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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 Ca(2+)-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 Ca(2+)-bound crystal structure of alpha-14 giardin, refined to 1.9, 1.6 and 1.65 A, 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 Ca(2+)-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.

Alpha-11 giardin is a member of the multi-gene alpha-giardin family in the intestinal protozoan, Giardia lamblia. This gene family shares an ancestry with the annexin super family, whose common characteristic is calcium-dependent binding to membranes that contain acidic phospholipids. Several alpha giardins are highly expressed during parasite-induced diarrhea in humans. Despite being a member of a large family of proteins, little is known about the function and cellular localization of alpha-11 giardin, although giardins are often associated with the cytoskeleton. It has been shown that Giardia exhibits high levels of alpha-11 giardin mRNA transcript throughout its life cycle; however, constitutive over-expression of this protein is lethal to the parasite. Determining the three-dimensional structure of an alpha-giardin is essential to identifying functional domains shared in the alpha-giardin family. Here we report the crystal structures of the apo and Ca(2+)-bound forms of alpha-11 giardin, the first alpha giardin to be characterized structurally. Crystals of apo and Ca(2+)-bound alpha-11 giardin diffracted to 1.1 A and 2.93 A, respectively. The crystal structure of selenium-substituted apo alpha-11 giardin reveals a planar array of four tandem repeats of predominantly alpha-helical domains, reminiscent of previously determined annexin structures, making this the highest-resolution structure of an annexin to date. The apo alpha-11 giardin structure also reveals a hydrophobic core formed between repeats I/IV and II/III, a region typically hydrophilic in other annexins. Surprisingly, the Ca(2+)-bound structure contains only a single calcium ion, located in the DE loop of repeat I and coordinated differently from the two types of calcium sites observed in previous annexin structures. The apo and Ca(2+)-bound alpha-11 giardin structures assume overall similar conformations; however, Ca(2+)-bound alpha-11 giardin crystallized in a lower-symmetry space group with four molecules in the asymmetric unit. Vesicle-binding studies suggest that alpha-11 giardin, unlike most other annexins, does not bind to vesicles composed of acidic phospholipids in a calcium-dependent manner.

A protein with a relative molecular mass of 31 kDa was specifically extracted by EGTA from a detergent-insoluble fraction of Giardia lamblia. N-terminal sequencing showed this protein to be identical to alpha 1-giardin, a component of the ventral disc which, based on its predicted amino acid sequence, has been classified as annexin XIX. Purified alpha 1-giardin associated with multilamellar phosphatidyl serine-containing vesicles in a Ca(2+)-dependent manner, confirming that it is a functional annexin. Molecular modelling of the amino acid sequence of the giardial annexin into the X-ray structure of annexin V suggests that the Ca(2+)-binding sites, which, as in other annexins, are all located on the convex surface of the molecule, are of the low-affinity type III.

The homologous repeats of annexin tetrads are believed to have originated by successive duplication and fusion from a putative monomeric precursor, but neither the nature of their ancestor nor the events leading to the formation of different subfamilies have been elucidated. We have performed molecular phylogenetic analysis of aligned annexin nucleotide and amino acids sequences to characterize subfamily branching, to delineate the temporal order of appearance of individual repeat units, and to gain insight into the origin and nature of the primordial unit. All extant annexins appear to have a common tetrad precursor that may have originated from a progenitor unit resembling repeat 3, followed by the generation of repeats 4, 1, and 2 from a more evolved progenitor with subsequent fusion. Repeat sequences of the earliest human annexins VII and XIII were used to identify alpha-giardin genes as primitive homologues from the unicellular protozoan Giardia lamblia, which diverged from eukaryote lineage 1-1.5 billion yr ago. The significant homology between alpha-giardins and annexins suggested that the cell membrane adhesive role of these proteins may be a common, fundamental property of the annexin C-terminal core region. Purported annexin VII of Dictyostelium discoideum was reclassified as new annexin XIV, three Caenorhabditis elegans genes were assigned to new subfamilies XV, XVI, and XVII, and plant annexin XVIII from Medicago sativa was among the earliest diverging subfamilies. Annexins I and II were found to be closely related, but analysis of protein mutation rates confirmed that the former is evolving up to three times more rapidly. The inclusion of early phyla in annexin taxonomy provides a useful basis for assessing the structural and functional changes associated with annexin evolution.