Biomedical subjects

J Rizo

Publications and source records attributed to J Rizo.

At least 19 recordsLinked to original sources

PubMed2001

Three-dimensional structure of the synaptotagmin 1 C2B-domain: synaptotagmin 1 as a phospholipid binding machine.

Synaptotagmin 1 probably functions as a Ca2+ sensor in neurotransmitter release via its two C2-domains, but no common Ca2+-dependent activity that could underlie a cooperative action between them has been described. The NMR structure of the C2B-domain now reveals a beta sandwich that exhibits striking similarities and differences with the C2A-domain. Whereas the bottom face of the C2B-domain has two additional alpha helices that may be involved in specialized Ca2+-independent functions, the top face binds two Ca2+ ions and is remarkably similar to the C2A-domain. Consistent with these results, but in contrast to previous studies, we find that the C2B-domain binds phospholipids in a Ca2+-dependent manner similarly to the C2A-domain. These results suggest a novel view of synaptotagmin function whereby the two C2-domains cooperate in a common activity, Ca2+-dependent phospholipid binding, to trigger neurotransmitter release.

Amino Acid Sequence↗

PubMed2001

The top loops of the C(2) domains from synaptotagmin and phospholipase A(2) control functional specificity.

The phospholipid-binding specificities of C(2) domains, widely distributed Ca(2+)-binding modules, differ greatly despite similar three-dimensional structures. To understand the molecular basis for this specificity, we have examined the synaptotagmin 1 C(2)A domain, which interacts in a primarily electrostatic, Ca(2+)-dependent reaction with negatively charged phospholipids, and the cytosolic phospholipase A(2) (cPLA(2)) C(2) domain, which interacts by a primarily hydrophobic Ca(2+)-dependent mechanism with neutral phospholipids. We show that grafting the short Ca(2+)-binding loops from the tip of the cPLA(2) C(2) domain onto the top of the synaptotagmin 1 C(2)A domain confers onto the synaptotagmin 1 C(2)A domain the phospholipid binding specificity of the cPLA(2) C(2) domain, indicating that the functional specificity of C(2) domains is determined by their short top loops.

Amino Acid Sequence↗

PubMed2001

The C2B domain of synaptotagmin I is a Ca2+-binding module.

Synaptotagmin I is a synaptic vesicle protein that contains two C(2) domains and acts as a Ca(2+) sensor in neurotransmitter release. The Ca(2+)-binding properties of the synaptotagmin I C(2)A domain have been well characterized, but those of the C(2)B domain are unclear. The C(2)B domain was previously found to pull down synaptotagmin I from brain homogenates in a Ca(2+)-dependent manner, leading to an attractive model whereby Ca(2+)-dependent multimerization of synaptotagmin I via the C(2)B domain participates in fusion pore formation. However, contradictory results have been described in studies of Ca(2+)-dependent C(2)B domain dimerization, as well as in analyses of other C(2)B domain interactions. To shed light on these issues, the C(2)B domain has now been studied using biophysical techniques. The recombinant C(2)B domain expressed as a GST fusion protein and isolated by affinity chromatography contains tightly bound bacterial contaminants despite being electrophoretically pure. The contaminants bind to a polybasic sequence that has been previously implicated in several C(2)B domain interactions, including Ca(2+)-dependent dimerization. NMR experiments show that the pure recombinant C(2)B domain binds Ca(2+) directly but does not dimerize upon Ca(2+) binding. In contrast, a cytoplasmic fragment of native synaptotagmin I from brain homogenates, which includes the C(2)A and C(2)B domains, participates in a high molecular weight complex as a function of Ca(2+). These results show that the recombinant C(2)B domain of synaptotagmin I is a monomeric, autonomously folded Ca(2+)-binding module and suggest that a potential function of synaptotagmin I multimerization in fusion pore formation does not involve a direct interaction between C(2)B domains or requires a posttranslational modification.

Amino Acid Motifs↗

PubMed2001

Functional analysis of conserved structural elements in yeast syntaxin Vam3p.

