title: N-Ethylmaleimide-sensitive fusion protein contains high and low affinity ATP-binding sites that are functionally distinct.
abstract: N-Ethylmaleimide-sensitive factor (NSF) has been shown to be involved in numerous intracellular membrane fusion events of both the regulated and constitutive secretory pathways. Sequence analysis indicates that the NSF subunit contains two nucleotide-binding sites, both with the classical Walker A and B motifs. In this report, we examine the nucleotide binding properties of NSF. The homotrimer contains three high affinity ATP-binding sites with Kd = 30-40 nM for ATP and Kd = 2 microM for ADP. This class of binding sites did not bind AMP, adenine, or GTP. A second class of lower affinity nucleotide binding sites with a Kd = 15-20 microM was also detected. Using various mutant forms of NSF, the high affinity nucleotide-binding sites were localized to the D2 domains and the low affinity sites were localized to the D1 domains. Functionally it is these lower affinity sites in D1 that are crucial for NSF activity. Nucleotide concentration greatly affected the ability of NSF to interact with alpha-SNAP.SNARE (soluble NSF attachment protein-SNAP receptor) complex, suggesting that only when the D1 domain ATP-binding sites are occupied does NSF bind to the alpha-SNAP.SNARE complex.
authors.affiliation: Department of Biochemistry, Chandler Medical Center, University of Kentucky College of Medicine, Lexington, Kentucky 40536-0084, USA.
authors.forename: E A
authors.initials: EA
authors.lastname: Matveeva
authors.forename: P
authors.initials: P
authors.lastname: He
authors.forename: S W
authors.initials: SW
authors.lastname: Whiteheart
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D052066:N-Ethylmaleimide-Sensitive Proteins; D019920:Photoaffinity Labels; D033921:Vesicular Transport Proteins
chemical_list: D002352:Carrier Proteins; D019920:Photoaffinity Labels; D033921:Vesicular Transport Proteins; D000255:Adenosine Triphosphate; D052066:N-Ethylmaleimide-Sensitive Proteins