This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and

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1 This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier s archiving and manuscript policies are encouraged to visit:

2 doi: /j.jmb J. Mol. Biol. (2009) 385, Available online at Apo and Calcium-Bound Crystal Structures of Cytoskeletal Protein Alpha-14 Giardin (Annexin E1) from the Intestinal Protozoan Parasite Giardia lamblia Puja Pathuri 1, Emily Tam Nguyen 1, Gabriel Ozorowski 1, Staffan G. Svärd 2,3 and Hartmut Luecke 1,4,5,6 1 Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA 2 Department of Cell and Molecular Biology, Uppsala University, SE Uppsala, Sweden 3 Microbiology and Tumor Biology Center, Karolinska Institute, SE 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 Received 13 August 2008; received in revised form 10 November 2008; accepted 11 November 2008 Available online 20 November 2008 Edited by R. Huber 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 phospholipidbinding 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 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 Ca 2+ -bound structure we detected five bound calcium ions, one of which is a novel, highly coordinated calciumbinding site not previously observed in annexin structures. This novel highaffinity calcium-binding site is composed of seven protein donor groups, a feature rarely observed in crystal structures. In addition, phospholipidbinding assays suggest that alpha-14 giardin exhibits calcium-dependent binding to phospholipids that coordinate cytoskeletal disassembly/ assembly during differentiation of the parasite Elsevier Ltd. All rights reserved. Keywords: Giardia; annexin; calcium binding; flagella; cytoskeleton *Corresponding author. Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA. address: hudel@uci.edu. Abbreviations used: PEG, polyethylene glycol; EDTA, ethylenediaminetetraacetic acid; PI, phosphatidylinositol; PtdIns (4)P, phosphatidylinositol 4-phosphate; PtdIns(3,4,5)P 3, 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 -tetraacetic acid; FOM, figure of merit; BSA, bovine serum albumin; MAD, multiwavelength anomalous dispersion /$ - see front matter 2008 Elsevier Ltd. All rights reserved.

3 Crystal Structures of Apo and Calcium-Bound Alpha-14 Giardin 1099 Introduction Giardia lamblia (syn. Giardia intestinalis, Giardia duodenalis) is a flagellated intestinal protozoan that triggers a form of diarrhea called giardiasis in humans and other mammals worldwide. 1,2 Its life cycle alternates between a dormant, waterresistant cyst that is able to survive the acidity of the stomach and an actively swimming trophozoite that attaches, colonizes and divides in the upper small intestine of the host. 3 Infection typically starts when cysts are ingested with contaminated water, food or through direct fecal oral transfer. 4 After the cysts pass through the stomach, the parasite undergoes excystation, a process whereby the cysts transform into flagellated trophozoites. 3 The trophozoites attach themselves to the epithelial cells lining the small intestine with the help of an adhesive disk that functions as a suction cup. 5 To ensure transmission, Giardia completes its life cycle when it undergoes encystation into cysts and is excreted through the feces. 6 Giardia is able to maneuver within the small intestine to locate an optimal environment with the aid of four pairs of flagella (anterior, posteriorlateral, caudal and ventral) and subsequently attaches itself to the epithelial cells in the intestine to avoid being swept away via the adhesive disk. 7 Since the flagella and adhesive disk are essential for survival, there is a strong connection between the cytoskeleton and Giardia virulence. 7 Previous studies have identified the association of a class of cytoskeletal proteins called alpha giardins to the Giardia cytoskeleton. 6 Alpha giardins, a multigene family consisting of 21 members, show a common ancestry with the annexins, a family of cytosolic proteins that bind to phospholipids in a Ca 2+ -dependent manner and are implicated in numerous membrane-related processes. 6 9 A phylogenetic analysis of all alpha giardin sequences showed that the alpha giardins are early representatives of the annexins; therefore, it is expected that key structural motifs are conserved between the alpha giardins and the rest of the annexin family, suggesting that they share similar biological functions. 6 Alpha giardins have been shown to localize to cytoskeletal components such as the flagella, the adhesive disk and the plasma membrane and may be active participants in the highly organized membrane and cytoskeletal rearrangements during excystation and encystation. 6 At Fig. 1. Ribbon diagrams of the apo R3 crystal form (a) and Ca 2+ - bound form of alpha-14 giardin (b). Repeat I is shown in green, repeat II in blue, repeat III in yellow, repeat IV in orange and the C- terminus in magenta. The N- and C-termini are labeled as Nt and Ct, respectively. Calcium ions bound to the AB and DE loops in the Ca 2+ - bound alpha-14 giardin structure are illustrated as red spheres. The figures were produced with the programs PyMOL ( sourceforge.net/) and POVRAY (

4 Author's personal copy 1100 Crystal Structures of Apo and Calcium-Bound Alpha-14 Giardin least six members of the alpha giardin family (alpha-2, 5, 9, 10, 14 and 17 giardins) have been shown to localize to either the ventral pair or all four pairs of flagella.6,10 Recently, immunocytochemical colocalization studies showed alpha-14 giardin forms local slubs in the flagella and Fig. 2. Sequence alignment of alpha-14 giardin and the other 20 members of the alpha giardin family. The α-helical segments for alpha-14 giardin are indicated above the sequence alignment and colored according to repeats I (residues 10 81), II (residues ), III (residues ) and IV (residues ). Residues denoted in red on a white background have the same conserved physicochemical property, while residues denoted in black on a white background are conserved in this group. Calcium-coordinating residues in the alpha-14 giardin sequence are highlighted in a green background (DE loop of repeat I), cyan background (AB loop of repeat II), yellow background and boxed in blue (AB loop of repeat III), yellow background (DE loop repeat III) and orange background (AB loop of repeat IV). The sequence alignment was performed using CLUSTALW14 and the figure was prepared with the program ESPript.15

5 Author's personal copy Crystal Structures of Apo and Calcium-Bound Alpha-14 Giardin 1101 Fig. 2 (legend on previous page) localizes to the proximal and distal sections of all eight giardial flagella.11 In 2007, we reported the crystal structures of the apo and calcium-bound forms of alpha-11 giardin [Protein Data Bank (PDB) codes 2II2 and 2IIC, respectively], the first three-dimensional structure of a member of the alpha giardin family12. Here we report the three-dimensional structures of apo and calcium-bound alpha-14 giardin, the only member of the alpha giardin family that localizes to all flagella in Giardia. The overall topology of the structure is similar to that of the previously determined alpha-11 giardin 12 structure and annexin structures in general; 9 however, several novel features were detected. In addition, phospholipid-binding studies revealed insights into alpha-14 giardin's lipid-binding properties and its possible role during differentiation in Giardia.

