Structural and Functional Highlights of Vacuolar Soluble Protein 1 from Pathogen T. Brucei. Brucei

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Structural and Functional highlights of Vacuolar Soluble Protein 1 from pathogen T. brucei. brucei Abhishek Jamwal, Adam Round, Ludovic Bannwarth, Catherine Vénien-Bryan, Hassan Belrhali, Manickam Yogavel, Amit Sharma

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Abhishek Jamwal, Adam Round, Ludovic Bannwarth, Catherine Vénien-Bryan, Hassan Belrhali, et al.. Structural and Functional highlights of Vacuolar Soluble Protein 1 from pathogen T. brucei. brucei. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2015, 290 (51), pp.30498-30513. 10.1074/jbc.M115.674176. hal-02545122

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Structural and Functional Highlights of Vacuolar Soluble Protein 1 from Pathogen Trypanosoma brucei brucei* Received for publication, June 24, 2015, and in revised form, September 28, 2015 Published, JBC Papers in Press, October 22, 2015, DOI 10.1074/jbc.M115.674176 Abhishek Jamwal‡1, Adam R. Round§¶, Ludovic Bannwarthʈ, Catherine Venien-Bryanʈ, Hassan Belrhali§¶, Manickam Yogavel‡, and Amit Sharma‡2 From the ‡Structural and Computational Biology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, India, the §European Molecular Biology Laboratory, Grenoble Outstation, 38042 Grenoble, France, the ¶Unit for Virus Host-Cell Interactions, University Grenoble Alpes-EMBL-CNRS, 38042 Grenoble, France, and the ʈ IMPMC, UMR 7590, CNRS-UPMC-IRD, 75252 Paris, France

Trypanosoma brucei (T. brucei) is responsible for the fatal lethal (5). A newer and much more expensive drug, eflorni- human disease called African trypanosomiasis, or sleeping sick- thine, is effective only against T. brucei gambiense. (6). A com- ness. The causative parasite, Trypanosoma, encodes soluble bination of another drug, nifurtimox, with eflornithine has versions of inorganic pyrophosphatases (PPase), also called vac- been used for treatment, but unfortunately it is not effective uolar soluble proteins (VSPs), which are localized to its acido- against T. brucei gambiense (6). Therefore, there is a pressing calcisomes. The latter are acidic membrane-enclosed organelles case to find new, safe, inexpensive, and broad spectrum drugs rich in polyphosphate chains and divalent cations whose signif- for treating sleeping sickness in humans and livestock. icance in these parasites remains unclear. We here report the Soluble inorganic pyrophosphatase (PPase, EC 3.6.1.1)3 is a

crystal structure of T. brucei brucei acidocalcisomal PPases in a ubiquitous and essential enzyme that hydrolyzes the PPi generated ؉ ternary complex with Mg2 and imidodiphosphate. The crystal during cellular processes such as DNA replication and protein structure reveals a novel structural architecture distinct from translation (7–9). Soluble PPases comprise two families that differ known class I PPases in its tetrameric oligomeric state in which in both sequence and structure (10, 11). Family/class I PPases a fused EF hand domain arranges around the catalytic PPase occur in all types of cells from bacteria to humans, whereas class II domain. This unprecedented assembly evident from TbbVSP1 occurs exclusively in bacteria (11–12). Both classes of enzymes crystal structure is further confirmed by SAXS and TEM data. require divalent metal cations for catalysis (13–15). PPases from SAXS data suggest structural flexibility in EF hand domains wide sources have been characterized, but those from Saccharo- indicative of conformational plasticity within TbbVSP1. myces cerevisiae and Escherichia coli are most well characterized in terms of three-dimensional structures and function. Acidocalcisomes are acidic organelles rich in short and African trypanosomiasis, commonly known as sleeping sick- long chain polyphosphates complexed with Caϩ2 and other ness, affects ϳ50,000 inhabitants of sub-Saharan Africa yearly divalent cations (16). Trypanosomes carry a subset of soluble (1) with 60 million people at risk of infection (2). Sleeping sick- PPases called vacuolar soluble proteins (VSPs) that localize to ness is caused by two subspecies of T. brucei: T. brucei gam- acidocalcisomes and are known for their roles in regulation of biense and T. brucei rhodesiense. The former alone accounts for acidocalcisome phosphate pool via their PPase and trip- ϳ98% of the cases in humans and livestock (1). T. brucei brucei olyphosphatase activities (17). There is increasing evidence for is another subspecies of T. brucei that is used as an experimen- the functional importance of VSPs for growth and survival of tal model in laboratory to study sleeping sickness, and this par- parasites inside their host (17–19). This has resulted in investi- asite infects animals causing a disease called nagana (3). Left gation of VSP as potential inhibitor target and has also high- untreated, sleeping sickness is fatal, and there are currently two lighted the importance of acidocalcisomal proteins in general drugs used to treat the initial phase of the disease: suramin and (20). A study has shown that small molecule inhibitors of VSP pentamidine. These are employed in treatment of trypanoso- tripolyphosphatase activity can provide protective effects against miasis caused by T. brucei rhodesiense and T. brucei gambiense T. brucei infection in a mouse model (21). However, these inhibi-

