Zhao Chunxia, Smith Everett C, Whiteheart Sidney W
Department of Cellular and Molecular Biochemistry, University of Kentucky Medical Center, Lexington, KY 40536-0509, USA.
Biochim Biophys Acta. 2012 Jan;1823(1):159-71. doi: 10.1016/j.bbamcr.2011.06.003. Epub 2011 Jun 13.
The N-ethylmaleimide-Sensitive Factor (NSF) was one of the initial members of the ATPases Associated with various cellular Activities Plus (AAA(+)) family. In this review, we discuss what is known about the mechanism of NSF action and how that relates to the mechanisms of other AAA(+) proteins. Like other family members, NSF binds to a protein complex (i.e., SNAP-SNARE complex) and utilizes ATP hydrolysis to affect the conformations of that complex. SNAP-SNARE complex disassembly is essential for SNARE recycling and sustained membrane trafficking. NSF is a homo-hexamer; each protomer is composed of an N-terminal domain, NSF-N, and two adjacent AAA-domains, NSF-D1 and NSF-D2. Mutagenesis analysis has established specific roles for many of the structural elements of NSF-D1, the catalytic ATPase domain, and NSF-N, the SNAP-SNARE binding domain. Hydrodynamic analysis of NSF, labeled with (Ni(2+)-NTA)(2)-Cy3, detected conformational differences in NSF, in which the ATP-bound conformation appears more compact than the ADP-bound form. This indicates that NSF undergoes significant conformational changes as it progresses through its ATP-hydrolysis cycle. Incorporating these data, we propose a sequential mechanism by which NSF uses NSF-N and NSF-D1 to disassemble SNAP-SNARE complexes. We also illustrate how analytical centrifugation might be used to study other AAA(+) proteins.
N - 乙基马来酰亚胺敏感因子（NSF）是与多种细胞活动相关的ATP酶（AAA(+)）家族的最初成员之一。在本综述中，我们讨论了关于NSF作用机制的已知信息以及它与其他AAA(+)蛋白机制的关系。与其他家族成员一样，NSF与一种蛋白质复合物（即SNAP - SNARE复合物）结合，并利用ATP水解来影响该复合物的构象。SNAP - SNARE复合物的拆解对于SNARE的循环利用和持续的膜运输至关重要。NSF是一种同型六聚体；每个亚基由一个N端结构域NSF - N和两个相邻的AAA结构域NSF - D1和NSF - D2组成。诱变分析确定了NSF - D1（催化性ATP酶结构域）和NSF - N（SNAP - SNARE结合结构域）的许多结构元件的特定作用。用（Ni(2+)-NTA）(2)-Cy3标记的NSF的流体动力学分析检测到NSF的构象差异，其中ATP结合构象比ADP结合形式显得更紧凑。这表明NSF在其ATP水解循环过程中经历了显著的构象变化。结合这些数据，我们提出了一种NSF利用NSF - N和NSF - D1拆解SNAP - SNARE复合物的顺序机制。我们还说明了分析离心法可如何用于研究其他AAA(+)蛋白。