6 1102 Crystal Structures of Apo and Calcium-Bound Alpha-14 Giardin Results and Discussion Apo structure of alpha-14 giardin The apo crystal structures of alpha-14 giardin determined in two crystal forms (R3 and P crystal forms) are very similar and have an all-atom r.m.s.d. value of 0.69 Å. The overall apo structure of alpha-14 giardin forms a curved, two-sided disk with a concave and a convex face with short loops connecting the helices, similar to alpha-11 giardin and other annexin structures (Fig. 1a). 9,12 The core domain comprises four tandem repeats (I, II, III and IV), each composed of approximately 75 residues; each repeat is composed of five α-helices (A E) stabilized by hydrophobic side-chain interactions. Unlike most annexin structures, alpha-14 giardin lacks an N-terminal domain preceding the core domain on the concave face of the molecule. 9 In contrast to alpha-11 giardin and other annexins, alpha-14 giardin has a C-terminal extension composed of 26 amino acids located on the concave side of the core domain where approximately half of the C-terminal domain is visible in the electron density maps, while the extreme C-terminal portion is disordered. The function of the C-terminal extension of alpha-14 giardin is currently unknown; however, it has been proposed that it may serve as a ligand for cytoskeletal binding proteins in the flagella of Giardia. 10 In addition to the possibility of forming protein protein interaction sites, the C-terminal extension could serve as a membrane-binding site for membrane aggregation or bridging and also may be subject to posttranslational modifications, such as phosphorylation, which have been reported for the N-terminal domain of annexins. 13 Recently, affinity chromatography, electron spray ionization mass spectrometric analyses and the two-hybrid system revealed that alpha-14 giardin interacts with a multiple ankyrin repeat of a Ser/Thr kinase in a calcium-dependent manner. 11 Ser320, Ser327 and Thr334 in alpha-14 giardin could be subject to phosphorylation via the multiple ankyrin repeat of this Ser/Thr kinase and this may regulate protein protein interactions or could lead to altered calcium affinities, as has been shown for the core domain in annexin A2. 9 The presence of a C-terminal extension is a special feature of the alpha giardin family. The alpha-11 giardin structure reported previously contains only four C-terminal amino acids located on the concave face of the core domain and most of the Fig. 3. Comparison of the apo form of alpha-14 giardin (R3 crystal form) and alpha-11 giardin structures. (a) Ribbon representation of the alpha-14 giardin structure (purple) superimposed on the alpha-11 giardin (cyan) crystal structure. (b) The alpha-14 giardin structure is colored from blue to red according to its r.m.s.d. from alpha-11 giardin structure, with regions with the largest deviations depicted in red (C α r.m.s.d., 1.78 Å; all-atom r.m.s.d., 6.18 Å). Superposition of the alpha-14 giardin and alpha-11 giardin structures was performed in Coot. 16 The N- and C-termini are labeled as Nt and Ct, respectively. The figures were prepared with the molecular graphics program PyMOL ( pymol.sourceforge.net/).

7 Crystal Structures of Apo and Calcium-Bound Alpha-14 Giardin 1103 Fig. 4. A plot of all-atom r.m.s.d. (angstroms) between the structures of alpha-14 giardin and alpha-11 giardin as a function of the amino acid residue number in the alpha-14 giardin sequence (repeat I, 10 81; repeat II, ; repeat III, ; repeat IV, ). The two arrows indicate the gap (residues ) in the sequence in repeat III, which could not be superposed. The figure was prepared in Excel. remaining members of the alpha giardin family are composed of C-terminal extensions that range from two to eight residues. Of the 21 members of the alpha giardin family, only 2 other members in addition to alpha-14 giardin have long C-terminal extensions that range in size from 17 (alpha-8 giardin) to 77 amino acids (alpha-19 giardin) (Fig. 2). Superposition of the structures of alpha-11 giardin and alpha-14 giardin hints at additional differences between these two alpha giardins and why extensive molecular replacement trials failed (Fig. 3a). The r.m.s.d. of the C α atoms and all atoms between alpha-14 giardin and alpha-11 giardin are 1.76 and 6.18 Å, respectively, indicating that there are significant differences between these two structures. The individual all-atom r.m.s.d. values for repeats I, II and IVare 1.7, 12.2 and 4.3 Å, respectively. The allatom r.m.s.d. value for repeat III could not be calculated, since nearly half of the residues (41 residues) in this repeat do not superimpose well (Fig. 3b). Based on the r.m.s.d. values and superposed alpha-11 giardin and alpha-14 giardin structures, the largest differences occur in repeats II, while the smallest differences are found in repeat I (Fig. 4). The superposition of the alpha-11 giardin and alpha-14 giardin structures reveals that the large deviations in repeats II and III are due to differences in the relative orientation of the helices and loops. In particular, there are large differences in the AB loops of repeats II and III located on the convex face of the core domain, which is not surprising, since calcium ions were detected in these loops in the Ca 2+ -bound alpha-14 giardin structure (Fig. 1b). Structure of Ca 2+ -bound alpha-14 giardin Crystals of Ca 2+ -bound alpha-14 giardin were grown in 50 mm calcium chloride, 100 mm bis-tris, ph 6.5, and 24% (w/v) polyethylene glycol (PEG) 4000 in space group P2 1 with one molecule per asymmetric unit. In contrast, the two apo crystal forms of alpha-14 giardin were grown under different conditions and yielded different space groups, suggesting that alpha-14 giardin is capable of binding calcium. A superposition of the Ca 2+ - bound alpha-14 giardin structure with the two apo forms of alpha-14 giardin (space groups R3 and P ) resulted in C α r.m.s.d. values of 0.92 and 0.85 Å, respectively, with most of the differences occurring in the C-terminal extension located on the concave face and in the calcium-binding loops located on the convex face of the core domain. The possibility that alpha-14 giardin is capable of binding to calcium is not a surprising feature, since we previously reported the crystal structure of alpha-11 giardin with one calcium ion bound. 