Characterization of interactions formed by the SNARE Syntaxin4 protein and the Sec/Munc protein Munc18c

Abstract

The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a family of proteins essential for vesicle fusion. SNAREs mediate fusion by the formation of the SNARE ternary complex whereby SNARE motifs of cognate SNARE proteins interact. Central to the work described in this thesis is the SNARE protein, Syntaxin (Sx). Most Sxs can exist in two different conformations: a fusion competent open conformation and a closed conformation that prevents the interaction of Syntaxin with its SNARE partners to form the ternary complex. SNARE proteins alone do not mediate vesicle fusion; many other proteins are involved in fusion regulation. The SM family of proteins are a group of regulatory proteins that regulate fusion, at least in part, via their interaction with Sxs. Several binding modes for the SM:Sx interaction have been reported: binding to Sx in the closed conformation; binding to the N-terminus of Sx; or binding to assembled SNARE ternary complexes. SM proteins act as negative regulators when bound to Sx in the closed conformation, whereas binding to the N-terminal 10 residues (the N-terminal peptide) of Sx is thought to facilitate SNARE complex formation. In the case of M18c and Sx4, M18c has been shown to be a positive regulator of fusion by up regulating SNARE complex assembly through its interaction with Sx4. A structure for M18c in complex with full length Sx4 has not been previously reported. Small angle scattering experiments were undertaken to obtain a model for this protein complex. Neutron contrast variation experiments were undertaken to determine the conformation of Sx4 whether open or closed, in the complex. As part of this work, I established a protocol for the expression and purification of deuterated Sx4 (DSx4). Sx4 obtained by this method was shown to be 87% deuterated by mass spectrometry analysis. Scattering experiments along with cross-linking studies were used to obtain a model for M18c:Sx4 complex. The model showed M18c bound to Sx4 in an open conformation. In contrast, M18a, the neuronal homolog of M18c has been shown to bind to its cognate Sx, Sx1a, when Sx1a is in a closed conformation. The M18c:Sx4 structure obtained here provides additional evidence to support a positive regulatory role in fusion for M18c. The SM:Sx complex is thought to be highly specific between cognate SM:Sx partners. However recent work on the crystal structure of M18c:Sx4 N-terminal peptide showed that the peptide binding pocket in SM proteins and the residues that interact with it on the Sx N-terminal peptide were highly conserved. Sequence analysis showed that M18a also had the conserved N-terminal peptide binding pocket. Thus M18a and M18c along with their cognate Syntaxin N-terminal peptides, Sx1a and Sx4 were used to investigate the specificity of the interaction using ITC and crystallization experiments. ITC experiments showed that M18c and M18a bind to both cognate and non-cognate Sx N-terminal peptides with micromolar affinity. The affinity of M18c to Sx N-terminal peptide was found to be slightly higher because M18c made more contacts with the peptide. I was also able to obtain crystals grown from M18c:Sx1a1-10 N-terminal peptide and M18a:Sx1a1-10 N-terminal peptide. Some of these crystals diffracted to 4.5 A. Previous work had shown that Sx4 interacts with metal ions. Pulldown experiments were carried out to confirm the metal binding ability of Sx4 using metal chelated resins (Co2+ and Ni2+). These experiments showed that Sx4 did bind metal ions as previously described. Pulldown experiments to determine the region on Sx4 that binds metal ions showed that full length Sx4 and the SNARE motif bound metal ions. The SNARE motif is therefore likely to be the region that binds to metal chelated resin. However a Sx4 construct with 29 N-terminal residues deleted did not bind metal ions despite containing the SNARE motif. I suggest that Sx4 was in a closed conformation in this construct and that the conformation of Sx4 may play a role in the ability of Sx4 to bind metal ions. Pulldown experiments also showed that Sx1a, the neuronal counterpart of Sx4 did not bind metal ions. Further experiments will be required to confirm these observations. Overall I was able to obtain a structure for M18c:Sx4 full-length complex showing for the first time that an SM protein, M18c can interact with a Sx, (Sx4) in an open conformation. This is consistent with a positive role for M18c in fusion regulation. I was also able to show that the SM:Sx N-terminal peptide interaction does not contribute toward the specificity of SM:Sx interactions and that the SNARE motif of Sx4 interacts with metal ions. Collectively this work has added to the overall knowledge on the mechanism governing GLUT4 transport and sheds light on the interaction between SM:Sx proteins in general.