Vam3p, a syntaxin-like SNARE protein involved in yeast vacuole fusion, is composed of a three-helical N-terminal domain, a canonical SNARE motif, and a C-terminal transmembrane region (TMR). Surprisingly, we find that the N-terminal domain of Vam3p is not essential for fusion, although analogous domains in other syntaxins are indispensible for fusion and/or protein-protein interactions. In contrast to the N-terminal domain, mutations in the SNARE motif of Vam3p or replacement of the SNARE motif of Vam3p with the SNARE motif from other syntaxins inhibited fusion. Furthermore, the precise distance between the SNARE motif and the TMR was critical for fusion. Insertion of only three residues after the SNARE motif significantly impaired fusion and insertion of 12 residues abolished fusion. As judged by co-immunoprecipitation experiments, the SNARE motif mutations and the insertions did not alter the association of Vam3p with Vam7p, Vti1p, Nyv1p, and Ykt6p, other vacuolar SNARE proteins implicated in fusion. In contrast, the SNARE motif substitutions interfered with the stable formation of Vam3p complexes with Nyv1p and Vti1p, although Vam3p complexes with Vam7p and Ykt6p were still present. Our data suggest that in contrast to previously characterized syntaxins, Vam3p contains only two domains essential for fusion, the SNARE motif and the TMR, and these domains have to be closely coupled to function in fusion.

Conserved Sequence↗

PubMed2001

An unusual C(2)-domain in the active-zone protein piccolo: implications for Ca(2+) regulation of neurotransmitter release.

Ca(2+) regulation of neurotransmitter release is thought to require multiple Ca(2+) sensors with distinct affinities. However, no low-affinity Ca(2+) sensor has been identified at the synapse. We now show that piccolo/aczonin, a recently described active-zone protein with C-terminal C(2)A- and C(2)B-domains, constitutes a presynaptic low-affinity Ca(2+) sensor. Ca(2+) binds to piccolo by virtue of its C(2)A-domain via an unusual mechanism that involves a large conformational change. The distinct Ca(2+)-binding properties of the piccolo C(2)A- domain are mediated by an evolutionarily conserved sequence at the bottom of the C(2)A-domain, which may fold back towards the Ca(2+)-binding sites on the top. Point mutations in this bottom sequence inactivate it, transforming low-affinity Ca(2+) binding (100-200 microM in the presence of phospholipids) into high-affinity Ca(2+) binding (12-14 microM). The unusual Ca(2+)-binding mode of the piccolo C(2)A-domain reveals that C(2)-domains are mechanistically more versatile than previously envisaged. The low Ca(2+) affinity of the piccolo C(2)A-domain suggests that piccolo could function in short-term synaptic plasticity when Ca(2+) concentrations accumulate during repetitive stimulation.

Amino Acid Sequence↗

PubMed2001

Synaptotagmin I functions as a calcium regulator of release probability.

In all synapses, Ca2+ triggers neurotransmitter release to initiate signal transmission. Ca2+ presumably acts by activating synaptic Ca2+ sensors, but the nature of these sensors--which are the gatekeepers to neurotransmission--remains unclear. One of the candidate Ca2+ sensors in release is the synaptic Ca2+-binding protein synaptotagmin I. Here we have studied a point mutation in synaptotagmin I that causes a twofold decrease in overall Ca2+ affinity without inducing structural or conformational changes. When introduced by homologous recombination into the endogenous synaptotagmin I gene in mice, this point mutation decreases the Ca2+ sensitivity of neurotransmitter release twofold, but does not alter spontaneous release or the size of the readily releasable pool of neurotransmitters. Therefore, Ca2+ binding to synaptotagmin I participates in triggering neurotransmitter release at the synapse.

Animals↗

PubMed2001

Vam3p structure reveals conserved and divergent properties of syntaxins.

