GTPases in the ARF family regulate vesicle transport events that underlie a broad range physiological processes, including secretion and endocytosis and the biogenesis of intracellular organelles. Research in this proposal focuses on structural characteriztion, by X-ray crystallography, of threee ligands for ARF1 GTPase; the positive regulator SEC7 domain, the negative regulator ARFGAP, and the effector protein Beta-COP; Additionally, the moleuclar basis for ARF conformational switching will be studiedby a comparative analysis of the GTP-analog- and GDP-bound forms of human ARF1. Consistent with the view that guanine-nucleotide exchange factors (GEFs) act by stabilizing a nucleotide-free form of the GTPase, preliminary work has demonstrated a stable complex between the Sec7 domain and nucleotide-free ARF1 core. Crystals of the complex have been obatined that diffract to at least 2.0 A resolution the structure will be determined by a combination of MR and MIR methods. Comparison of this structure with that of RasGAP, which is unrealated in amino-acid sequence, will identify common features that enable GAPs to accelerate the GTPase reaction . The analog, GppNHp. Crystals have been obtained that diffrract to very high resolution ( at least 1.4 A), and the structure solved by molecular replacement; crystallographic refinement is in progress. Comparison of the structures of GppNHp- and GDP-bound ARF will exstablish the mechanism of ARF conformational swithching leads to GTP-dependent interactions with the effector molecule. Overall these studies will provide insight into both the specific mechanism of information transfer through ARF, ARFGAP and Sec7 domain, and into the gneral structural principles that govern the regulation of Ras-related GTPases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059187-03
Application #
6386440
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Lewis, Catherine D
Project Start
1999-05-01
Project End
2004-04-30
Budget Start
2001-05-01
Budget End
2002-04-30
Support Year
3
Fiscal Year
2001
Total Cost
$233,389
Indirect Cost
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065