Three-Dimensional Structure of the Complexin/SNARE Complex

During neurotransmitter release, the neuronal SNARE proteins synaptobrevin/VAMP, syntaxin, and SNAP-25 form a four-helix bundle, the SNARE complex, that pulls the synaptic vesicle and plasma membranes together possibly causing membrane fusion. Complexin binds tightly to the SNARE complex and is essential for efficient Ca2+-evoked neurotransmitter release. A combined X-ray and TROSY-based NMR study now reveals the atomic structure of the complexin/SNARE complex. Complexin binds in an antiparallel α-helical conformation to the groove between the synaptobrevin and syntaxin helices. This interaction stabilizes the interface between these two helices, which bears the repulsive forces between the apposed membranes. These results suggest that complexin stabilizes the fully assembled SNARE complex as a key step that enables the exquisitely high speed of Ca2+-evoked neurotransmitter release

Computational de novo design of a four-helix bundle protein - DND-4HB

The de novo design of proteins is a rigorous test of our understanding of the key determinants of protein structure. The helix bundle is an interesting de novo design model system due to the diverse topologies that can be generated from a few simple α-helices. Previously, non-computational studies demonstrated that connecting amphipathic helices together with short loops can sometimes generate helix bundle proteins, regardless of the bundle's exact sequence. However using such methods, the precise positions of helices and side-chains cannot be predetermined. Since protein function depends on exact positioning of residues, we examined if sequence design tools in the program Rosetta could be used to design a four-helix bundle with a predetermined structure. Helix position was specified using a folding procedure that constrained the design model to a defined topology, and iterative rounds of rotamer-based sequence design and backbone refinement were used to identify a low energy sequence for characterization. The designed protein, DND_4HB, unfolds cooperatively (Tm >90°C) and a NMR solution structure shows that it adopts the target helical bundle topology. Helices 2, 3 and 4 agree very closely with the design model (backbone RMSD = 1.11 Å) and >90% of the core side-chain χ1 and χ2 angles are correctly predicted. Helix 1 lies in the target groove against the other helices, but is displaced 3 Å along the bundle axis. This result highlights the potential of computational design to create bundles with atomic-level precision, but also points at remaining challenges for achieving specific positioning between amphipathic helices

Structural Basis for a Munc13–1 Homodimer to Munc13–1/RIM Heterodimer Switch

C (2) domains are well characterized as Ca (2+)/phospholipid-binding modules, but little is known about how they mediate protein–protein interactions. In neurons, a Munc13–1 C (2)A-domain/RIM zinc-finger domain (ZF) heterodimer couples synaptic vesicle priming to presynaptic plasticity. We now show that the Munc13–1 C (2)A domain homodimerizes, and that homodimerization competes with Munc13–1/RIM heterodimerization. X-ray diffraction studies guided by nuclear magnetic resonance (NMR) experiments reveal the crystal structures of the Munc13–1 C (2)A-domain homodimer and the Munc13–1 C (2)A-domain/RIM ZF heterodimer at 1.44 Å and 1.78 Å resolution, respectively. The C (2)A domain adopts a β-sandwich structure with a four-stranded concave side that mediates homodimerization, leading to the formation of an eight-stranded β-barrel. In contrast, heterodimerization involves the bottom tip of the C (2)A-domain β-sandwich and a C-terminal α-helical extension, which wrap around the RIM ZF domain. Our results describe the structural basis for a Munc13–1 homodimer–Munc13–1/RIM heterodimer switch that may be crucial for vesicle priming and presynaptic plasticity, uncovering at the same time an unexpected versatility of C (2) domains as protein–protein interaction modules, and illustrating the power of combining NMR spectroscopy and X-ray crystallography to study protein complexes

Crystal Structure of the Formin mDia1 in Autoinhibited Conformation

Formin proteins utilize a conserved formin homology 2 (FH2) domain to nucleate new actin filaments. In mammalian diaphanous-related formins (DRFs) the FH2 domain is inhibited through an unknown mechanism by intramolecular binding of the diaphanous autoinhibitory domain (DAD) and the diaphanous inhibitory domain (DID).Here we report the crystal structure of a complex between DID and FH2-DAD fragments of the mammalian DRF, mDia1 (mammalian diaphanous 1 also called Drf1 or p140mDia). The structure shows a tetrameric configuration (4 FH2 + 4 DID) in which the actin-binding sites on the FH2 domain are sterically occluded. However biochemical data suggest the full-length mDia1 is a dimer in solution (2 FH2 + 2 DID). Based on the crystal structure, we have generated possible dimer models and found that architectures of all of these models are incompatible with binding to actin filament but not to actin monomer. Furthermore, we show that the minimal functional monomeric unit in the FH2 domain, termed the bridge element, can be inhibited by isolated monomeric DID. NMR data on the bridge-DID system revealed that at least one of the two actin-binding sites on the bridge element is accessible to actin monomer in the inhibited state.Our findings suggest that autoinhibition in the native DRF dimer involves steric hindrance with the actin filament. Although the structure of a full-length DRF would be required for clarification of the presented models, our work here provides the first structural insights into the mechanism of the DRF autoinhibition

