TRIM5α SPRY/coiled-coil interactions optimize avid retroviral capsid recognition.

Restriction factors are important components of intrinsic cellular defense mechanisms against viral pathogens. TRIM5α is a restriction factor that intercepts the incoming capsid cores of retroviruses such as HIV and provides an effective species-specific barrier to retroviral infection. The TRIM5α SPRY domain directly binds the capsid with only very weak, millimolar-level affinity, and productive capsid recognition therefore requires both TRIM5α dimerization and assembly of the dimers into a multivalent hexagonal lattice to promote avid binding. Here, we explore the important unresolved question of whether the SPRY domains are flexibly linked to the TRIM lattice or more precisely positioned to maximize avidity. Biochemical and biophysical experiments indicate that the linker segment connecting the SPRY domain to the coiled-coil domain adopts an α-helical fold, and that this helical portion mediates interactions between the two domains. Targeted mutations were generated to disrupt the putative packing interface without affecting dimerization or higher-order assembly, and we identified mutant proteins that were nevertheless deficient in capsid binding in vitro and restriction activity in cells. Our studies therefore support a model wherein substantial avidity gains during assembly-mediated capsid recognition by TRIM5α come in part from tailored spacing of tethered recognition domains.

Munc18-1 and the Syntaxin-1 N Terminus Regulate Open-Closed States in a t-SNARE Complex.

Neuronal exocytosis is mediated by SNARE proteins, which assemble into a highly stable four-helical bundle in a process that is not well understood. Here, electron paramagnetic resonance spectroscopy was used to examine how the t-SNAREs syntaxin and SNAP25 assemble in the presence and absence of the regulatory protein Munc18-1. Syntaxin and SNAP25 form a 2:1 complex, which is structurally heterogeneous and persists in the presence of excess SNAP25. Munc18-1 dissociates this 2:1 complex, but a 1:1 complex is retained where syntaxin is in a closed state. In the absence of an N-terminal fragment of syntaxin, Munc18-1 also stabilizes a 1:1 complex of sytaxin/SNAP25; however, syntaxin now samples an open state. These data demonstrate that the open-closed syntaxin equilibrium is shifted toward the open state when syntaxin and Munc18-1 are associated with SNAP25, and the results indicate that a syntaxin/SNAP25:Munc18-1 complex is a likely starting point for SNARE assembly.

The SNARE motif of synaptobrevin exhibits an aqueous-interfacial partitioning that is modulated by membrane curvature.

The structure and interfacial association of the full-length vesicle SNARE, synaptobrevin, were compared in four different lipid environments using nuclear magnetic resonance and electron paramagnetic resonance spectroscopy. In micelles, segments of the SNARE motif are helical and associated with the interface. However, the fraction of helix and interfacial association decreases as synaptobrevin is moved from micelle to bicelle to bilayer environments, indicating that the tendency toward interfacial association is sensitive to membrane curvature. In bilayers, the SNARE motif of synaptobrevin transiently associates with the lipid interface, and regions that are helical in micelles are in conformational and environmental exchange in bicelles and bilayers. This work demonstrates that the SNARE motif of synaptobrevin has a significant propensity to form a helix and exchange with the membrane interface prior to SNARE assembly. This transient interfacial association and its sensitivity to membrane curvature are likely to play a role in SNARE recognition events that regulate membrane fusion.

Allosteric control of syntaxin 1a by Munc18-1: characterization of the open and closed conformations of syntaxin.

Syntaxin 1a is a plasma membrane soluble N-ethylmaleimide-sensitive factor attachment receptor protein (SNARE) that contains an H3 domain (SNARE motif) and a regulatory Habc domain. These regions associate to produce a closed state, which is generally thought to suppress assembly of syntaxin into the SNARE complex. However, the molecular nature of the closed and open states of syntaxin is not well defined. Here, we use electron paramagnetic resonance spectroscopy to characterize conformational exchange in syntaxin. The data indicate that the H3 segment is in equilibrium between ordered and disordered states that have significant populations. In solution, the central region of the H3 segment is positioned close to the Habc domain and the configuration of syntaxin 1a is dominated by a closed state. However, an open state is enhanced in full-length membrane reconstituted syntaxin. Munc18-1 binding alters the equilibrium along H3 to favor the ordered, folded state. Munc18 also suppresses the minor open population and narrows the distance distributions between H3 and Habc. The allosteric control exhibited by Munc18 on the H3 segment and the suppression of the minor open component may both play a role in regulating membrane fusion by controlling the assembly of syntaxin into the SNARE complex.