Limits...
The role of the N-D1 linker of the N-ethylmaleimide-sensitive factor in the SNARE disassembly.

Liu CC, Sun S, Sui SF - PLoS ONE (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biomembrane and Membrane Biotechnology, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China.

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.

Show MeSH

Related in: MedlinePlus

Domain sketch of NSF protein and the amino acid conservation of the N-D1 linker.The diagram shows the structural domains of NSF: the N-terminal domain, D1 and D2 AAA+ modules. The position of the N-D1 linker and D1-D2 linker are indicated. Sequence alignment of the N-D1 linker from different eukaryotic species shows that the N-D1 linker is highly conserved. The numbers at the two ends of the sequence indicate the amino acid residues in NSF. Residues are colored by similarity. The letters under the sequence indicate the amino acid substitutions constructed in this study.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3646813&req=5

pone-0064346-g001: Domain sketch of NSF protein and the amino acid conservation of the N-D1 linker.The diagram shows the structural domains of NSF: the N-terminal domain, D1 and D2 AAA+ modules. The position of the N-D1 linker and D1-D2 linker are indicated. Sequence alignment of the N-D1 linker from different eukaryotic species shows that the N-D1 linker is highly conserved. The numbers at the two ends of the sequence indicate the amino acid residues in NSF. Residues are colored by similarity. The letters under the sequence indicate the amino acid substitutions constructed in this study.

Mentions: There are two linker regions connecting the N and D1 domains, and the D1 and D2 domains, called the N-D1 linker and the D1-D2 linker, respectively. The NSF N-D1 linker is a highly conserved 20-residue linker, consisting of amino acid residues 203–222 (Fig. 1). Two conserved glycine residues (G221 and G222), present at the C-terminal region of the NSF N-D1 linker, can potentially provide the N-D1 linker with dynamic properties similar to the features seen in the linker of p97 [21], [22]. Structurally the glycine residues are in close proximity to the nucleotide-binding site in p97-D1 domain and could be sensitive to the state of the bound nucleotide [21], [23]. Structural studies of NSF in different nucleotide states showed that the protein undergoes conformation changes during nucleotide binding and hydrolysis, most notably with NSF-N domain changing its disposition relative to the rest of the NSF hexamer [24], [25]. Presumably, communication between the N and D1 domains couples complex assembly/disassembly with the ATP hydrolytic cycle. Despite the functions of the N and D1 domains are clearly demonstrated, little is known about the roles of N-D1 linker for NSF function.


The role of the N-D1 linker of the N-ethylmaleimide-sensitive factor in the SNARE disassembly.

Liu CC, Sun S, Sui SF - PLoS ONE (2013)

Domain sketch of NSF protein and the amino acid conservation of the N-D1 linker.The diagram shows the structural domains of NSF: the N-terminal domain, D1 and D2 AAA+ modules. The position of the N-D1 linker and D1-D2 linker are indicated. Sequence alignment of the N-D1 linker from different eukaryotic species shows that the N-D1 linker is highly conserved. The numbers at the two ends of the sequence indicate the amino acid residues in NSF. Residues are colored by similarity. The letters under the sequence indicate the amino acid substitutions constructed in this study.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646813&req=5

pone-0064346-g001: Domain sketch of NSF protein and the amino acid conservation of the N-D1 linker.The diagram shows the structural domains of NSF: the N-terminal domain, D1 and D2 AAA+ modules. The position of the N-D1 linker and D1-D2 linker are indicated. Sequence alignment of the N-D1 linker from different eukaryotic species shows that the N-D1 linker is highly conserved. The numbers at the two ends of the sequence indicate the amino acid residues in NSF. Residues are colored by similarity. The letters under the sequence indicate the amino acid substitutions constructed in this study.
Mentions: There are two linker regions connecting the N and D1 domains, and the D1 and D2 domains, called the N-D1 linker and the D1-D2 linker, respectively. The NSF N-D1 linker is a highly conserved 20-residue linker, consisting of amino acid residues 203–222 (Fig. 1). Two conserved glycine residues (G221 and G222), present at the C-terminal region of the NSF N-D1 linker, can potentially provide the N-D1 linker with dynamic properties similar to the features seen in the linker of p97 [21], [22]. Structurally the glycine residues are in close proximity to the nucleotide-binding site in p97-D1 domain and could be sensitive to the state of the bound nucleotide [21], [23]. Structural studies of NSF in different nucleotide states showed that the protein undergoes conformation changes during nucleotide binding and hydrolysis, most notably with NSF-N domain changing its disposition relative to the rest of the NSF hexamer [24], [25]. Presumably, communication between the N and D1 domains couples complex assembly/disassembly with the ATP hydrolytic cycle. Despite the functions of the N and D1 domains are clearly demonstrated, little is known about the roles of N-D1 linker for NSF function.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biomembrane and Membrane Biotechnology, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China.

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.

Show MeSH
Related in: MedlinePlus