The Longin SNARE VAMP7/TI-VAMP Adopts a Closed Conformation

Sandro Vivona,Corey W Liu,Pavel Strop,Valeria Rossi,Francesco Filippini,Axel T Brunger

doi:10.1074/jbc.m110.120972

Abstract

SNARE protein complexes are key mediators of exocytosis by juxtaposing opposing membranes, leading to membrane fusion. SNAREs generally consist of one or two core domains that can form a four-helix bundle with other SNARE core domains. Some SNAREs, such as syntaxin target-SNAREs and longin vesicular-SNAREs, have independent, folded N-terminal domains that can interact with their respective SNARE core domains and thereby affect the kinetics of SNARE complex formation. This autoinhibition mechanism is believed to regulate the role of the longin VAMP7/TI-VAMP in neuronal morphogenesis. Here we use nuclear magnetic resonance spectroscopy to study the longin-SNARE core domain interaction for VAMP7. Using complete backbone resonance assignments, chemical shift perturbations analysis, and hydrogen/deuterium exchange experiments, we conclusively show that VAMP7 adopts a preferentially closed conformation in solution. Taken together, the closed conformation of longins is conserved, in contrast to the syntaxin family of SNAREs for which mixtures of open and closed states have been observed. This may indicate different regulatory mechanisms for SNARE complexes containing syntaxins and longins, respectively.

Highlights

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)3 complex is the centerpiece of all intracellular membrane fusion reactions
The longin v-SNAREs Ykt6, VAMP7, and Sec22b are characterized by an autoinhibitory intramolecular interaction between the longin domain (LD) and the SNARE core domain
We conclusively show that VAMP7 adopts a stable, closed conformation in solution

Summary

Introduction

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)3 complex is the centerpiece of all intracellular membrane fusion reactions. Neuronal Syntaxin 1A and yeast Sso1 have been observed in a closed conformation in vitro (i.e. with the N-terminal domain interacting with the SNARE core domain) [10, 11], other syntaxins, such as the endosomal Tlg2p and Pep12p [12] or the yeast, vacuolar Vam3p, are predominantly open [13]. In contrast to Ykt6, Sec22b, and VAMP7, a predominantly open conformation was found for the yeast-specific longin Nyv1p as no significant LD-SNARE core domain interaction was observed in NMR studies [34].

Results

Conclusion