12 In addition, the alpha giardin family has a common ancestry with annexins, a family of proteins whose members are known to bind calcium. 8,9 To date, numerous annexins have been crystallized with 2 to 10 calcium ions bound to the convex face of the core domain. 13 Calcium coordination in annexins is achieved via type II or type III binding sites (both non-ef hand). Type II binding sites are formed by AB loops via three backbone carbonyl oxygen atoms within the (M,L)-XG-X-G sequence (coordinating residues are highlighted in bold and X is a variable residue), the bidentate carboxylate ligand from either an aspartic acid or glutamic acid residue ( cap residue ) located approximately 44 residues downstream in sequence and one or two water molecules in a pentagonal bipyramidal or octacoordination sphere. 17 Type III binding sites are formed by DE loops via two backbone carbonyl oxygen atoms, one acidic side chain located approximately eight residues downstream in sequence and three water molecules in a pentagonal bipyramidal coordination sphere. 17 More recently, a novel calcium-binding site termed type IIIb was identified in the DE loop of repeat I in the Ca 2+ - bound alpha-11 giardin structure, the first alpha giardin structure solved. 12 In this type IIIb binding site, the calcium ion is coordinated by two backbone carbonyls, the carboxylate of a glutamic acid and the side-chain carbonyl of an asparagine residue. Since

8 1104 Crystal Structures of Apo and Calcium-Bound Alpha-14 Giardin the presence of a polar, uncharged side chain (asparagine residue) in the calcium coordination sphere had not been reported in previously published calcium-bound annexin structures, the term type IIIb calcium-binding site was proposed. All three types of calcium-binding sites (type II, III and IIIb) mentioned above were detected in the Ca 2+ - bound alpha-14 giardin structure. Fig. 5. Coordination of the calcium ions in the AB and DE loops in the Ca 2+ -bound alpha-14 giardin structure. Calcium ions are represented as red spheres, water molecules as yellow spheres and the coordination between the calcium ion and oxygen atoms is indicated by cyan dotted lines. (a) Calcium coordination in the DE loop of repeat I. The coordination distances between the calcium ion and the coordinating oxygen atoms (Ca O) are as follows: Ca O Lys64, 2.35 Å; Ca O Leu67, 2.34 Å; Ca OE1 Glu72, 2.38 Å; Ca O HOH515, 2.38 Å; Ca O HOH615, 2.61 Å; Ca O HOH742, 2.59 Å; and Ca O HOH830, 2.09 Å. (b) Calcium coordination in the DE loop of repeat III has the following Ca O bond distances: Ca O Glu222, 2.31 Å; Ca O Phe225, 2.38 Å; Ca OD1 Gln226, 2.87 Å; Ca OE1 Glu230, 2.37 Å; Ca O HOH637, 2.57 Å; Ca O HOH695, 1.93 Å; and Ca O HOH757, 2.80 Å. (c) Calcium coordination in the AB loop of repeat II has the following Ca O bond distances: Ca O Thr94, 2.28 Å; Ca O Gly96, 2.35 Å; Ca O Gly98, 2.29 Å; Ca OD1 Asp138, 2.41 Å; Ca OD2 Asp138, 2.58 Å; Ca O HOH647, 2.37 Å; and Ca O HOH726, 2.41 Å. (d) Calcium coordination in the AB loop of repeat IV has the following Ca O bond distances: Ca O Phe255, 2.30 Å; Ca O Gly257, 2.37 Å; Ca O Gly259, 2.38 Å; Ca OE1 Glu298, 2.60 Å; Ca OE2 Glu298, 2.66 Å; and Ca O HOH621, 2.41 Å. (e) Calcium coordination in the AB loop of repeat III has the following Ca O bond distances: Ca O Ala175, 2.32 Å; Ca O Thr178, 2.50 Å; Ca OG1 Thr178, 2.34 Å; Ca O Gly180, 2.34 Å; Ca OE1 Glu185, 2.40 Å; Ca OE1 Glu224, 2.57 Å; and Ca OE2 Glu224, 2.41 Å. The figures were produced with the programs PyMOL ( and POVRAY (

9 Crystal Structures of Apo and Calcium-Bound Alpha-14 Giardin 1105 The crystal structure of alpha-14 giardin with bound calcium consists of 303 amino acid residues (out of 337, the first 10 N-terminal residues and the last 26 C-terminal residues were not visible in the electron density maps). The five calcium ions detected on the convex face of the core domain are located in repeat I (DE loop), repeat II (AB loop), repeat III [one each in the AB (new type) and DE loops], and repeat IV (AB loop). The calcium ion in the DE loop of repeat I of alpha-14 giardin is coordinated in a traditional type III binding site by the backbone carbonyls of Lys64 and Leu67, the unidentate carboxylate group of Glu72 and four water molecules in a pentagonal bipyramidal coordination sphere (Fig. 5a), and the calcium ion detected in the DE loop of repeat III is coordinated in a type IIIb binding site by the backbone carbonyl oxygen atoms of Glu222 and Phe225, the unidentate carboxylate oxygen of Glu230, three water molecules and the side-chain carbonyl of Gln226 in a pentagonal bipyramidal coordination sphere (Fig. 5b). Slight differences were detected in the coordination of the calcium ions in the AB loops of repeats II and IV in Ca 2+ -bound alpha-14 giardin in comparison to the calcium coordination observed in the AB loops in annexins. In alpha-14 giardin, the calcium ion located in repeat II is coordinated by the backbone carbonyl oxygen atoms of Thr94, Gly96 and Gly98, the bidentate carboxylate oxygen atoms of Asp138 and two water molecules, and the calcium ion located in repeat IV is coordinated by the backbone carbonyl oxygen atoms of Phe255, Gly257 and Gly259, the bidentate carboxylate oxygen atoms of Glu298 and one water molecule in a pentagonal bipyramidal and hexagonal coordination sphere, respectively (Fig. 5c and d). The traditional aliphatic residues in the (M,L)-X-G-X-G sequence motif of the AB loops (type II binding site) observed in annexins are substituted by either a polar residue (Thr94) or a hydrophobic residue (Phe255) in the Ca 2+ -bound alpha-14 giardin structure. Sequence alignment of alpha-14 giardin with other alpha giardins reveals that the types II and III calcium-binding sites detected in the Ca 2+ -bound alpha-14 giardin structure may not be conserved in all members of the alpha giardin family (Fig. 2). In the AB loop (type II binding site) of repeat II, the two glycine residues in the (M,L)-X-G-X-G sequence motif and the acidic residue located approximately 44 residues downstream in sequence are strictly conserved in alpha-7.1, -7.2, -7.3 and -10 giardins; however, the other members of the alpha giardin family have various amino acid substitutions in these regions and may coordinate the calcium ion differently or not at all. Surprisingly, the traditional residues involved in calcium coordination in the AB loop (type II binding site) of repeat IV do not appear to be conserved in any of the alpha giardins except in