Syntaxins and Sec1/munc18 proteins are central to intracellular membrane fusion. All syntaxins comprise a variable N-terminal region, a conserved SNARE motif that is critical for SNARE complex formation, and a transmembrane region. The N-terminal region of neuronal syntaxin 1A contains a three-helix domain that folds back onto the SNARE motif forming a 'closed' conformation; this conformation is required for munc18-1 binding. We have examined the generality of the structural properties of syntaxins by NMR analysis of Vam3p, a yeast syntaxin essential for vacuolar fusion. Surprisingly, Vam3p also has an N-terminal three-helical domain despite lacking apparent sequence homology with syntaxin 1A in this region. However, Vam3p does not form a closed conformation and its N-terminal domain is not required for binding to the Sec1/munc18 protein Vps33p, suggesting that critical distinctions exist in the mechanisms used by syntaxins to govern different types of membrane fusion.

Amino Acid Sequence↗

PubMed2000

Selective interaction of complexin with the neuronal SNARE complex. Determination of the binding regions.

Complexins are evolutionarily conserved proteins that specifically bind to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and thus may regulate SNARE function. Using purified proteins, we have performed a detailed analysis of the structure of complexin and of its interaction with SNARE proteins. NMR spectroscopy revealed that isolated complexins have no tertiary structure but contain an unusual alpha-helical middle domain of approximately 58 amino acids that overlaps with the most highly conserved region of the molecules. Complexins form a stable stoichiometric complex with the central domain of the ternary SNARE complex, whereas no binding was observed to monomeric SNAREs. Using a combination of limited proteolysis, deletion mutagenesis, and NMR spectroscopy, we found that the helical middle region of complexin is responsible for binding to the SNARE complex. Binding was highly sensitive to substitution of syntaxin 1 or synaptobrevin 2 with other SNARE homologs but less sensitive to substitution of SNAP-25. In addition, a stretch of 12 amino acids in the middle of the SNARE motif of syntaxin 1A was able to confer binding activity to the non-binding relative syntaxin 4. Furthermore, disassembly of ternary complexes is not affected by complexins. We conclude that complexins are specific ligands of the neuronal core complex that bind with a central alpha-helical domain, probably to the middle of the surface groove formed by synaptobrevin and syntaxin. Complexins may regulate the function of ternary complexes and control membrane fusion through this interaction.

Adaptor Proteins, Vesicular Transport↗

PubMed2000

Consensus bioactive conformation of cyclic GnRH antagonists defined by NMR and molecular modeling.

Little is known of the conformation of peptide hormones as they interact with their receptors for a number of reasons: peptide hormones are notoriously flexible in solution, their receptors are particularly complex, and there is strong evidence that receptor-ligand interaction leading to activation is a dynamic process. Insights into the active conformation of the decapeptide gonadotropin releasing hormone (GnRH) have been obtained previously from the solution structures of four constrained GnRH antagonists ¿cyclo(1-10)[Ac-Delta(3)-Pro(1),DCpa(2),DTrp(3,6),NMeLeu+ ++(7), betaAla(10)]GnRH (1), cyclo(4-10)[Ac-Delta(3)Pro(1),DFpa(2),DTrp(3), Asp(4),DNal(6),Dpr(10)]GnRH (2), dicyclo(4-10/5-8)[Ac-DNal(1), DCpa(2),DTrp(3),Asp(4),Glu(5),DArg(6),Lys(8),Dpr (10)]GnRH (3), and dicyclo(4-10/5-5'-8)[Ac-DNal(1),DCpa(2),DPal(3), Asp(4),Glu(5)(Gly), DArg(6),Dbu(8),Dpr(10)]GnRH (4)¿. However, the precise location of the N-terminal tripeptide in the highly potent (K(i) < 0.4 nM) 2-4 remained unclear due to the lack of constraints in this region. The NMR structure of the newly discovered and potent (K(i) = 0.24 nM) dicyclo(1-1'-5/4-10)[Ac-Glu(1)(Gly),DCpa(2),DTrp(3),As p(4),Dbu(5), DNal(6),Dpr(10)]GnRH (5) now allows the definition of the conformation of this region. A combined computational analysis (consensus forcing) of compounds 2-5, designed to explore the common conformations available to them that are simultaneously consistent with the NMR data corresponding to each compound, leads to a consensus structural model for the GnRH pharmacophore. This model shares some common features with the structure of the nonpeptidic GnRH mimetic T-98475. In the course of that comparative study, two additional contact points to those proposed by the authors are identified, suggesting that this model has predictive value.

