Energetics of (Dis)Assembly of the Ternary SNARE Complex

Abstract

The soluble N-ethylmaleimidesensitive fusion protein attachment protein receptor (SNARE) complex (Sollner et al., 1993) plays a central role in the process of exocytosis whereby vesicles fuse with the plasma membrane to release their cargo of transmitter molecules into the extracellular space. In the majority of neurons, this complex is composed of the vesicular protein synaptobrevin 2 (Sb2), and two proteins located at the plasma membrane, syntaxin (Sx) and synaptosome-associated protein of 25 kDa (SNAP25). The energetics of (dis)assembly of the ternary SNARE complex is critical for understanding of exocytosis, in particular to their role in mediating vesicular fusions to and/or pinching off the plasma membrane. The energy required for disassembly of the ternary SNARE complex has been recently assessed by two different groups (Li et al., 2007; Liu et al., 2009). In both studies SNARE proteins were immobilized to surfaces. One surface contained a SxSNAP25 binary complex, while the other Sb2. These surfaces were brought into contact allowing for the formation of the ternary complex, before the surfaces were pulled apart to dismantle the complex. Using surface force apparatus (SFA), Li et al. (2007) revealed a change in free, presumably Gibbs (ΔG), energy of 21 kcal mol (35 k B T) assigned to a disassembly of single SNARE complex. Liu et al. (2009) using Atomic Force Microscopy (AFM) in force spectroscopy mode reported the enthalpic changes (ΔH) of 25.7 kcal mol (43 k B T), as well changes in free energy (ΔG) of 13.8–18.0 kcal mol (23–30 k B T) and entropy (−TΔS) for a disassembly of single ternary SNARE complex (Table 1). Both SFA and AFM approaches, however, could not be used to measure the energetics of the assembly of the complex. Wiederhold and Fasshauer (2009) investigated the ternary SNARE complex assembly by isothermal titration calorimetry (ITC). Various combinations of SNARE proteins were put in a thermally insulated cell and syringe, and then were mixed by injection from the syringe to the cell, while measuring the thermodynamic properties. To avoid formation of the Sx1-SNAP25 binary complex with 2:1 stoichiometry, referred to as a “dead-end species” (Weninger et al., 2008) since it does not represent a reactive Sb2 binding site (Pobbati et al., 2006), SNAP25A was injected into a mixture of Sx1A (H3 domain) and Sb2 [cytosolic domain; amino acids (aa) 1–96] to form the ternary SNARE complex. In these conditions there was extremely large favorable ΔH of −112.8 kcal mol recorded with the positive entropy changes (102.4 kcal mol), refl ecting the major conformation change during complex assembly, and resulting in ΔG of −10.4 kcal mol (−17.4 k B T) (Table 1). The ITC measurements above represent energetics of a non-sequential ternary SNARE complex formation, rather than the sequential interactions in which Sb2 binds to a preformed Sx1-SNAP25 binary complex with 1:1 stoichiometry. To addrsess this issue the authors cleverly designed experiments using so-called “ΔN complex”(Pobbati et al., 2006). Here, the 1:1 Sx-SNAP25 binary complex can be stabilized by addition of C-terminal fragment of Sb2 SNARE domain (aa 49–96), and then purifi ed. ΔN complex was titrated by injection of the entire cytosolic domain of Sb2 1–96, which binds to the complex and displaces Sb2 49–96 as confi rmed by fl uorescence anisotropy measurements in