alpha-14 giardin. Although the downstream aspartic acid or glutamic acid residue is conserved in some members of the alpha giardin family (alpha- 1, -2, -4, -5, -6 and -9 giardins), the two glycine residues in the (M,L)-X-G-X-G sequence motif are not present and have different amino acid substitutions in repeat IV. Calcium coordination in the DE loop (type III binding site) of repeat I in alpha-14 giardin and in several annexins (annexin A1, A2 and A5) occurs via two backbone carbonyl oxygen atoms within the K-X-X-(L/I) sequence motif (coordinating residues are highlighted in bold and X is a variable residue), a unidentate carboxylate ligand from a glutamic acid residue located approximately eight residues downstream in sequence and two to three water molecules. This calcium coordination sequence motif in the DE loop of repeat I is strictly conserved in alpha-1 and alpha- 2 giardins, while in alpha-5, -6, -9, -11 and -17 giardins, the amino acid residues in the calcium coordination sequence are conserved, but with some modifications in the consensus sequence. Although the traditional type II and type III calcium-binding sites present in annexins are conserved in alpha-14 giardin, the unique tryptophan residue (Trp187) that plays a role in calciumdependent membrane association of annexin A5 is not conserved in alpha-14 giardin. 20,21 The crystal structures of annexin A5 and UV-difference spectroscopy, circular dichroism, and steady-state and time-resolved fluorescence studies revealed that Trp187 in the loop region of repeat III is solventexposed in the presence of high concentrations of calcium and plays a crucial role in calcium-dependent membrane association, while at low calcium concentrations, Trp187 is buried in the hydrophobic core In alpha-14 giardin it is unlikely that a tryptophan residue is crucial for calcium-dependent membrane association, since this unique tryptophan residue present in annexin A5 is substituted by a glycine residue. In addition, the tryptophan residues (Trp145 in repeat II, Trp189 in repeat III and Trp311 in repeat IV) in alpha-14 giardin are all buried in the hydrophobic core in the apo and calcium-bound structures and do not undergo a major conformational change in the presence of calcium (C α r.m.s.d. values: Trp145, 0.28 Å; Trp189, 0.40 Å; Trp311, 0.52 Å). Furthermore, Trp187 of annexin A5, which plays a role in its calcium-dependent membrane association, is also not conserved in any of the other giardins, indicating that if the other giardins associate with membranes it may be by another mechanism. New calcium-binding site A new calcium-binding site was observed in the AB loop of repeat III of alpha-14 giardin, one in which all seven coordinating oxygen atoms are provided by the protein, something that has not been reported previously in annexin structures. In the crystal structures of annexins A1, A2, A3 and A6 (PDB codes 1MCX, 18 1XJL, 19 1AXN 27 and 1AVC, 28 respectively), the calcium ion is coordinated via a traditional type II binding site by the backbone carbonyl oxygen atoms of two glycines and either a lysine residue or arginine residue, the carboxylates of a glutamic acid residue and two water molecules.

10 1106 Crystal Structures of Apo and Calcium-Bound Alpha-14 Giardin In the Ca 2+ -bound alpha-14 giardin structure, the calcium ion is coordinated by the backbone carbonyl oxygen atoms of Ala175, Thr178 and Gly180, the hydroxyl of Thr178, the unidentate carboxylate group of Glu185 and the bidentate carboxylate oxygen atoms of Glu224 in a pentagonal bipyramidal coordination sphere (Fig. 5e). There are significant differences in the coordination observed in the alpha-14 giardin structure in the AB loop of repeat III. In particular, one of the two glycine residues in the traditional type II calcium coordination sequence (M,L)-X-G-X-G is absent in the coordination sphere in alpha-14 giardin. The presence of two carboxylate ligands and the side-chain hydroxyl from a threonine residue in the calcium coordination is highly unusual and has not been reported in any of the calcium-binding sites reported in the annexin structures. Another highly unusual feature is the absence of water molecules from the calcium coordination sphere. All seven ligands participating in the calcium coordination sphere are backbone carbonyl or side-chain oxygen atoms, indicating that this may be a very high affinity calcium-binding site. Harding et al. report that the presence of seven protein donor groups interacting with a calcium ion in a protein structure is quite rare. 29 A set of tables detailing the architecture of metal-coordination groups in proteins shows that only 1.4% (5/359) of the protein structures with calcium deposited in the PDB by 2004 contain seven protein donor groups coordinating a calcium ion (PDB codes 1K12, 30 1K3I, 31 1D2V, 32 1LYC 33 and 1GWU 29 ). Interestingly, the protein structures in this list are either oxidoreductases or sugar-binding proteins There is a possibility that alpha-14 giardin is a sugar-binding protein, since previous studies have shown that both alpha-1 giardin and alpha-2 giardin display calcium-dependent binding to glycosaminoglycans and may initiate the first host parasite contact with the microvilli in the small intestine. 34 Based on the observations described above, we propose the term type IIb` calciumbinding site for the arrangement observed in the AB loop of repeat III in alpha-14 giardin. Initially, we suspected the type IIb calciumbinding site to be occupied in both apo crystal forms (R3 and P ) of alpha-14 giardin due to the presence of strong positive difference electron density in the AB loop of repeat III. The difference electron density (5.9 sigma) for the R3 crystal form was suspected to be a magnesium ion, since the crystals were grown in a crystallization buffer containing 100 mm magnesium chloride. However, the difference electron density (4.8 sigma) in the P crystal form was unexpected, since these crystals were grown in crystallization conditions without salt plus 1 mm ethylenediaminetetraacetic acid (EDTA), a divalent cation chelator. To determine whether the difference electron density corresponded to a magnesium ion or a calcium ion, crystallographic refinement was performed on the P crystal form by testing either cation as part of the model. The positive difference electron density in the AB loop of repeat III was no longer present after modeling a calcium ion; furthermore, negative difference electron density in the AB loop of repeat III appeared after modeling in a magnesium ion, suggesting that this site is occupied by a calcium ion. In addition, the typical coordination number for a magnesium ion is six, while the typical coordination number for a calcium ion can be either six or seven. 