Annexins share the property of Ca2+-dependent phospholipid binding and are involved in a number of membrane related events including membrane aggregation and fusion as well as anti-inflammatory activity. Although numerous high-resolution crystal structures of annexins have been published, none contained an N-terminal domain that is thought to be primarily responsible for the diversity of function among different annexins. Annexins I and II are members of the protein family that exhibit membrane aggregation properties, which are attributed to their N-terminal domains. These harbor phosphorylation sites as well as binding sites for members of the S100 protein family. A peptide of the N-terminal domain of annexin I activates the formyl peptide receptor (FPR) on human neutrophils. Annexin II has been found on the surface of endothelial cells as a component of the fibrinolysis machinery where it is thought to colocalize tissue plasminogen activator (t-PA) and plasminogen. Based on existing biochemical evidence in combination with our recently solved crystal structure of full-length annexin I in the absence of Ca 2+ we have proposed a novel model for membrane aggregation by annexins. Our model postulates initial Ca2+-dependent binding of annexin to one membrane surface that in turn expels the amphipathic N-terminal domain, which is previously buried inside the core domain, thereby creating a second membrane-binding site that is able to bind to a second membrane. In order to obtain detailed structural information about the secondary membrane binding region of annexin I and to test this model of annexin-mediated membrane aggregation we will (1) Crystallize full-length annexin I in the presence of Ca2+and determine the crystal structure. (2) Produce a fusion protein with the N-terminal domain of annexin I fused to the N-terminus of Red Fluorescent protein and compare its membrane-binding and its effect on the phase behavior of phospholipid monolayers with those of wild-type annexin I and Red Fluorescent protein. 3) Employ tryptophan fluorescence spectroscopy including acrylamide quenching and steady-state anisotropy measurements to analyze conformational changes as well as membrane interaction of the N-terminal domain. The second major goal is the elucidation of the role of the N-terminal domain of annexin II in t-PA binding. In order to determine the requirements for complex formation of annexin H with t-PA we will (4) Crystallize full-length annexin II in the presence and absence of Ca to elucidate the x-ray structure of its N-terminal domain. (5) Co-crystallize annexin with t-PA and solve the structure of this complex after characterizing complex formation in solution.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM056445-05A2
Application #
6572882
Study Section
Special Emphasis Panel (ZRG1-SSS-B (02))
Program Officer
Chin, Jean
Project Start
1997-01-01
Project End
2007-05-31
Budget Start
2003-06-01
Budget End
2004-05-31
Support Year
5
Fiscal Year
2003
Total Cost
$300,554
Indirect Cost
Name
University of California Irvine
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
Pathuri, Puja; Vogeley, Lutz; Luecke, Hartmut (2008) Crystal structure of metastasis-associated protein S100A4 in the active calcium-bound form. J Mol Biol 383:62-77
Luecke, Hartmut; Schobert, Brigitte; Stagno, Jason et al. (2008) Crystallographic structure of xanthorhodopsin, the light-driven proton pump with a dual chromophore. Proc Natl Acad Sci U S A 105:16561-5
Pathuri, Puja; Nguyen, Emily Tam; Svard, Staffan G et al. (2007) Apo and calcium-bound crystal structures of Alpha-11 giardin, an unusual annexin from Giardia lamblia. J Mol Biol 368:493-508
Stagno, Jason; Aphasizheva, Inna; Aphasizhev, Ruslan et al. (2007) Dual role of the RNA substrate in selectivity and catalysis by terminal uridylyl transferases. Proc Natl Acad Sci U S A 104:14634-9
Stagno, Jason; Aphasizheva, Inna; Rosengarth, Anja et al. (2007) UTP-bound and Apo structures of a minimal RNA uridylyltransferase. J Mol Biol 366:882-99
Trakhanov, Sergei; Vyas, Nand K; Luecke, Hartmut et al. (2005) Ligand-free and -bound structures of the binding protein (LivJ) of the Escherichia coli ABC leucine/isoleucine/valine transport system: trajectory and dynamics of the interdomain rotation and ligand specificity. Biochemistry 44:6597-608
Freites, J Alfredo; Ali, Shahla; Rosengarth, Anja et al. (2004) Annexin A1 interaction with a zwitterionic phospholipid monolayer: a fluorescence microscopy study. Langmuir 20:11674-83
Rosengarth, Anja; Luecke, Hartmut (2003) A calcium-driven conformational switch of the N-terminal and core domains of annexin A1. J Mol Biol 326:1317-25
Prosise, Glen L; Luecke, Hartmut (2003) Crystal structures of Tritrichomonasfoetus inosine monophosphate dehydrogenase in complex with substrate, cofactor and analogs: a structural basis for the random-in ordered-out kinetic mechanism. J Mol Biol 326:517-27
Tran, Jacqueline T; Rosengarth, Anja; Luecke, Hartmut (2002) Cloning, purification and crystallization of full-length human annexin 2. Acta Crystallogr D Biol Crystallogr 58:1854-7

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