Sequence-specific assignment of methyl groups from the neuronal SNARE complex using lanthanide-induced pseudocontact shifts.

Yun-Zu Pan,Josep Rizo,Kyle D Brewer,Bradley Quade,Monika Szabo,James D Swarbrick,Bim Graham

doi:10.1007/s10858-016-0078-1

Abstract

Neurotransmitter release depends critically on the neuronal SNARE complex formed by syntaxin-1, SNAP-25 and synaptobrevin, as well as on other proteins such as Munc18-1, Munc13-1 and synaptotagmin-1. Although three-dimensional structures are available for these components, it is still unclear how they are assembled between the synaptic vesicle and plasma membranes to trigger fast, Ca2+-dependent membrane fusion. Methyl TROSY NMR experiments provide a powerful tool to study complexes between these proteins, but assignment of the methyl groups of the SNARE complex is hindered by its limited solubility. Here we report the assignment of the isoleucine, leucine, methionine and valine methyl groups of the four SNARE motifs of syntaxin-1, SNAP-25 and synaptobrevin within the SNARE complex based solely on measurements of lanthanide-induced pseudocontact shifts. Our results illustrate the power of this approach to assign protein resonances without the need of triple resonance experiments and provide an invaluable tool for future structural studies of how the SNARE complex binds to other components of the release machinery.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-016-0078-1) contains supplementary material, which is available to authorized users.

Highlights

The development of methyl-TROSY techniques using highly deuterated 13CH3-labeled (2H,13CH3-labeled) proteins (Tugarinov et al 2004) has greatly facilitated the application of NMR spectroscopy to analyze the structure and dynamics of macromolecular protein complexes, helping to elucidate how they mediate biological processes (Rosenzweig and Kay 2014)
The four-helix bundle of the neuronal SNARE complex is formed by one SNARE motif from each synaptobrevin (Syb) and syntaxin-1 (Syx), and two SNARE motifs from SNAP-25
The abundance of aliphatic residues and paucity of aromatic side chains leads to poor spectral dispersion, the resonances are broader than expected for a 32 kDa species because of the elongated shape of the complex, and resonances from nuclei near a polar layer formed by three buried glutamines (Q226 from syntaxin-1, Q53 from SNN and Q174 from SNC) and one buried arginine from synaptobrevin (R56) (Sutton et al 1998) exhibit additional broadening due to chemical exchange (Chen et al 2002)

Summary

Introduction

The development of methyl-TROSY techniques using highly deuterated 13CH3-labeled (2H,13CH3-labeled) proteins (Tugarinov et al 2004) has greatly facilitated the application of NMR spectroscopy to analyze the structure and dynamics of macromolecular protein complexes, helping to elucidate how they mediate biological processes (Rosenzweig and Kay 2014) These studies are normally more informative when sequence-speciic resonance assignments of methyl groups are available. Since 1H-13C HMQC spectra of 2H,13CH3labeled-proteins ofers high sensitivity even for species in the 1 MDa range (Sprangers and Kay 2007), methyl resonance assignments can be obtained for large systems using these spectra in combination with systematic mutagenesis (Amero et al 2011).

Methods

Results

Conclusion