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.
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