29 Since there are seven ligands contributing to the coordination sphere, we strongly believe that the positive difference electron density in the AB loop of repeat III is a calcium ion. We suspect that a calcium ion must have been retained in the P crystal form despite having 1 mm EDTA present in all purification buffers. Thus, this is likely a high-affinity calcium-binding site; however, additional biochemical studies will need to be performed to determine the dissociation constant. Our results indicate that the metal ion binding site in the AB loop of repeat III is interchangeable between calcium and magnesium if the appropriate crystallization conditions are used. Phospholipid binding assay Since five calcium-binding sites were detected in the alpha-14 giardin structure, we wanted to test whether alpha-14 giardin exhibits the same calciumdependent phospholipid-binding property featured in annexins. 35 Various biologically active lipids were screened to determine the preferential phospholipidbinding properties of alpha-14 giardin and possible biological roles were proposed based on these results. The phospholipid binding assay was carried out using membrane lipid strips prespotted with a variety of phospholipid species triglyceride, phosphatidylinositol (PI), phosphatidylinositol 4-phosphate [PtdIns(4)P], phosphatidylinositol (4,5) bisphosphate [PtdIns(4,5)P 2 ], phosphatidylinositol (3,4,5) trisphosphate [PtdIns(3,4,5)P 3 ], phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylethanolamine (PE), diacylglycerol (DAG), cholesterol, phosphatidylcholine (PC), sphingomyelin, phosphatidylglycerol (PG), 3-sulfogalactosylceramide (sulfatide) and cardiolipin which are important in several signaling pathways. The assay, which is essentially a Western blot, was performed by incubating glutathione S-transferase (GST) alpha-14 giardin fusion protein in the presence of either 2 mm ethylene glycol bis(β-aminoethyl ether) N,N -tetraacetic acid (EGTA; results not shown) or 1 mm CaCl 2 (Fig. 6) with the membrane lipid strips. For detection of protein binding, the membrane lipid strips were first incubated with a primary antibody (anti-gst antibody) to bind to the GST tag and then a secondary antibody (anti-mouse antibody) conjugated with horseradish peroxidase directed against the primary antibody. The phospholipid binding profile for alpha-14 giardin was then detected using a

11 Crystal Structures of Apo and Calcium-Bound Alpha-14 Giardin 1107 Fig. 6. Phospholipid binding assay. Membrane lipid strips prespotted with 15 different biologically active lipids were incubated with GST alpha-14 giardin fusion protein in the presence of 1 mm CaCl 2. No binding was observed in the presence of 2 mm EGTA or with the GST tag alone (not shown). chemiluminescent reagent. Alpha-14 giardin associated strongly with PS, cardiolipin, PtdIns(4)P and PtdIns(4,5)P 2 in a calcium-dependent manner. No binding of GST alpha-14 giardin fusion protein in the presence of 2 mm EGTA was observed (control not shown). The association of alpha-14 giardin with PS was not a surprise, since previous vesicle pull-down assays revealed the calcium-dependent binding of this giardin to multilamellar liposomes from brain extract. 10 Surprisingly, alpha-14 giardin associated with cardiolipin, a lipid that is an important component of the inner mitochondrial membrane where it serves as an insulator and stabilizes the activity of protein complexes important to the electron transport chain. 36 This was an interesting result, since the anaerobic protist Giardia lacks mitochondria. 7 However, mitosomes, which appear to be highly reduced mitochondria, were recently identified in G. lamblia. 37,38 These double-membrane-bound organelles devoid of DNA have also been found in other unicellular eukaryotes such as the amoeba Entamoeba histolytica, the microsporidian Trachipleistophora hominis and the alveolate Cryptosporidium parvum. 37 It has been hypothesized that mitosome origins date back to the endosymbiotic event leading to the acquisition of mitochondria in other eukaryotes, as in the case of hydrogenosomes found in Trichomonas and in anaerobic ciliates. 39,40 Cardiolipin has been found in hydrogenosomes, 41 and potentially this lipid also exists in mitosomes. This result suggests that alpha-14 giardin may be involved in mitosome function. The results also reveal the calcium-dependent association of alpha-14 giardin to PtdIns(4)P and PtdIns(4,5)P 2. PtdIns(4)P is prevalent in the membrane of the Golgi apparatus and is also the precursor to PtdIns(4,5)P Previous studies have shown that the Golgi apparatus is required to build a cyst wall when the Giardia encysts and alpha-14 giardin may have a direct role during this process, since our results show calcium-dependent binding to PtdIns(4)P. 7 In addition, we also demonstrate the calcium-dependent binding of alpha-14 giardin to PtdIns(4,5)P 2, a signaling molecule in the membrane that has recently been shown to be important in flagellar outgrowth and proper axoneme architecture in developing Drosophila sperm. 43 The direct binding of alpha-14 giardin to PtdIns(4,5)P 2 indicates that this protein may have a pivotal role during the highly dynamic processes of encystation and excystation. During encystation, when the Giardia transforms from trophozoites to cysts, the flagella are broken into several large fragments and stored in the cytoplasm of the cyst until excystation. 44 When excystation is triggered by alterations in calcium levels, the flagella are reassembled. 45 Alpha-14 giardin may have a direct role in this process, since it has been shown to localize to all flagella and bind specifically to tubulin, 11 and our results show calcium-dependent binding to PtdIns(4,5)P 2. The data shown here would fit in with a model in which binding of calcium to the convex face of alpha-14 giardin increases affinity to PtdIns(4,5)P 2 in the membrane, while the long C- terminal region of the protein interacts with a binding partner such as tubulin. Interestingly, alpha-14 giardin binding to tubulin was shown to be a calcium-dependent event but calcium was not observed in the ordered portion of the C-terminal extension of the protein. 