Structure/Function of the N-ethylmaleimide Sensitive Factor

Statement of the Problem: N-Ethylmaleimide Sensitive Factor (NSF) is essential to all physiological processes that involve membrane fusion and secretion (e.g. neurotransmission, reproduction, and hemostasis). Each protomer of the NSF homohexamer is divided into three contiguous domains (NSF-N, NSF-D1, and NSF-D2) and each domain contributes uniquely to NSF activity. NSF is a "protein helicase" unwinding spent SNARE (SNAP receptor) complexes after membrane fusion. SNAREs are integral membrane proteins which facilitat~ fusion by forming heteromeric complexes that span the two fusing bilayers. A major function of NSF is to insure that individual SNARE proteins are released from the stable SNARE complexes so each can be recycled. While the role for NSF has been the subject of many studies, little is known about how it uses ATP hydrolysis to disassemble SNARE complexes. Active NSF must be hexameric and must bind and hydrolyze ATP. NSF also requires adaptor proteins, called SNAPs (Soluble NSF Attachment Proteins) to bind SNARE complexes and to stimulate ATP hydrolysis. Despite our advances, a detailed picture of the structural elements of NSF that are required for SNAP binding, ATP hydrolysis and SNARE complex disassembly is still lacking. These questions will be directly addressed by the two Aims listed below. The data gained from this analysis will more precisely define the molecular mechanisms of this central facilitator of all membrane trafficking events. 1) To determine which structural features of NSF-N are required for SNAP-SNARE complex binding and SNAP-mediated stimulation of the A TPase activity of NSF. Our previous work demonstrated that the NSF-N domain is required for binding to the SNAPSNARE complex. The goal of this aim is to determine which structural elements of NSF-N are critical for SNAP-SNARE binding. We will employ structure-based, site-directed mutagenesis and focus on potential binding-site surfaces and residues that could playa role in SNAPSNARE binding and in stimulation of NSF's ATPase. All of the mutants will be tested for binding to SNARE/SNARE complexes using pull-down assays. Mutants will also be tested for both basal and SNAP/SNARE-stimulated ATPase activity. 2) To determine what structural elements of NSF-D1 promote SNAP-dependent enhancement of nucleotide hydrolysis and facilitate the conformational changes needed for SNAP-SNARE complex disassembly. The experiments in Aim 2 address four specific questions: What residues are responsible for: 1) Nucleotide Binding 2) Nucleotide Hydrolysis 3) Stimulation of Nucleotide Hydrolysis 4) Propagation of Nucleotide-Dependent Conformational Changes. This aim's specific focus will be on the Sensor 1 and 2 regions, the adenine binding pocket, and the residues that form the central pore of the NSF-D1 hexamer. Additional mutations will focus on the linker region which connects NSF-N and 01. Sitedirected mutants will be analyzed for nucleotide binding, hydrolysis, and SNAP-SNARE complex binding and disassembly. These data will specifically identify regions of NSF that convert the chemical energy of ATP hydrolysis into physical work for SNARE complex disassembly. Significance: The experiments of this proposal will determine which structural elements of the NSF hexamer are required for substrate binding and which are necessary for disassembly of SNAP-SNARE complexes. This data will not only yield significant insight into the mechanisms of this specific element of the membrane trafficking machinery but will also elucidate the mechanisms of other AAA proteins which carry out a myriad of cellular functions.s