infections (4). For the treatment of second or neurological tors suffer from poor IC50 values, and further improvement has phase, an arsenic-based drug called melarsopol is used. How- been suggested for generating more potent compounds. VSPs ever, this drug causes severe side effects and can sometimes be have mostly been explored from cellular and biochemical perspec- tives to date, despite the pivotal role of structure-based drug development in modern infectious disease drug discovery (22). * This work was supported by Outstanding Scientist Research Programme Grants PR6303 (to A. S.) and PR3084 (to A. S. and M. Y.) from the Depart- Here, we present the crystal structure of T. brucei brucei ment of Biotechnology of the Government of India. The authors declare ϩ2 VSP1 (TbbVSP1) at 2.35 Å resolution in complex with Mg that they have no conflicts of interest with the contents of this article. The atomic coordinates and structure factors (code 5C5V) have been deposited in the Protein Data Bank (http://wwpdb.org/). 1 Supported by Indian Council of Medical Research-Senior Research 3 The abbreviations used are: PPase, inorganic pyrophosphatase; VSP, vacuo- Fellowship. lar soluble protein; SAXS, small angle x-ray scattering; TEM, transmission 2 Supported by the J. C. Bose fellowship. To whom correspondence should be electron microscopy; ITC, isothermal calorimetry; rmsd, root mean square

addressed. E-mail: [email protected]. deviation; PDB, Protein Data Bank; polyP3, tripolyphosphate.

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FIGURE 1. Domain organization and crystal structure of TbbVSP1. a, schematic representation of domain structures of VSPs and other class I PPases. The EF hand domain (yellow), PPase domain (blue), and interdomain region (red) are colored; domain boundaries are based on x-ray crystal structures. b, top panel depicts size exclusion chromatography elution profile/peaks of full-length TbbVSP1, monitored by absorbance at 280 nm. Peaks are color-coded according to divalent cations mixed with the protein: red, mixed with Caϩ2; brown, mixed with Mgϩ2; and blue, with EDTA/no divalent cation. Standard molecular mass markers are indicated by green arrows on elution volume axis. The middle panel shows the line curve obtained with protein standard markers, which was fitted to a linear equation for calculating molecular masses (see “Experimental Procedures”). Kav values for full-length TbbVSP1 (red) and its EF hand deletion mutant (blue) are indicated. The bottom panel depicts size exclusion chromatography profile of EF hand deletion TbbVSP1. The deletion mutant is shown by domain schematic labeled with boundaries, and the tetrameric peak is indicated with the molecular masses noted in parentheses. c, ribbon representation of crystal ␤ structure of TbbVSP1 monomer with individual domains colored same as in the schematic. Secondary structures elements are labeled: h for helix and for strand. The lower panel shows conformational difference in the bridge helix, revealed upon superposition of individual subunits in the asymmetric unit. and IDP. Our analyses reveal an unusual tetrameric quaternary Experimental Procedures association of TbbVSP1 when compared with other known class I PPases that adopt dimeric or hexameric states. Our Cloning, Overexpression, and Purification of TbbVSP1—The ORF corresponding to full-length Tb VSP1 containing EF hand TbbVSP1 tetramer structure has been confirmed by SAXS and b EM experiments, which indicate flexibility in the EF hands within domain (residues 1–160) and PPase domain (residues 161– this protein. This is the first study of structural characterization of 414) was optimized for expression in E. coli and cloned into an acidocalcisomal protein from protozoan parasites to our pETM11 vector using NcoI and KpnI restriction sites. Briefly, knowledge, and provides new structural insights into class I bacterial cells were lysed by sonication in a buffer containing PPases. Our work provides a foundation for further functional dis- protease inhibitor mixture. Affinity purification was performed section and pharmacological exploitation of VSPs. on a nickel-nitrilotriacetic acid (His-Trap FF; GE Healthcare)

