? The homohexameric N-ethylmaleimide Sensitive Factor (NSF) is a key element in most physiologically important vesicular trafficking and exocytosis events (i.e. neurotransmission, hemostasis, immune responses). As an AAA ATPase, NSF acts as a """"""""protein helicase"""""""" unwinding spent SNARE (SNAP receptor) complexes after they have mediated membrane fusion. Dysfunction of NSF leads to stark phenotypes such as paralysis or growth arrest. NSF protomers have three contiguous domains (NSF-N, NSF-D1, and NSF-D2); each contributes uniquely to activity. Despite our advances, little is known about which structural elements are required for NSF to bind its adaptor protein, alpha SNAP (Soluble NSF Attachment Proteins), and to use the chemical energy from ATP hydrolysis to disassemble SNARE complexes. This proposal's Specific Aims focus on these key unanswered questions.
The Aims are: 1) To determine which structural features of NSF-N are required for SNAP-SNARE complex binding and SNAP-mediated stimulation of the ATPase activity of NSF. 2) To determine what structural elements of NSF-D1 promote SNAP-dependent enhancement of nucleotide hydrolysis and facilitate the conformational changes needed for SNAP-SNARE binding and disassembly. 3) To determine the conformational changes that occur in NSF as it progresses through the different nucleotide states of its ATP hydrolysis cycle. For the first two aims, structure-based mutagenesis will be combined with a battery of functional assays to assess the importance of specific regions of NSF. For the third aim, cryo-electron microscopy and single particle image analysis will be used to generate 3D maps of the NSF hexamer in the different nucleotide-induced conformations (ADP, ATP, and ADP-Pi). These structures will then be compared to determine what parts of the NSF hexamer change and how they might shift conformations during the ATP hydrolysis cycle. From the knowledge generated by the experiments in these three specific aims, it will be possible to better understand the catalytic mechanism of NSF as well as that of a number of other cellular ATPases of the AAA family. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS046242-04
Application #
7256296
Study Section
Synapses, Cytoskeleton and Trafficking Study Section (SYN)
Program Officer
Talley, Edmund M
Project Start
2004-09-20
Project End
2009-06-30
Budget Start
2007-07-01
Budget End
2009-06-30
Support Year
4
Fiscal Year
2007
Total Cost
$269,839
Indirect Cost
Name
University of Kentucky
Department
Biochemistry
Type
Schools of Medicine
DUNS #
939017877
City
Lexington
State
KY
Country
United States
Zip Code
40506
Moeller, Arne; Zhao, Chunxia; Fried, Michael G et al. (2012) Nucleotide-dependent conformational changes in the N-Ethylmaleimide Sensitive Factor (NSF) and their potential role in SNARE complex disassembly. J Struct Biol 177:335-43
Zhao, Chunxia; Smith, Everett C; Whiteheart, Sidney W (2012) Requirements for the catalytic cycle of the N-ethylmaleimide-Sensitive Factor (NSF). Biochim Biophys Acta 1823:159-71
Hellman, Lance M; Zhao, Chunxia; Melikishvili, Manana et al. (2011) Histidine-tag-directed chromophores for tracer analyses in the analytical ultracentrifuge. Methods 54:31-8
Zhao, Chunxia; Hellman, Lance M; Zhan, Xin et al. (2010) Hexahistidine-tag-specific optical probes for analyses of proteins and their interactions. Anal Biochem 399:237-45
Zhao, Chunxia; Slevin, John T; Whiteheart, Sidney W (2007) Cellular functions of NSF: not just SNAPs and SNAREs. FEBS Lett 581:2140-9
Matveeva, Elena A; Vanaman, Thomas C; Whiteheart, Sidney W et al. (2007) Asymmetric accumulation of hippocampal 7S SNARE complexes occurs regardless of kindling paradigm. Epilepsy Res 73:266-74