Vesicle-fusing ATPase

 

N-ethylmaleimide sensitive factor is responsible for the destabilisation and disassembly of the proteins in the SNARE complex thus has a key role in vesicular trafficking inside the cell. The structure described in this pdb code is of the ATP binding domain which contains the active site necessary for ATP hydrolysis: conformational changes that occur as a result of the hydrolysis lead to the breaking up of the SNARE complex. The ATP binding domain of the protein shares homology with other Rossmann fold containing proteins, particularly with E. coli DNA polymerase III.

 

Reference Protein and Structure

Sequence
P18708 UniProt (3.6.4.6) IPR027417 (Sequence Homologues) (PDB Homologues)
Biological species
Cricetulus griseus (Chinese hamster) Uniprot
PDB
1nsf - D2 HEXAMERIZATION DOMAIN OF N-ETHYLMALEIMIDE SENSITIVE FACTOR (NSF) (1.9 Å) PDBe PDBsum 1nsf
Catalytic CATH Domains
1.10.8.60 CATHdb 3.40.50.300 CATHdb (see all for 1nsf)
Cofactors
Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:3.6.4.6)

ATP(4-)
CHEBI:30616ChEBI
+
water
CHEBI:15377ChEBI
→
hydron
CHEBI:15378ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
+
ADP(3-)
CHEBI:456216ChEBI

Enzyme Mechanism

Introduction

Hydrolysis of ATP is facilitated by a Mg2+ ion and two Lysine residues (Lys 549 and Lys 708) and Thr 550 which contact the beta and gamma phosphates of the ATP and Asp 504 and Mg2+ aid in the deprotonation of water so that it can nucleophilically attack the gamma phosphate of ATP. This enables the conformational changes that cause the breaking up of the hexameric protein and the collapse of the SNARE complex. However, in order to reduce the rate that this occurs to allow the protein to interact with the SNARE complex before breaking up, Lys 631 acts to reduce the rate of hydrolysis so that ATP stays bound for as long as possible.

Catalytic Residues Roles

UniProt PDB* (1nsf)
Thr550 Thr550(77)A Coordinates to Mg2+ metal ligand
Thr550 (main-N) Thr550(77)A (main-N) Forms electrostatic contacts to the beta phosphate of ATP, thus stabilising the pentavalent phosphate intermediate thus facilitating hydrolysis of ATP. electrostatic stabiliser
Asp603 Asp603(130)A Forms an electrostatic interaction with a water molecule that is coordinated to Mg2+ electrostatic stabiliser
Asp604 Asp604(131)A Activates water by deprotonating it so it can nucleophilically attack the gamma phosphate of ATP proton acceptor
Lys549 Lys549(76)A Forms electrostatic contacts to the gamma and beta phosphate of ATP, thus stabilising the pentavalent phosphate intermediate thus facilitating hydrolysis of ATP. electrostatic stabiliser
Lys708 Lys708(235)A Forms electrostatic contact with the gamma phosphate of ATP thus stabilises the pentavalent phosphate that forms when hydrolysis occurs. electrostatic stabiliser
Lys631 Lys631(158)A(AD) Acts to reduce the rate at which incoming water molecules are deprotonated, thus reducing the rate of nucleophilic attack on the ATP. This has the overall effect of speeding up the dissociation of the SNARE complex because it allows the hexamer to remain intact whilst binding to the SNARE complex. electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, bimolecular nucleophilic substitution, overall reactant used, overall product formed, native state of enzyme is not regenerated

References

  1. Yu RC et al. (1998), Nat Struct Biol, 5, 803-811. Structure of the ATP-dependent oligomerization domain of N-ethylmaleimide sensitive factor complexed with ATP. DOI:10.1038/1843. PMID:9731775.
  2. Zhao C et al. (2012), Biochim Biophys Acta, 1823, 159-171. Requirements for the catalytic cycle of the N-ethylmaleimide-Sensitive Factor (NSF). DOI:10.1016/j.bbamcr.2011.06.003. PMID:21689688.

Step 1. Asp 604 deprotonates the polarised water molecule which activates it to nucleophilically attack the gamma phosphate which results in the cleavage of the phosphoanhydride bond and release of the gamma phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys708(235)A electrostatic stabiliser
Lys631(158)A(AD) electrostatic stabiliser
Lys549(76)A electrostatic stabiliser
Lys549(76)A (main-N) electrostatic stabiliser
Thr550(77)A (main-N) electrostatic stabiliser
Asp603(130)A electrostatic stabiliser
Thr550(77)A metal ligand
Asp604(131)A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, overall reactant used, overall product formed, native state of enzyme is not regenerated
Download: Image, Marvin File

Step 1. Asp 604 deprotonates the polarised water molecule which activates it to nucleophilically attack the gamma phosphate which results in the cleavage of the phosphoanhydride bond and release of the gamma phosphate.

Products.

Contributors

Peter Sarkies, Gemma L. Holliday, Charity Hornby