N-ethylmaleimide-sensitive fusion protein contains high and low affinity ATP-binding sites that are functionally distinct
N-ethylmaleimide-sensitive fusion protein contains high and low affinity ATP-binding sites that are functionally distinct
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DOI：
10.1074/jbc.272.42.26413
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发表时间：
1997-10-17
影响因子：
4.8
通讯作者：
Whiteheart, SW
中科院分区：
文献类型：
--
作者：
Matveeva, EA;He, P;Whiteheart, SW
n -乙基马来酰亚胺敏感因子（NSF）已被证明参与了许多细胞膜内融合事件的调节和组成分泌途径。序列分析表明，NSF亚基包含两个核苷酸结合位点，都具有经典的Walker A和B基序。在本报告中，我们研究了NSF的核苷酸结合特性。该三聚体含有3个高亲和力ATP结合位点，ATP的K-d = 30-40 nM， ADP的K-d = 2 μ M。这类结合位点不结合AMP、腺嘌呤或GTP。第二类低亲和力核苷酸结合位点，K-d = 15-20 μ M。在不同的NSF突变体中，高亲和力的核苷酸结合位点定位在D2结构域，低亲和力的核苷酸结合位点定位在D1结构域。从功能上讲，正是D1上的这些低亲和力位点对NSF的活性至关重要。核苷酸浓度极大地影响了NSF与α - snap SNARE（可溶性NSF附着蛋白- snap受体）复合物相互作用的能力，这表明只有当D1结构域atp结合位点被占据时，NSF才能与α - snap SNARE复合物结合。
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 K-d = 30-40 nM for ATP and K-d = 2 mu M 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 K-d = 15-20 mu M 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.