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column using AKTA-FPLC system (GE Healthcare). The His6 TABLE 1 tag of the protein was removed with TEV protease followed by Data collection and refinement statistics dialysis in low salt buffer (30 mM HEPES, pH 7.2, 25 mM NaCl, The values in parentheses are for the highest resolution shell. Tb VSP1-Mg-PNP and1mM DTT), and it was subsequently applied to Q-Sephar- b ose (GE Healthcare) for further purification and removal of Data collection Space group P42212 TEV protease by anion exchange chromatography. Purest frac- a, b, c (Å) 104.2, 104.2, 215.5 tions were pooled and saturated with ammonium sulfate to a ␣, ␤, ␥ (°) 90, 90, 90 Rmerge(%) 0.07 (0.69) final concentration of 1 M, and further polishing and purifica- I/␴(I) 32.8 (2.71) tion was achieved by hydrophobic interaction chromatography Completeness 98.1 (97.2) on a phenyl FF 16/10 column (GE Healthcare). Finally, purest Redundancy 10.9 (11.0) Ϫ1 Refinement fractions were pooled and concentrated to 10 mg ml with Resolution 39.8–2.35 10-kDa cutoff centrifugal devices (Millipore) followed by gel No. of Reflections 49,471 (3366) R /R (%) 19.9/23.8 permeation chromatography on S200–16/60 column (GE work free No. of atoms M Healthcare) in a buffer containing 30 m Na-HEPES, pH 7.2, 50 Protein 5580 mM NaCl, and 1 mM DTT. Ligand/Ions 20/15 Analytical Size Exclusion Chromatography—Oligomeriza- Water 200 2 tion of Tb VSP1 was examined using analytical size exclusion B-factor (Å ) b Protein 64 chromatography (Superdex 200 HR 10/300; GE Healthcare) in Ligand/Ions 56/59 30 mM HEPES, pH 7.2, 500 mM NaCl, and 5 mM ␤-mercapto- Water 59 ␮ rmsd ethanol. A total of 500 l of full-length TbbVSP1 at concentra- Ϫ1 Bond length (Å) 0.0073 tion of 4 mg ml was applied to the column; separately, 100 ␮l Bond angle (°) 1.061

of N-terminally truncated TbbVSP1 at concentration of 0.5 mg/ml was injected into size exclusion chromatography column. The molecular mass standards used for this study (blue dextran (ϳ2000 kDa), ferritin (ϳ400 kDa), aldolase (158 kDa), conalbumin (74 kDa), ovalbumin (66 kDa), carbonic anhydrase using COOT and phenix.refine respectively. An EF hand (30 kDa), ribonuclease (12 kDa), and apoprotinin (6 kDa) were domain was built manually into electron density maps. Imi- purchased from GE Healthcare. A standard curve was gener- dodiphoshate and coordinating Mgϩ2 ions were added dur-

