Uncoupling the ATPase activity of the N-ethylmaleimide sensitive factor (NSF) from 20S complex disassembly.

The N-ethylmaleimide sensitive factor (NSF) plays a critical role in intracellular trafficking by disassembling soluble NSF attachment protein receptor (SNARE ) complexes. The NSF protomer consists of three domains (NSF-N, NSF-D1, and NSF-D2). Two domains (NSF-D1 and NSF-D2) contain a conserved approximately 230 amino acid cassette, which includes a distinctive motif termed the second region of homology (SRH) common to all ATPases associated with various cellular activities (AAA). In hexameric NSF, several SRH residues become trans elements of the ATP binding pocket. Mutation of two conserved arginine residues in the NSF-D1 SRH (R385A and R388A) did not effect basal or soluble NSF attachment protein (SNAP)-stimulated ATPase activity; however, neither mutant underwent ATP-dependent release from SNAP-SNARE complexes. A trans element of the NSF-D2 ATP binding site (K631) has been proposed to limit the ATPase activity of NSF-D2, but a K631D mutant retained wild-type activity. A mutation of the equivalent residue in NSF-D1 (D359K) also did not affect nucleotide hydrolysis activity but did limit NSF release from SNAP-SNARE complexes. These trans elements of the NSF-D1 ATP binding site (R385, R388, and D359) are not required for nucleotide hydrolysis but are important as nucleotide-state sensors. NSF-N mediates binding to the SNAP-SNARE complex. To identify the structural features required for binding, three conserved residues (R67, S73, and Q76) on the surface of NSF-N were mutated. R67E completely eliminated binding, while S73R and Q76E showed limited effect. This suggests that the surface important for SNAP binding site lies in the cleft between the NSF-N subdomains adjacent to a conserved, positively charged surface.