Animals↗

PubMed2000

The relation of protein binding to function: what is the significance of munc18 and synaptotagmin binding to syntaxin 1, and where are the corresponding binding sites?

The Q-SNARE syntaxin 1 is a central component of the synaptic membrane fusion machinery. Syntaxin probably interacts with multiple proteins during synaptic vesicle exocytosis. In vitro, the tightest binding partners for syntaxin 1 are other SNAREs (synaptobrevin/VAMP and SNAP-25) and munc18-1 (also known as rbsec1/nsec1). Recent studies on Drosophila syntaxin led to the surprising finding that a syntaxin mutant which does not bind the munc18-homolog Rop nevertheless functionally substitutes for wild-type syntaxin in membrane fusion (Wu et al., Neuron 23, 593-605, 1999). This observation suggested that syntaxin 1 binding to munc18-1 is not essential for fusion, a puzzling conclusion in view of the tight binding of munc18 and syntaxin homologs in all organisms. To address this issue, we have now reinvestigated the interaction of syntaxin with munc18 and Rop. We find that the syntaxin sequence that was mutated in the Drosophila studies is not essential for munc18/Rop binding, and that the mutant is in fact able to bind to munc18/Rop. Thus the fact that the mutant syntaxin rescues release cannot be used as an argument that munc18 binding is not essential. In addition to munc18 and SNAREs, several other proteins have been suggested to interact with various domains of syntaxin 1, notably the calcium-sensor synaptotagmin and the vesicle protein CSP. Our results confirm that the SNARE motif in syntaxin binds to synaptotagmin, but this interaction does not require the very C-terminus of the motif. Interestingly, binding of synaptotagmin appears to be decreased in the closed conformation of syntaxin. In contrast, no interaction of CSP with syntaxin was detected even under low-stringency conditions. Our data suggest 1., that assays measuring protein/protein interactions that involve syntaxin may be more difficult to evaluate than is often assumed because of the sticky nature of the proteins involved, and 2., that it is currently not possible to draw conclusions about the importance of the various interactions with the available data from Drosophila or vertebrates.

Amino Acid Motifs↗

PubMed1999

A conformational switch in syntaxin during exocytosis: role of munc18.

Syntaxin 1, an essential protein in synaptic membrane fusion, contains a helical autonomously folded N-terminal domain, a C-terminal SNARE motif and a transmembrane region. The SNARE motif binds to synaptobrevin and SNAP-25 to assemble the core complex, whereas almost the entire cytoplasmic sequence participates in a complex with munc18-1, a neuronal Sec1 homolog. We now demonstrate by NMR spectroscopy that, in isolation, syntaxin adopts a 'closed' conformation. This default conformation of syntaxin is incompatible with core complex assembly which requires an 'open' syntaxin conformation. Using site-directed mutagenesis, we find that disruption of the closed conformation abolishes the ability of syntaxin to bind to munc18-1 and to inhibit secretion in PC12 cells. These results indicate that syntaxin binds to munc18-1 in a closed conformation and suggest that this conformation represents an essential intermediate in exocytosis. Our data suggest a model whereby, during exocytosis, syntaxin undergoes a large conformational switch that mediates the transition between the syntaxin-munc18-1 complex and the core complex.

Animals↗

PubMed1999

Evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome.

The dose-limiting toxicity of interleukin-2 (IL-2) and immunotoxin (IT) therapy in humans is vascular leak syndrome (VLS). VLS has a complex etiology involving damage to vascular endothelial cells (ECs), extravasation of fluids and proteins, interstitial edema, and organ failure. IL-2 and ITs prepared with the catalytic A chain of the plant toxin, ricin (RTA), and other toxins, damage human ECs in vitro and in vivo. Damage to ECs may initiate VLS; if this damage could be avoided without losing the efficacy of ITs or IL-2, larger doses could be administered. In this paper, we provide evidence that a three amino acid sequence motif, (x)D(y), in toxins and IL-2 damages ECs. Thus, when peptides from RTA or IL-2 containing this sequence motif are coupled to mouse IgG, they bind to and damage ECs both in vitro and, in the case of RTA, in vivo. In contrast, the same peptides with a deleted or mutated sequence do not. Furthermore, the peptide from RTA attached to mouse IgG can block the binding of intact RTA to ECs in vitro and vice versa. In addition, RTA, a fragment of Pseudomonas exotoxin A (PE38-lys), and fibronectin also block the binding of the mouse IgG-RTA peptide to ECs, suggesting that an (x)D(y) motif is exposed on all three molecules. Our results suggest that deletions or mutations in this sequence or the use of nondamaging blocking peptides may increase the therapeutic index of both IL-2, as well as ITs prepared with a variety of plant or bacterial toxins.