ated by plotting relative elution volume Kav against log10 molec- ing the manual model building, and ligand density was verified ular mass for each standard marker. The curve was fitted to a using SA-OMIT maps. The overall stereochemical quality of linear equation, and the apparent molecular masses were the built model was assessed using MolProbity (26) and with

deduced from Kav for respective proteins. PDB validation server. All structural figures were generated Crystallization of TbbVSP1—A single peak corresponding with PyMOL and Chimera (27). The coordinates for ϩ2 to tetramer in gel permeation chromatography was collected TbbVSP1.PNP.Mg complex have been deposited with Pro- followed by the addition of 3 mM MgCl2 and 0.5 mM imido- tein Data Bank with accession code 5C5V. diphosphate (Sigma-Aldrich) to protein (concentrated to 30 Small Angle X-ray Scattering Experiments—SAXS experi- mg mlϪ1) solution for co-crystallization. Initial crystallization ments were conducted at the European Synchrotron Radiation trials were performed at 293 K by the hanging drop vapor dif- Facility BioSAXS Beamline BM29 in Grenoble, France (28). fusion method using commercially available crystallization Highly purified protein (Ͼ95% purity) was used, measurements screens. Single cuboid shaped crystals were obtained within 4 for 30 ␮l of protein solution at five different concentrations (8.6, days from MORPHEUS screen (Molecular Dimensions) in a 4.3, 2.1, 1.08, and 0.54 mg mlϪ1) for each sample (and buffer) solution containing 10% PEG 8000, 20% ethylene glycol, 0.03 M were defined using the ISPyB BioSAXS interface (29), and the halide, and 0.1 M bicine/trizma pH 8.3. Further optimization of sequence was triggered via BsxCuBE. Ten individual frames solution conditions to 9% PEG 8000, 18% ethylene glycol, 0.02 M were collected for every exposure, each2sinduration using the halide, 0.1 M bicine/trizma, pH 8.0, and 0. 1 M spermine tetra- Pilatus 1 M detector (Dectris). Individual frames were processed hydrochloride produced diffraction quality crystals. automatically and independently within the EDNA framework, Data Collection, Structure Solution, and Analysis—X-ray yielding individual radially averaged curves of normalized diffraction experiments were conducted at BM14 European intensity versus scattering angle s ϭ 4␲ sin (␪)/␭. The data were Synchrotron Radiation Facility (Grenoble, France), and data processed and merged using PRIMUS (30) and ATSAS package were collected using synchrotron radiation (␭ ϭ 0.953 Å) on a (31), and pair distribution function was computed using MAR CCD225 detector at 100 K. A total of 270 images were GNOM (32). The ab initio models were calculated for each collected and processed to 2.35 Å using HKL2000 (23). The construct using DAMMIF (33) and then averaged, aligned, and

PPase domain of TbbVSP1 shares 65% sequence homology with compared (which showed minimal variation) using yeast PPase, and therefore the structure of TbbVSP1 was solved DAMAVER (34). Rigid body modeling of the complex was com- by molecular replacement using PHASER (24) with yeast PPase plicated by the internal cavities that caused artifacts (PDB code 1M38) as a template. Initially, model was built using in the calculation of theoretical experimental curves. There- AutoBuild in PHENIX (25), which was further subjected to sev- fore, ensemble modeling (35) was used to generate 10,000 mod- eral rounds of manual building and refinement performed els allowing random movements, and the resulting models were

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FIGURE 2. Dimer of dimers assembly of TbbVSP1. a, two views of the dimer in asymmetric unit with 2-fold noncrystallographic (NCS) axis colored green. The PPase domain (blue), EF hand domain (yellow), and interdomain region (red) are highlighted. b, schematic representation of tetrameric TbbVSP1 assembly containing two dimers: A-B and AЈ-BЈ. A-B is highlighted domain-wise as in panel a, and similarly AЈ-BЈ domains (orange), the EF hand domain (cyan), and the interdomain region (pink) are colored. The 2-fold crystallographic symmetry (CS) axis is highlighted in purple. c, multiple molecular surface views of the tetramer. The colors of different domains are same as in Fig. 2b.

