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Apo and Calcium-Bound Crystal Structures of Cytoskeletal Protein Alpha-14 Giardin (Annexin E1) from the Intestinal Protozoan Parasite Giardia lamblia |
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| Authors: | Puja Pathuri Gabriel Ozorowski Hartmut Luecke |
| Affiliation: | 1 Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA 2 Department of Cell and Molecular Biology, Uppsala University, SE-751 24 Uppsala, Sweden 3 Microbiology and Tumor Biology Center, Karolinska Institute, SE-171 77 Stockholm, Sweden 4 Department of Physiology and Biophysics, University of California, Irvine, CA 92697, USA 5 Department of Information and Computer Sciences, University of California, Irvine, CA 92697, USA 6 Center for Biomembrane Systems, University of California, Irvine, CA 92697, USA |
| Abstract: | Alpha-14 giardin (annexin E1), a member of the alpha giardin family of annexins, has been shown to localize to the flagella of the intestinal protozoan parasite Giardia lamblia. Alpha giardins show a common ancestry with the annexins, a family of proteins most of which bind to phospholipids and cellular membranes in a Ca2+-dependent manner and are implicated in numerous membrane-related processes including cytoskeletal rearrangements and membrane organization. It has been proposed that alpha-14 giardin may play a significant role during the cytoskeletal rearrangement during differentiation of Giardia. To gain a better understanding of alpha-14 giardin's mode of action and its biological role, we have determined the three-dimensional structure of alpha-14 giardin and its phospholipid-binding properties. Here, we report the apo crystal structure of alpha-14 giardin determined in two different crystal forms as well as the Ca2+-bound crystal structure of alpha-14 giardin, refined to 1.9, 1.6 and 1.65 Å, respectively. Although the overall fold of alpha-14 giardin is similar to that of alpha-11 giardin, multiwavelength anomalous dispersion phasing was required to solve the alpha-14 giardin structure, indicating significant structural differences between these two members of the alpha giardin family. Unlike most annexin structures, which typically possess N-terminal domains, alpha-14 giardin is composed of only a core domain, followed by a C-terminal extension that may serve as a ligand for binding to cytoskeletal protein partners in Giardia. In the Ca2+-bound structure we detected five bound calcium ions, one of which is a novel, highly coordinated calcium-binding site not previously observed in annexin structures. This novel high-affinity calcium-binding site is composed of seven protein donor groups, a feature rarely observed in crystal structures. In addition, phospholipid-binding assays suggest that alpha-14 giardin exhibits calcium-dependent binding to phospholipids that coordinate cytoskeletal disassembly/assembly during differentiation of the parasite. |
| Keywords: | PEG, polyethylene glycol EDTA, ethylenediaminetetraacetic acid PI, phosphatidylinositol PtdIns(4)P, phosphatidylinositol 4-phosphate PtdIns(3,4,5)P3, phosphatidylinositol (3,4,5) trisphosphate PS, phosphatidylserine PA, phosphatidic acid PE, phosphatidylethanolamine DAG, diacylglycerol PC, phosphatidylcholine PG, phosphatidylglycerol GST, glutathione S-transferase EGTA, ethylene glycol bis(β-aminoethyl ether) N,N&prime -tetraacetic acid FOM, figure of merit BSA, bovine serum albumin MAD, multiwavelength anomalous dispersion |
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