ADP Ribose Transferases↗

PubMed1999

NMR analysis of the structure of synaptobrevin and of its interaction with syntaxin.

Synaptobrevin is a synaptic vesicle protein that has an essential role in exocytosis and forms the SNARE complex with syntaxin and SNAP-25. We have analyzed the structure of isolated synaptobrevin and its binary interaction with syntaxin using NMR spectroscopy. Our results demonstrate that isolated synaptobrevin in largely unfolded in solution. The entire SNARE motif of synaptobrevin is capable of interacting with the isolated C-terminal SNARE motif of syntaxin but only a few residues bind to the full-length cytoplasmic region of syntaxin. This result suggests an interaction between the N- and C-terminal regions of syntaxin that competes with core complex assembly.

Amino Acid Sequence↗

PubMed1999

Structure of the Janus-faced C2B domain of rabphilin.

C2 domains are widespread protein modules that often occur as tandem repeats in many membrane-trafficking proteins such as synaptotagmin and rabphilin. The first and second C2 domains (C2A and C2B, respectively) have a high degree of homology but also specific differences. The structure of the C2A domain of synaptotagmin I has been extensively studied but little is known about the C2B domains. We have used NMR spectroscopy to determine the solution structure of the C2B domain of rabphilin. The overall structure of the C2B domain is very similar to that of other C2 domains, with a rigid beta-sandwich core and loops at the top (where Ca2+ binds) and the bottom. Surprisingly, a relatively long alpha-helix is inserted at the bottom of the domain and is conserved in all C2B domains. Our results, together with the Ca(2+)-independent interactions observed for C2B domains, indicate that these domains have a Janus-faced nature, with a Ca(2+)-binding top surface and a Ca(2+)-independent bottom surface.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Solution structures of the Ca2+-free and Ca2+-bound C2A domain of synaptotagmin I: does Ca2+ induce a conformational change?

C2 domains are widespread Ca2+-binding modules that are particularly abundant in proteins involved in membrane traffic and signal transduction. The C2A domain of synaptotagmin I is believed to play a key role in neurotransmitter release through its Ca2+-dependent interactions with syntaxin and phospholipids. Elucidating the structural consequences of Ca2+ binding to the C2A domain is critical for understanding its mechanism of action and for models of the functions of other C2 domains. We have determined the solution structure of the Ca2+-free and Ca2+-bound forms of the C2A domain of synaptotagmin I by NMR spectroscopy. Our data represent the first structure determination of a C2 domain in its Ca2+-free and Ca2+-bound forms. Three Ca2+ ions were included in the Ca2+-bound structure, yielding a Ca2+-binding motif that involves five aspartate side chains and one serine side chain. Ca2+ immobilizes the structure of the C2A domain but does not produce a significant conformational change from a well-defined conformation to another. Thus, the mechanism of action of the C2A domain of synaptotagmin I is different from that used by Ca2+-binding proteins of the EF-hand family. The main effect of Ca2+ binding on the C2A domain is to change its electrostatic potential rather than its structure. These results support a model whereby the C2A domain functions as an electrostatic switch in neurotransmitter release. The similarity between the structures of the synaptotagmin I C2A domain and the PLC-delta1 C2 domain suggests that the latter binds four Ca2+ ions and acts by a similar mechanism. This mechanism may also be valid for other C2 domains that share the unusual ability to bind multiple Ca2+ ions in a tight cluster at the tip of the domain.