screened using Pepsi-SAXS.4 The resulting representative using SPIDER (37) and K-means classification. The average model and ab initio models for each construct selected by classes obtained were compared with the two-dimensional pro-

DAMAVER were overlaid using PyMOL. The fits to the exper- jections of three-dimensional crystal structure of TbbVSP1 fil- imental data of the models and the theoretical scattering of the tered at 30 Å obtained using the PJ 3Q function in SPIDER. structures were prepared with SAXSVIEW. Isothermal Titration Calorimetry—All ITC experiments Electron Microscopy—Small aliquots (3.5 ␮l) of diluted pro- were performed on a GE-Micro Cal ITC 200, and Origin was Ϫ1 tein (0.025 mg ml ) were deposited on carbon-coated electron used for data analysis. Direct titration of EF hand domain of microscope grids and stained with 2% uranyl acetate. Speci- ϩ2 Tb VSP1 with Ca was done in 50 mM Na-HEPES, pH 7.2, and mens were observed using a JEOL JEM2100 LaB6 at a b 50 mM NaCl. Before titration, the protein was made divalent nominal magnification of ϫ39600 with a pixel size of 3.5 Å at cation free and decalcified using sequential treatment of first 10 the given magnification. Pictures were recorded using a GATAN 2K ϫ 2K cameras. A total of 5268 particles were mM EDTA followed by dialysis against decreasing concentra- selected, normalized using EMAN software (36), and classified tions of EGTA (20 to 0.5 mM). Finally, the protein was dialyzed against experimental buffer (30 mM Na-HEPES, pH 7.2, and 100 ϩ2 4 A. Jamwal, A. R. Round, L. Bannwarth, C. V. Bryan, H. Belrhali, M. Yogavel, and mM NaCl) prepared in deionized water. Ca solutions were A. Sharma, unpublished observations. prepared by diluting 1 M CaCl2 stock solution with dialysate/

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FIGURE 3. Intermolecular interactions within TbbVSP1 tetramer subunits. a, schematic of the tetramer of TbbVSP1 showing interfaces in the crystal lattice. Circles showing interface regions are color-coded: blue as hydrophilic interface and green as hydrophobic interface. b–e, view of intersubunit contacts with important residues shown as sticks. Interactions are shown as dotted lines, and residues in salt bridges are marked with asterisks. The stacking/␲ ...␲ edge to face, face to face interactions and N-H...␲ interactions were calculated/measured from centroids of aromatic rings that are shown as green spheres.

experimental buffer. A typical titration was carried over 40 and domain analysis shows that TbbVSP1 belongs to class I injections with initial injection of 0.4 ␮l followed by 39 injec- PPases with best identity with animal/fungal PPases in the ϩ2 tions of 1 ␮l of 0.2 mM Ca into protein solution (50 ␮M) with C-terminal region, but it also contains an EF hand domain in 150-s intervals between injections and 750 rpm stirring speed. the N-terminal region (Fig. 1a). In particular, this EF hand Independent titrations were performed at 25 and 30 °C. All domain is present in all VSPs of kinetoplastida parasites (Fig. titrations were done twice, and the values are reported in 1a). TbbVSP1 ORF encodes a protein of 414 amino acids with Table 3. relative molecular mass of 47.2 kDa. Nevertheless, native size Results analysis by gel permeation chromatography indicated that both full-length and N-terminal truncated TbbVSP1 (residues 160– Domain Analysis and Characterization of TbbVSP1—The T. brucei brucei genome contains two isoforms of VSPs present 414; PPase domain) predominantly elute as tetramers with molecular masses of 183 and 138 kDa respectively, suggesting on chromosome XI: TbbVSP1 (TriTrypDB ID Tb927.11.7060, that PPase domain is sufficient for tetramer formation in solu- characterized in this study) and TbbVSP2 (TriTrypDB ID Tb927.11.7080). Both forms are nearly identical and diverge tion (Fig. 1b). This is in contrast to animal/fungal PPases that only in three amino acids (F32V, S32L, and L33H). Sequence associate as dimers (38). Because of the putative EF hand