11 This may indicate that the interaction between alpha-14 giardin and tubulin is more complex and may require a linker protein or posttranslational modification such as phosphorylation. 11 Further experiments are required to determine the interaction mechanism of alpha-14 giardin and tubulin. Another model we propose based on the above results would involve the calcium-dependent association of alpha-14 giardin with PtdIns(4,5)P 2 in the membrane, while the long C-terminal region of the protein would interact with actin. Currently, it is unclear whether alpha-14 giardin harbors an actinbinding site. Interestingly, annexin A2 has been shown to interact with PtdIns(4,5)P 2 and actin, and the actin-binding site was mapped to the last nine residues at the annexin A2 C-terminus (LLYLCG- GDD). 46 The C-terminal actin-binding motif of annexin A2 is also present in annexin A4 but absent in certain plant annexins. Surprisingly, actin binding has not been observed for annexin A4 but has been reported for certain plant annexins, suggesting that the mechanism for actin binding is complex The C-terminal actin-binding motif observed in annexin A2 is not conserved in alpha-14 giardin; however, actin-binding domains present in other proteins are known to be variable in length with no consensus sequence, so it is possible that alpha-14 giardin has a yet unidentified actin-binding domain in its C- terminal extension. Conclusions Presented here are the crystal structures of alpha- 14 giardin in two apo crystal forms (R3 and P ) and in the Ca 2+ -bound form, the second alpha

12 1108 Crystal Structures of Apo and Calcium-Bound Alpha-14 Giardin giardin from the intestinal protozoan G. lamblia to be characterized structurally. The crystal structure of alpha-14 giardin reveals a curved disk with a concave face and convex face composed of four tandem repeats similar to that of the previously determined alpha-11 giardin structure and annexin structures. 9,12 Unlike most annexin structures, which have long N-terminal domains, 13 alpha-14 giardin has a long C-terminal extension that snakes across the concave face of the protein. Although alpha-14 giardin exhibits the same overall topology as alpha-11 giardin, there were several significant differences in the orientations of the helical and loop regions particularly in repeats II and III, which made molecular replacement using alpha-11 giardin as the search model difficult. The structure of Ca 2+ -bound alpha-14 giardin reveals five calcium ions bound in the AB and DE loops on the convex face of the core domain. Calcium ions were detected in the AB loops of repeats II, III and IVand in the DE loops of repeats I and III. The calcium ion in the DE loop of repeat I is coordinated in a traditional type III binding site, while the calcium ion in the DE loop of repeat III is coordinated in a type IIIb binding site. The calcium coordination observed in the AB loops of repeats II and IV are in a traditional type II binding site with a few amino acid substitutions in the calcium coordination sequence. We observed significant differences in the calcium coordination sequence in the AB loop of repeat III in comparison to the traditional type II binding site observed in the Ca 2+ -bound annexin structures. 26 In Ca 2+ -bound alpha-14 giardin, two carboxylate ligands and the side-chain hydroxyl from a threonine residue were observed in the calcium coordination sphere in the AB loop of repeat III. In addition, the calcium ion is coordinated only by amino acids and no water molecules in a pentagonal bipyramidal coordination sphere, a feature rarely observed in protein structures and not previously reported for any Ca 2+ -bound annexin structure. Thus, the term type IIb binding site is proposed, in contrast to the traditional type II binding site. Surprisingly, the highly coordinated calcium ion in the AB loop of repeat III was observed in the P apo crystal form of alpha-14 giardin, indicating that this must be a site of very high affinity for calcium. In addition, our results demonstrate that alpha-14 giardin exhibits the calciumdependent phospholipid-binding property exhibited in annexins. A phospholipid binding assay revealed the calcium-dependent binding of alpha-14 giardin to PS, cardiolipin, PtdIns(4)P and PtdIns(4,5) P 2. Unexpectedly, alpha-14 giardin interacted with a component of the inner mitochondrial membrane. Also, the phospholipid binding assay results show alpha-14 giardin directly interacting with phospholipids involved in cytoskeletal rearrangements and motility. Thus, alpha-14 giardin may have a calciumregulated role as a membrane-cytoskeletal linker during the disassembly and reassembly of the flagella in the encystation and excystation of Giardia. In this article, we propose possible biological roles of alpha-14 giardin based on our phospholipid binding assay results; however, further experiments will need to be conducted to confirm our results and proposed roles of alpha-14 giardin in cytoskeletal rearrangements in G. lamblia. Materials and Methods Protein expression and purification Recombinant native, selenomethionine-substituted alpha-14 giardin and GST alpha-14 giardin fusion protein were expressed as previously described for alpha-11 giardin. 51 N-terminal GST-tagged alpha-14 giardin was purified on a preequilibrated [50 mm Tris HCl, ph 7.4, 1 mm EDTA, 100 mm NaCl, 1% (v/v) NP40, 10% (v/v) glycerol] 20-ml bed volume GSTrap Fast Flow column (Amersham). The cell lysate was incubated on the GST column for 1.5 h, washed with 10 column volumes of 1 M NaCl and 10 column volumes of 50 mm Tris HCl, ph 8.0, and 150 mm NaCl to remove any unbound protein and impurities. The column was then equilibrated with 10 column volumes of buffer A (10 mm Tris HCl, ph 8.0, and 150 mm NaCl) and the GST alpha-14 giardin fusion protein was eluted in buffer A containing 10 mm reduced glutathione. Fractions containing GST alpha-14 giardin fusion protein were collected and dialyzed against buffer A before being concentrated in a Centricon 10-kDa cutoff concentrator (Millipore) to 1 mg/ml for the phospholipid binding assay. To obtain recombinant native and selenomethionine-substituted protein for crystallization, GSTfusion protein was dialyzed in buffer B (50 mm Tris HCl, ph 8.0, 150 mm NaCl, 