Exocytosis requires asymmetry in the central layer of the SNARE complex

Assembly of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment pr otein receptors) mediates membrane fusions in all eukaryotic cells. The syn aptic SNARE complex is represented by a twisted bundle of four a-helices, L eucine zipper-like layers extend through the length of the complex except f or an asymmetric and ionic middle layer formed by three glutamines (Q) and one arginine (R), We have examined the functional consequences of Q-R excha nges in the conserved middle layer using the exocytotic SNAREs of yeast as a model. Exchanging Q for R in Sso2p drastically reduces cell growth and pr otein secretion. When a 3Q/1R ratio is restored by a mirror R-->Q substitut ion in the R-SNARE Snc2p, wild-type functionality is observed. Secretion is near normal when all four helices contain Q, but defects become apparent w hen additional mutations are present in other layers. Using molecular dynam ics free energy perturbation simulations, these findings are rationalized i n structural and energetic terms. We conclude that the asymmetric arrangeme nt of the polar amino acids in the central layer is essential for normal fu nction of SNAREs in membrane fusion.