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FIGURE 4. SAXS analysis of TbbVSP1 solution structure indicates flexibility. a, Guinier plot from raw data recorded at a range of protein concentrations. b, Individual domains of homodimer subunits in the asymmetric unit are superimposed. EF hand domains show more conformational variation relative to the PPase domains. c, SAXS curve showing logarithm of scattered intensity plotted as a function of momentum transfer, s ϭ 4␲ sin (␪) ␭Ϫ1, where ␪ is the scattering ␭ angle, and is the x-ray wavelength. d, two views of TbbVSP1 model (PPase domains (blue sticks) and EF hand domain (orange sticks)) in solution by SAXS overlaid on ab initio low resolution envelope colored in green. e, left panel shows temperature factor variation in TbbVSP1 crystal structure colored from low to high values (40–105 Å2) where arrows indicate the bridge helix. The right panel shows sequence conservation in the bridge helix region where residue positions with highest variability are boxed.

domain in the N-terminal region of the protein, we tested effect traceable in the model because of poor or no electron density. of divalent cations on protein oligomerization state. Gel perme- The final refinement and statistical parameters are stereo- ϩ2 ation chromatography data indicated that addition of Ca chemically sound (Table 1). The crystal structure of TbbVSP1 and Mgϩ2 had no influence on oligomeric stoichiometry of reveals bidomain architecture (Fig. 1c). A DALI search against

TbbVSP1 (Fig. 1b, top panel). Thus, collectively these data sug- PDB using the complete structure of TbbVSP1 failed to identify

gested that TbbVSP1 possesses an unusual and specific struc- any structure with significant similarities over entire polypep- tural organization, which is different from conventional class I tide, suggesting a unique overall architecture. However, PPases. searches using individual domains revealed closest structural Crystal Structure of Tb VSP1—The Tb VSP1 crystallized in homolog of the N-terminal domain (residues 72–142) as EF b b ϭ tetragonal space group (P42212), and its structure was solved hand calcium binding protein (PDB code 2BE4; Z 8.2), and using molecular replacement method with yeast PPase as tem- for C-terminal domain (residues 167–414) the yeast PPase plate (PDB code 1M38). Electron density for most of the resi- (PDB code 1E6A; Z ϭ 33.3). Therefore, hereon we refer to the dues was generally good; however, residues 1–71 were not N- and C-terminal domains as EF hand and PPase domains

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FIGURE 5. TEM studies. a, typical micrograph as observed in TEM (negative staining) with nominal magnification of 39,600ϫ where the scale bar represents 50 nm. b, class averages of TbbVSP1. c, corresponding 2D projections. d, volume view of the three-dimensional crystal structure filtered at 30 Â. The box size is 180 Å. e, molecular surface view with the coloring scheme as in Fig. 2c.

respectively. The EF hand domain has a compact globular fold entation shift is observed at bridge helix (Fig. 1c, bottom panel), and consists of four ␣-helices, h1–h4 (residues 75–87, 98–108, which suggests flexibility in this region.

