hydrophobic membrane contact for Syt7 C2A. A strong hydrophobic contribution to the membrane docking mechanism of Syt7 C2A stands in contrast to the known electrostatic membrane interaction of Syt1 C2A, and is somewhat surprising given the 90% conserved amino acid polarity between the two domains. In order to test our proposed hydrophobic docking model for Syt7 C2A and probe its structural origins, we use a combination of site-directed mutagenesis, equilibrium and kinetic protein-membrane docking assays, and electron paramagnetic resonance-based depth measurements. In addition, single-molecule measurement of protein lateral diffusion on supported lipid bilayers is used to report on contributions of intra- and intermolecular protein-protein contact to the membrane-docked states of individual C2 domains and C2AB tandems. The results are interpreted to provide information on the structural origins of differences in function between these two isoforms.

Membrane targeting proteins are recruited to specific membranes during cell signaling events, including signals at the leading edge of chemotaxing cells. Recognition and binding to specific lipids play a central role in targeting reactions, but it remains difficult to analyze the molecular features of such protein-lipid interactions. We propose that the surface diffusion constant of peripheral membrane-bound proteins contains useful information about protein-lipid contacts and membrane dynamics. To test this hypothesis, we use single-molecule fluorescence microscopy to probe the effects of lipid binding stoichiometry on the diffusion constants of engineered proteins containing one to three pleckstrin homology domains coupled by flexible linkers. Within error, the lateral diffusion constants of these engineered constructs are inversely proportional to the number of tightly bound phosphatidylinositol-(3,4,5)-trisphosphate lipids. The same trend is observed in coarse-grained molecular dynamics simulations and hydrodynamic bead calculations of lipid multimers connected by model tethers. Overall, single molecule diffusion measurements are found to provide molecular information about protein-lipid interactions. Moreover, the experimental and computational results independently indicate that the frictional contributions of multiple, coupled but well-separated lipids are additive, analogous to the free-draining limit for isotropic fluids—an insight with significant implications for theoretical description of bilayer lipid dynamics.