1 mm EDTA and 1 mm DTT) and then incubated with 10 U PreScission Protease (Amersham) per milligram of GST fusion protein overnight at 4 C. The PreScission Protease (containing a GST tag) and the cleaved GST tag were separated from alpha-14 giardin after applying the protein onto a GSTrap Fast Flow column equilibrated with buffer B. During this purification step, the PreScission Protease and GST tag remained bound to the GSTrap Fast Flow column, while the alpha- 14 giardin eluted in the flow-through in buffer B. Crystallization Prior to crystallization, apo and selenomethionine-substituted alpha-14 giardin was concentrated to 10 mg/ml with a Centricon 10-kDa cutoff concentrator (Millipore). Using the sitting-drop vapor-diffusion method, 2 μl of protein solution was mixed with 2 μl of reservoir solution over wells containing 200 μl of reservoir solution. Apo alpha-14 giardin crystals were obtained in two different space groups (R3andP ) from two different conditions from the Protein Complex Suite (condition A) and the PEGS suite (condition B) from Nextal Biotechnologies. Condition A yielded bipyramidal crystals in 100 mm magnesium chloride, 100 mm sodium citrate, ph 5.0, and 15% (w/v) PEG 4000, and condition B yielded long rods in 100 mm sodium acetate, ph 4.6, and 15% (w/v) PEG at 4 C after 1 week. Ca 2+ -bound alpha-14 giardin crystals were obtained in 50 mm calcium chloride, 100 mm bis-tris, ph 6.5, and 24% (w/v) PEG 4000 at 22 C after a few days. Selenomethionyl-derivatized alpha-14 giardin crystals were obtained in condition A. Crystals were transferred to a solution containing the reservoir solution and 30% (v/v) glycerol as a cryoprotectant before being flash-cooled in liquid nitrogen for data collection.

13 Crystal Structures of Apo and Calcium-Bound Alpha-14 Giardin 1109 Data collection X-ray diffraction data on apo, Ca 2+ -bound and selenomethionyl-derivatized alpha-14 giardin were collected at beamlines 9-1 and 11-1 at the Stanford Synchrotron Radiation Laboratory (California, USA), using remote robotic data collection. The Q315 and Mar325 CCD area detectors were used for data collection. A total of 180 frames were collected with an oscillation angle of 1 (10 s exposure time) on the two apo crystals (conditions A and B) and Ca 2+ -bound alpha-14 giardin crystals with a crystal-to-detector distance of 200 mm. For conditions A and B, two high-resolution data sets were collected to 1.9 and 1.6 Å, respectively, and one data set was collected on a Ca 2+ -bound alpha-14 giardin crystal to 1.65 Å. An X-ray fluorescence scan of selenomethionylderivatized alpha-14 giardin crystal was collected near the selenium K absorption edge to select the appropriate wavelengths for multiwavelength anomalous dispersion (MAD) data collection. MAD diffraction data were collected on a single crystal at the inflection point, peak and high-energy remote wavelengths with a crystal-todetector distance of 300 mm, an oscillation angle of 1 and 10 s exposure time. For each wavelength, 360 data were collected in 30 wedges to 2.5 Å. The data sets collected on the two apo crystals (conditions A and B), the Ca 2+ -bound crystal and the selenomethionyl-derivatized crystal were processed with HKL in the rhombohedral space group R3 (condition A), primitive orthorhombic space group P222 (condition B), primitive monoclinic space group P2 and rhombohedral space group R3, respectively. Data collection statistics are listed in Table 1. Structure determination by MAD phasing and refinement Initially, molecular replacement trials were carried out to solve the structure of alpha-14 giardin with the program PHASER 52 using alpha-11 giardin (PDB code 2II2, % sequence identity) as the search model, either in its entirety or broken up into separate repeats. Since PHASER was unable to find a solution, the structure of alpha-14 giardin was determined by MAD phasing. The structure of apo-alpha-14 giardin was solved in the space group R3 using the data sets collected at the inflection point, peak and high-energy remote wavelengths. Seven out of nine selenium sites were located with the program SOLVE 53 with an overall Z score of The two remaining selenium sites that were not located in the program SOLVE were at the termini of alpha-14 giardin and were disordered in the electron density maps. The initial electron density map from SOLVE was improved using the program RESOLVE, 54 which increased the figure of merit (FOM) from 0.73 to Phases were extended to 1.9 Å using the high-resolution apo data set (space group R3) and the automated protein model-building program ARP/wARP. 55 The second apo alpha-14 giardin structure and Ca 2+ -bound alpha-14 giardin structure were solved in the space groups P and P2 1, respectively, via molecular replacement with the program PHASER and using apo alpha-14 giardin solved in the space group R3as the search model. The program REFMAC5 56 was used to refine the models until the R-factors could not be decreased further. Calcium ions and ordered solvent water molecules were added by visual inspection. Manual model building was Table 1. Data collection and MAD phasing statistics Native (apo) 1 Native (apo) 2 Ca 2+ -bound Selenomethionine MAD Edge Inflection Remote Data collection Wavelength (Å/eV) /12, /12, /13,600 f /f 8.31/ / /3.48 Resolution range (Å) a ( ) ( ) ( ) ( ) ( ) ( ) Space group R3 P P2 1 R3 R3 R3 Unit-cell parameters a (Å) b (Å) c (Å) α, β, γ ( ) 90.00, 90.0, , 90.0, , , , 90.0, , 90.0, , 90.0, Total observations 211, , , , , ,949 Unique reflections 36,608 42,635 38,460 30,552 31,538 30,752 Completeness (%) a 99.7 (99.5) (99.9) 97.0 (82.4) 96.8 (79.3) 99.0 (99.0) 97.4 (82.3) R merge (%) a,b 4.6 (48.6) 4.6 (48.6) 4.1 (25.5) 7.7 (19.3) 6.8 (16.0) 7.7 (19.2) Average I/σ(I) a 45.0 (3.5) 45.0 (3.5) 37.9 (4.0) 16.6 (3.2) 24.1 (6.7) 17.9 (3.3) Redundancy a 5.8 (5.4) 5.8 (5.4) 3.5 (2.8) 5.3 (3.2) 5.7 (5.0) 5.3 (3.3) Mosaicity ( ) MAD phasing Resolution range (Å) No. of selenomethionines per 7 asymmetric unit Solve peaks (peak heights/σ) S1=25.7, S2=24.2, S3=25.1, S4=23.4, S5=22.1, S6=21.1, S7=18.1 Overall SOLVE Z score FOM before density modification 0.73 FOM after density modification (RESOLVE) 0.81 a Values in parentheses are for the highest resolution shell. b R merge = j I j (hkl) I(hkl) hkl j I(hkl), where I j (hkl) and I(hkl) are the intensity of measurement j and the mean intensity for the reflection with indices hkl, respectively.