118–125, and 133–142) (Fig. 1c). The PPase domain consists of Quaternary Structure of TbbVSP1—The protomers in asym- a five-stranded ␤ barrel made from strands ␤4 and ␤7–␤10 in metric unit make contact via their PPase domain and extend EF the core region, and in addition a ␤ sheet is formed away from hand domain in opposite directions, thus presenting an inward the core by strands ␤1–␤3. The ␤-barrel is flanked by two long facing concave surface (Fig. 2a). The tetramer can be generated ␣ -helices, h8 and h11, three short 310 helices (residues 341–343 by application of 2-fold crystallographic operator resulting in a (h6), 370–372 (h9), and 380–382 (h10)) and helix h7, which is a dimer of dimers assembly (Fig. 2b). The assembly contains two ␣ Ј Ј combination of a 310 helix and helix. The h7 lies at the base of the dimers, AB and A B , where each uses its concave surface to ␤-barrel. The EF hand and PPase domain are bridged via a 24-res- intimately lock with another to form a tetramer of 84 ϫ 72 ϫ idue interdomain region (residue 145–165). The interdomain 120 Å dimensions. The central core of the tetramer contains region is composed of a helix (“bridge helix,” residues 145–159) PPase domains, whereas EF hand domains jut out in the oligo- present at the base of the EF hand domain followed by a small meric assembly (Fig. 2c). This assembly is stabilized by four unstructured region (residues 160–165) that leads into PPase unique intersubunit interfaces (interface I–IV). Interface I 2 2 domain (Fig. 1c). Overall folds of two TbbVSP1 protomers in the (1840 Å ) and II (700 Å ) are hydrophilic, whereas interface III asymmetric unit are similar. However, an approximately 12° ori- (825 Å2) and IV (300 Å2) are hydrophobic in nature (Fig. 3a). As

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FIGURE 6. Electrostatic surface potential. a, two views of TbbVSP1 tetramer surface potential where blue indicates positive and red indicates negative charge foci, displaying range of Ϯ 12 kT/e. b, zoomed view of basic pocket enveloped in yellow with underlying residues labeled. c, an alignment showing region of VSP sequences from T. brucei brucei (TbbVSP1), T. brucei gambiense (TbgVSP1), T. evansi (TeVSP), T. cruzi (TcVSP), and Leishmania major (LmVSP) containing conserved residues (blue) of the basic pocket; arrows indicate their position in the amino acid sequence.

indicated by buried surface areas, interface I is much more is another specific feature of TbbVSP1. Thus, these interactions extensive than other interfaces and involves interaction demonstrate extensive connections between the monomers of between residues from PPase domain and interdomain region the assembly. In line with above, the theoretical free energy of monomers A-AЈ or B-BЈ (Fig. 3b). Interface II is majorly value estimated for the assembly by PISA (⌬Gdiss ϭ 26.5 kcal formed by contacts between EF hand and PPase domains of two molϪ1) identified the tetramer as stable. This oligomer is fur- protomers (Fig. 3c). Crystal structure showed that His92 of EF ther confirmed by SAXS and EM (see below) data. 396 hand domain forms a salt bridge with Asp present on h11 of SAXS Reveals Conformational Flexibility of TbbVSP1—To PPase domain, and this could be an important contributing study solution structure of TbbVSP1, we conducted SAXS stud- factor to stability of this interface. In addition, the bridge helix ies with highly pure full-length protein. The Guinier plot of 158 also makes contribution to interface II stability via Asp , TbbVSP1 exhibits good linearity (Fig. 4a), suggesting the pro- which makes a salt bridge with Arg410 of h11 (Fig. 3c). Interface tein solution was free of large aggregates or higher molecular III involves interaction between monomer A-B or AЈ-BЈ, which mass contaminants. The molecular masses estimated from is braced by hydrophobic stacking interactions between Trp233 experimental volume and ab initio envelopes are 150–200 and and Trp268 (Fig. 3d) and further stabilized by hydrogen bonding 203 kDa respectively, which correlate well with values expected andN-H... ␲ between Arg235 and His263 residues. Further, for a tetrameric species. However, the experimental volume the aromatic ring stacking interactions between Phe343 and estimated from ab initio modeling (302 Ϯ 50 nm3) does not Phe353 are observed at interface IV (Fig. 3e). Phe343 and Phe353 correlate with volume from tetramer crystal structure (248 are highly conserved among VSPs, suggesting that interface IV nm3). This increase in volume is likely to be a result of flexibility

