title: The role of the N-D1 linker of the N-ethylmaleimide-sensitive factor in the SNARE disassembly.
abstract: N-ethylmaleimide-sensitive factor (NSF) is a member of the type II AAA+ (ATPase associated with various cellular activities) family. It plays a critical role in intracellular membrane trafficking by disassembling soluble NSF attachment protein receptor (SNARE) complexes. Each NSF protomer consists of an N-terminal domain (N domain) followed by two AAA ATPase domains (D1 and D2) in tandem. The N domain is required for SNARE/α-SNAP binding and the D1 domain accounts for the majority of ATP hydrolysis. Little is known about the role of the N-D1 linker in the NSF function. This study presents detailed mutagenesis analyses of NSF N-D1 linker, dissecting its role in the SNARE disassembly, the SNARE/α-SNAP complex binding, the basal ATPase activity and the SNARE/α-SNAP stimulated ATPase activity. Our results show that the N-terminal region of the N-D1 linker associated mutants cause severe defect in SNARE complex disassembly, but little effects on the SNARE/α-SNAP complex binding, the basal and the SNARE/α-SNAP stimulated ATPase activity, suggesting this region may be involved in the motion transmission from D1 to N domain. Mutating the residues in middle and C-terminal region of the N-D1 linker increases the basal ATPase activity, indicating it may play a role in autoinhibiting NSF activity until it encounters SNARE/α-SNAP complex substrate. Moreover, mutations at the C-terminal sequence GIGG exhibit completely abolished or severely reduced activities of the substrate binding, suggesting that the flexibility of N-D1 linker is critical for the movement of the N domain that is required for the substrate binding. Taken together, these data suggest that the whole N-D1 linker is critical for the biological function of NSF to disassemble SNARE complex substrate with different regions responsible for different roles.
authors.affiliation: State Key Laboratory of Biomembrane and Membrane Biotechnology, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China.
authors.forename: Cui-Cui
authors.initials: CC
authors.lastname: Liu
authors.forename: Shan
authors.initials: S
authors.lastname: Sun
authors.forename: Sen-Fang
authors.initials: SF
authors.lastname: Sui
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D017124:Conserved Sequence / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D052066:N-Ethylmaleimide-Sensitive Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D050600:SNARE Proteins / Q000378:metabolism; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D050505:Mutant Proteins; D050600:SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D000251:Adenosine Triphosphatases; D052066:N-Ethylmaleimide-Sensitive Proteins