14 1110 Crystal Structures of Apo and Calcium-Bound Alpha-14 Giardin Table 2. Refinement statistics Apo alpha-14 giardin (R3) Apo alpha-14 giardin (P ) Ca 2+ -bound alpha-14 giardin (P2 1 ) Refinement Resolution range (Å) No. of reflections (working set/test set) 32,915/ ,280/ ,517/1932 R work /R free (%) a,b 20.0/ / /23.7 Average B-factor (Å 2 ) Ramachandran plot c Most favorable regions (%) Allowed regions (%) Disallowed regions (%) r.m.s.d. from ideality Bond lengths (Å) Bond angles ( ) Model details Protein residues/water molecules 315/ / /367 No. of protein atoms No. of calcium ions No. of magnesium ions a R work = F obs F calc /( F obs ). b R free is the R-factor based on 5% of the data excluded from refinement. c Determined by PROCHECK. 57 performed with the program Coot 16 using 2F o F c and F o F c maps. Nearly the entire amino acid sequence of alpha-14 giardin (337 amino acid residues) was built into the electron density maps for both apo crystal forms and the calcium-bound form with the exception of extreme N- and C-terminal residues, which were not visible in the electron density maps. Missing from the electron density are nine N-terminal residues and 13 C-terminal residues in the R3 apo crystal form, 10 N-terminal residues and 16 C-terminal residues in the P apo crystal form and 10 N-terminal residues and 26 C-terminal residues in the calcium-bound form. The final model of the R3 crystal form of alpha-14 giardin includes one monomer in the asymmetric unit (residues ), 169 water molecules and one magnesium ion, and the final R work and R free values were 20.0% and 23.1%, respectively. The final model of the P crystal form of alpha-14 giardin includes one monomer in the asymmetric unit (residues ), 256 water molecules and one calcium ion, and the final R work and R free values were 19.8% and 22.8%, respectively. The final model of the Ca 2+ -bound form of alpha-14 giardin includes one monomer in the asymmetric unit (residues ), 367 water molecules and five calcium ions, and the final R work and R free values were 20.2% and 23.7%, respectively. Good stereochemistry for the two apo forms and Ca 2+ -bound alpha-14 giardin models was confirmed with the program PROCHECK. 57 Final refinement statistics for the two apo forms and Ca 2+ -bound alpha-14 giardin are listed in Table 2. Phospholipid binding assay Phospholipid binding assays were performed using membrane lipid strips (Echelon Research Laboratories, Salt Lake City, UT) that have been pre-spotted with 15 different biologically active lipids [triglyceride, PI, PtdIns (4,5)P 2, PtdIns(3,4,5)P 3 ), PS, PA, PE, DAG, cholesterol, PC, sphingomyelin, PG, 3-sulfogalactosylceramide (sulfatide) and cardiolipin]. Membranes were blocked for 1 h at 22 C in TBST [10 mm Tris, 150 mm NaCl, 0.1% (v/v) Tween 20, ph 8.0) with 3% (w/v) fatty-acid-free bovine serum albumin (BSA). The membranes were then incubated with 1 μg/ml of GST alpha-14 giardin fusion protein in TBST with 3% (w/v) BSA overnight at 4 C. After incubation, the membranes were washed three times for 10 min with gentle agitation in TBST and then incubated for 2 h with anti-gst monoclonal antibody (Sigma G- 1160) diluted 1:4000 in TBST with 3% (w/v) BSA at 22 C. After three additional 10-min washes in TBST, the membranes were incubated with horseradish-peroxidase-conjugated anti-mouse IgG antibody (Sigma A- 9917) diluted to 1:10,000 in TBST with 3% (w/v) BSA for 1.5 h at 22 C. Finally, the membranes were washed three times in distilled H 2 O and then treated with a chemiluminescence substrate (Western Lighting Chemiluminescence Reagent Plus, Perkin Elmer Life Sciences, Boston, MA) for 1 2 min to detect binding of GST alpha-14 giardin fusion protein to the prespotted lipids. All the solutions used throughout the procedure contained either 1 mm CaCl 2 or 2 mm EGTA. Protein Data Bank accession codes The atomic coordinates for the two apo crystal forms (R3 and P ) and Ca 2+ -bound alpha-14 giardin have been deposited in the Research Collaboratory for Structural Bioinformatics Protein Data Bank under accession codes 3CHL, 3CHJ and 3CHK, respectively. Acknowledgements We thank Dr. Imre Berger for recommending the phospholipid binding assay. This work was supported by NIH grant R01-GM References 1. Hunter, P. R. & Thompson, R. C. (2005). The zoonotic transmission of Giardia and Cryptosporidium. Int. J. Parasitol. 35, Gardner, T. B. & Hill, D. R. (2001). Treatment of giardiasis. Clin. Microbiol. Rev. 14,