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FIGURE 7. Substrate specificity and pyrophosphate binding pocket of TbbVSP1. a, Michaelis-Menten plot of initial velocity versus substrate concentration ⌬ 2 for TbbVSP1 (left panel) and 166 TbbVSP1 (right panel). Km and kcat values are indicated along with adjusted regression co-efficient (R ) for the curves. b, optimum pH measurement with polyP . The curves show variation in tripolyphosphatase activity of Tb VSP1 over a pH range of 5.2–9.0 and in the presence of ϩ3 ϩ ϩ b different transition metals (1 mM of Zn 2,Co 2, and Mn 2). A buffer with 30 mM sodium citrate tri-basic dihydrate (pH 5.2–5.8) and 30 mM Bis-Tris propane (pH ϩ2 6.0–9.0) was used in these experiments. c, inhibition of PPi hydrolysis by Ca . PPase activity was determined with increasing concentrations of CaCl2 with ␮ ϩ2 Ϫ substrate (PPi) concentration near Km value (25 M) and co-factor (3 mM of Mg ). d, stereo view of Fo Fc simulated annealing OMIT electron density map contoured at 6␴ showing IDP (blue mesh) and Mg2ϩ (orange mesh) in the active site. e, important interactions between IDP, Mgϩ2 ions, and protein side chains are shown as dashed lines. Dashed lines with shorter and longer width depict metal coordination and hydrogen bonding, respectively. Protein side chains and IDP are shown as sticks, whereas Mgϩ2 ions (chartreuse) and water (pale green) are shown as spheres.

in TbbVSP1 structure in solution state. We further noticed dif- gate the SAXS curves at molecular level, we probed the tetra- Ϯ ference in Dmax between SAXS (157 3 Å) and the crystal meric crystal structure model, but it did not fit with experimen- structure (120 Å), suggesting again that the crystal structure tal SAXS curves (the associated ␹2 were Ͼ10). Consistent with described a compact conformation for this protein. To investi- this observation, the overlay of tetrameric crystal structure

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form produced poor fits with the SAXS envelope. We further TABLE 2 noticed that PPase domains docked nicely into the core region Kinetic parameters of polyP3 hydrolysis in the presence of different of the envelope but the peripheral EF hand domains fit poorly transition metal co-factors Cofactor Concentration pH K k k /K and in fact protruded outside the envelope. Considering the m cat cat m Ϫ1 Ϫ1 Ϫ1 mM ␮M s M s increase in size observed by SAXS, we attributed this disagree- Znϩ2 0.5 6.2 44.8 Ϯ 6.7 15.8 Ϯ 3.7 3.69 ϫ 105 ment to absence of flexibility in our atomic model based on Coϩ2 1.0 7.1 96.8 Ϯ 11.2 7.3 Ϯ 1.2 0.75 ϫ 105 ϩ2 Ϯ Ϯ ϫ 4 crystal structure data. Mn 1.0 7.1 142.3 24.7 1.3 0.9 0.91 10 In line with the above, alignment of homodimer subunits in the asymmetric unit identified a significant difference in the consistent with the three-dimensional crystal structure pre- positioning of EF hand domains (rmsd 0.75 Å for 70 C␣ atoms) sented here (Fig. 5, d and e). and bridge helices (rmsd 1.3 Å, for 18 C␣ atoms; Figs. 4b and 1c, Electrostatic Potential—The molecular surface of TbbVSP1 bottom panel). However, PPase domains did not display signif- tetramer displays a bipolar distribution of electrostatic poten- icant deviation (rmsd 0.12 Å for 247 C␣ atoms; Fig. 4b). These tial. A highly negatively charged pocket concentrated on the observations collectively suggest inherent flexibility in EF hand distal face of the tetramer whereas positively charged pockets domain and bridge helix with respect to the PPase domain are formed near the tetramer inner core, toeing the tetramer backbone. Therefore, to assess the likelihood that crystal pack- midline (Fig. 6a). The negatively charged pocket consists of ing may stabilize a more flexible structure, we deployed an clusters of multiple aspartate residues (Asp291, