The long-term goal of this project is to understand the molecular mechanism of thin filament linked smooth muscle regulation in atomic detail. Malfunction in smooth muscle contraction may contribute to diseases such as asthma and hypertension. Two independent mechanisms, respectively responsible for switch and modulatory fine-tuning, seem to regulate smooth muscle contraction. The primary mechanism is switched on by phosphorylation of the myosin regulatory light chain. The other, less well-established mechanism, involves caldesmon , a protein found in association with the actin thin filaments in smooth muscle cells. Caldesmon is a flexible and elongated protein and its atomic structure is still unknown. The C-terminal domain of caldesmon, however, is more structured and is responsible for nearly all the functional interactions of the molecule including binding to actin, tropomyosin, and Ca2+-dependent regulatory proteins (such as calmodulin and S100 proteins). To understand caldesmon regulatory function there is an urgent need to know the atomic structure of this domain and its interactions with other modulatory proteins.
The specific aims of this proposal are 1 ) To determine the crystal structures of the complexes between Ca 2+- calmodulin and two caldesmon target peptides, 2) To determine the atomic structure of a complex between Ca2+-calmodulin and a proteolytic fragment from the C-terminal domain of caldesmon, 3) To determine the apo and Ca2+-bound crystal structures of human calcyclin (a S100 protein), 4) To crystallize and determine the structure of a complex between Ca2+-calcyclin and a caldesmon proteolytic fragment. The primary tool will be x-ray crystallography, but other techniques will be used as well for the preparation and characterization of the proteins. Accomplishment of these specific aims will considerably advance our understanding of thin filament linked Ca2+-modulated regulation of smooth muscle, and will unveil the differences and similarities in target recognition by calmodulin and S100 proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR046524-05
Application #
6721179
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Nuckolls, Glen H
Project Start
2000-03-01
Project End
2006-02-28
Budget Start
2004-03-01
Budget End
2006-02-28
Support Year
5
Fiscal Year
2004
Total Cost
$476,078
Indirect Cost
Name
Boston Biomedical Research Institute
Department
Type
DUNS #
058893371
City
Watertown
State
MA
Country
United States
Zip Code
02472
Soulsby, Matthew; Bennett, Anton M (2009) Physiological signaling specificity by protein tyrosine phosphatases. Physiology (Bethesda) 24:281-9
Roth, Rachel J; Le, Annie M; Zhang, Lei et al. (2009) MAPK phosphatase-1 facilitates the loss of oxidative myofibers associated with obesity in mice. J Clin Invest 119:3817-29
Ha, Chang Hoon; Bennett, Anton M; Jin, Zheng-Gen (2008) A novel role of vascular endothelial cadherin in modulating c-Src activation and downstream signaling of vascular endothelial growth factor. J Biol Chem 283:7261-70
Terrak, Mohammed; Rebowski, Grzegorz; Lu, Renne C et al. (2005) Structure of the light chain-binding domain of myosin V. Proc Natl Acad Sci U S A 102:12718-23
Terrak, Mohammed; Wu, Guanming; Stafford, Walter F et al. (2003) Two distinct myosin light chain structures are induced by specific variations within the bound IQ motifs-functional implications. EMBO J 22:362-71
Otterbein, Ludovic R; Kordowska, Jolanta; Witte-Hoffmann, Carlos et al. (2002) Crystal structures of S100A6 in the Ca(2+)-free and Ca(2+)-bound states: the calcium sensor mechanism of S100 proteins revealed at atomic resolution. Structure 10:557-67
Terrak, Mohammed; Otterbein, Ludovic R; Wu, Guanming et al. (2002) Crystallization, X-ray characterization and selenomethionine phasing of Mlc1p bound to IQ motifs from myosin V. Acta Crystallogr D Biol Crystallogr 58:1882-5
Otterbein, Ludovic R; Cosio, Christophe; Graceffa, Philip et al. (2002) Crystal structures of the vitamin D-binding protein and its complex with actin: structural basis of the actin-scavenger system. Proc Natl Acad Sci U S A 99:8003-8
Brown, J H; Kim, K H; Jun, G et al. (2001) Deciphering the design of the tropomyosin molecule. Proc Natl Acad Sci U S A 98:8496-501
Otterbein, L R; Graceffa, P; Dominguez, R (2001) The crystal structure of uncomplexed actin in the ADP state. Science 293:708-11