Calcium↗

PubMed1998

Three-dimensional structure of an evolutionarily conserved N-terminal domain of syntaxin 1A.

Syntaxin 1A plays a central role in neurotransmitter release through multiple protein-protein interactions. We have used NMR spectroscopy to identify an autonomously folded N-terminal domain in syntaxin 1A and to elucidate its three-dimensional structure. This 120-residue N-terminal domain is conserved in plasma membrane syntaxins but not in other syntaxins, indicating a specific role in exocytosis. The domain contains three long alpha helices that form an up-and-down bundle with a left-handed twist. A striking residue conservation is observed throughout a long groove that is likely to provide a specific surface for protein-protein interactions. A highly acidic region binds to the C2A domain of synaptotagmin I in a Ca2+-dependent interaction that may serve as an electrostatic switch in neurotransmitter release.

Animals↗

PubMed1998

Mechanism of phospholipid binding by the C2A-domain of synaptotagmin I.

Synaptotagmin I is a synaptic vesicle membrane protein that probably functions as a Ca2+ sensor in neurotransmitter release and contains two C2-domains which bind Ca2+. The first C2-domain of synaptotagmin I (the C2A-domain) binds phospholipids in a Ca2+-dependent manner similar to that of the C2-domains of protein kinase C, cytoplasmic phospholipase A2, and phospholipase Cdelta1. Although the tertiary structure of these C2-domains is known, the molecular basis for their Ca2+-dependent interactions with phospholipids is unclear. We have now investigated the mechanisms involved in Ca2+-dependent phospholipid binding by the C2A-domain of synaptotagmin I. Our data show that the C2A-domain binds negatively charged liposomes in an electrostatic interaction that is determined by the charge density of the liposome surface but not by the phospholipid headgroup. At the tip of the C2A-domain, three tightly clustered Ca2+-binding sites are formed by five aspartates and one serine. Mutations in these aspartate and serine residues demonstrated that all three Ca2+-binding sites are required for phospholipid binding. The Ca2+ binding sites at the top of the C2A-domain are surrounded by positively charged amino acids that were shown by mutagenesis to be also involved in phospholipid binding. Our results yield a molecular picture of the interactions between a C2-domain and phospholipids. Binding is highly electrostatic and occurs between the surfaces of the phospholipid bilayer and of the tip of the C2A-domain. The data suggest that the negatively charged phospholipid headgroups interact with the basic side chains surrounding the Ca2+-binding sites and with bound Ca2+ ions, thereby filling empty coordination sites and increasing the apparent affinity for Ca2+. In addition, insertion of hydrophobic side chains may contribute to phospholipid binding. This model is likely to be general for other C2-domains, with the relative contributions of electrostatic and hydrophobic interactions dictated by the exposed side chains surrounding the Ca2+-binding region.

Amino Acid Sequence↗

PubMed1998

Ca2+ binding to synaptotagmin: how many Ca2+ ions bind to the tip of a C2-domain?

C2-domains are widespread protein modules with diverse Ca2+-regulatory functions. Although multiple Ca2+ ions are known to bind at the tip of several C2-domains, the exact number of Ca2+-binding sites and their functional relevance are unknown. The first C2-domain of synaptotagmin I is believed to play a key role in neurotransmitter release via its Ca2+-dependent interactions with syntaxin and phospholipids. We have studied the Ca2+-binding mode of this C2-domain as a prototypical C2-domain using NMR spectroscopy and site-directed mutagenesis. The C2-domain is an elliptical module composed of a beta-sandwich with a long axis of 50 A. Our results reveal that the C2-domain binds three Ca2+ ions in a tight cluster spanning only 6 A at the tip of the module. The Ca2+-binding region is formed by two loops whose conformation is stabilized by Ca2+ binding. Binding involves one serine and five aspartate residues that are conserved in numerous C2-domains. All three Ca2+ ions are required for the interactions of the C2-domain with syntaxin and phospholipids. These results support an electrostatic switch model for C2-domain function whereby the beta-sheets of the domain provide a fixed scaffold for the Ca2+-binding loops, and whereby interactions with target molecules are triggered by a Ca2+-induced switch in electrostatic potential.

Animals↗