Mechanism of Strong Membrane Binding by Synaptotagmin 7 C2A Domain: Insight from Mutation and Lipid Composition Dependence

Abstract

Synaptotagmins are membrane trafficking proteins that contain two C-terminal C2 domains, C2A and C2B. In eight of the seventeen mammalian isoforms, the C2 domains serve as calcium-dependent membrane binding motifs that trigger exocytosis upon cellular calcium influx. C2 domains from the different isoforms display different kinetics and affinities for membranes, which reflect their functions in a variety of different cell types. For example, synaptotagmin-7 (Syt7) is mainly active in slow secretion events such as insulin release. Previous work in our lab has shown that the C2A domain of human Syt7 has membrane association and dissociation rates that are ∼2 and ∼60-fold slower than the corresponding domain of Syt1, an isoform involved in rapid neurotransmitter release [Brandt DS, et al., Biochemistry; 51(39):7654-7664.] The purpose of this study is to investigate the balance of forces governing the stronger membrane binding of Syt7 C2A. We have used kinetic measurements of association and dissociation to assess membrane affinity, while systematically varying buffer composition, liposome size, lipid composition, and mutant forms of the protein domain. The results indicate that this domain's unusually strong membrane binding strength is due to a combination of electrostatics and the hydrophobic effect. In particular, dissociation rates are governed mainly by the hydrophobic effect, as seen by slower dissociation in the presence of the kosmotropic agent trehalose. Furthermore, the F167 residue in the Syt7 C2A domain acts as a secondary hydrophobic anchor for the membrane-docked protein domain, as its mutation to methionine (as in Syt1) results in a reproducible 2-fold increase in off rate.