Structural basis for the evolutionary inactivation of Ca2+ binding to synaptotagmin 4

C 2 domains are the second most widespread Ca 2+ -binding modules present in nature according to analyses of the human genome 1 and Ca 2+ -dependent phospholipid binding constitutes their most general activity 2 . The sequence determinants of this activity have been particularly well characterized for the two C 2 domains that form most of the cytoplasmic region of synaptotagmin 1 (referred to as the C 2 A and C 2 B domains). This synaptic vesicle protein functions in triggering fast neurotransmitter release by binding Ca 2+ through both of its C 2 domains 3,4 , and is the most conserved among the synaptotagmins, a family of membrane-trafficking proteins with at least 8 isoforms in D. melanogaster and 15 in mammals Biochemical analyses of additional synaptotagmins have suggested that they form a hierarchy of Ca 2+ sensors with different Ca 2+ affinities RESULTS Intrinsic Ca 2+ binding to synaptotagmins 4 and 11 To analyze the intrinsic Ca 2+ -binding properties of the rat and D. melanogaster synaptotagmin 4 C 2 domains, we used 1 H-15 N heteronuclear single quantum correlation (HSQC) spectra. Ca 2+ induced The neuronal protein synaptotagmin 1 functions as a Ca 2+ sensor in exocytosis via two Ca 2+ -binding C 2 domains. The very similar synaptotagmin 4, which includes all the predicted Ca 2+ -binding residues in the C 2 B domain but not in the C 2 A domain, is also thought to function as a neuronal Ca 2+ sensor. Here we show that, unexpectedly, both C 2 domains of fly synaptotagmin 4 exhibit Ca 2+ -dependent phospholipid binding, whereas neither C 2 domain of rat synaptotagmin 4 binds Ca 2+ or phospholipids efficiently. Crystallography reveals that changes in the orientations of critical Ca 2+ ligands, and perhaps their flexibility, render the rat synaptotagmin 4 C 2 B domain unable to form full Ca 2+ -binding sites. These results indicate that synaptotagmin 4 is a Ca 2+ sensor in the fly but not in the rat, that the Ca 2+ -binding properties of C 2 domains cannot be reliably predicted from sequence analyses, and that proteins clearly identified as orthologs may nevertheless have markedly different functional properties

Crystal Structure of the Munc13–1 C ₂A Domain Homodimer

<div><p>(A) A region of the 2F₀-F_c electron density contoured at the 1σ level. </p> <p>(B) Ribbon diagram of the Munc13–1 C₂A domain homodimer formed by monomers A (blue) and C (orange) showing a top view of the β-barrel like structure. The β-strands are labeled with numbers, and the N- and C-termini are indicated with N and C, respectively. </p> <p>(C) Superposition of monomers A and C.</p> <p>(D) Superposition of monomer C of the Munc13–1 C₂A domain (orange) and the C₂ domain of PLC-δ1 (green). </p> <p>(E) Surface representation of the Munc13–1 C₂A-domain homodimer. </p> <p>(F) Ribbon diagram of the Munc13–1 C₂A-domain homodimer showing a view perpendicular to that of (B) and illustrating the intermolecular strand–strand interactions that close the β-barrel. The backbone atoms from strand 3 of monomer A and strand 6 of monomer C involved in strand–strand hydrogen bonds are shown as stick models. </p> <p>(G) Close-up view of the dimerization interface. The side chains from residues involved in intermolecular contacts and the Cα carbons of the same residues are shown as stick models, with oxygen atoms in red and nitrogen atoms in blue; Cα carbons are shown with the same color as the ribbon, and other carbons are shown in gray for monomer A and yellow for monomer C. The carbonyl groups of I70, which form hydrogen bonds (dotted lines) with the S33 hydroxyl groups are also shown as stick models. For simplicity, other hydrogen bonds are not shown.</p> <p>All diagrams were generated with Pymol (DeLano Scientific, San Carlos, California).</p></div

The Munc13–1 C ₂A Domain Homodimerizes

<div><p>(A) Domain structure of Munc13–1 and RIM2α. The calmodulin-binding sequence (CaMb) of Munc13–1 and the helices that flank the RIM2α ZF domain (labeled a1 and a2) are indicated below the diagrams, and residue numbers are indicated above them.</p> <p>(B) Gel filtration analysis of Munc13–1_3–150 (black), RIM2α_82–142 (blue), and the complex between them (red). Elution volumes of two molecular standards are indicated at the top. </p> <p>(C)¹H-¹⁵N HSQC spectrum of¹⁵N-labeled Munc13–1_3–150 at 500 MHz. </p> <p>(D)¹H-¹⁵N HSQC spectrum of¹⁵N-labeled Munc13–1_3–150 bound to unlabeled RIM2α_82–142 at 500 MHz. </p> <p>(E) Equilibrium sedimentation analysis of Munc13–1_3–128. The data were obtained at centrifugation speeds of 20,000 rpm (black), 25,000 rpm (red), 30,000 rpm (green), and 35,000 rpm (blue). Curves in the bottom panel were generated by fitting the data to a monomer-dimer equilibrium model. The top panel shows the residuals. </p> <p>mAU, milliabsorbance units; ppm, parts per million.</p></div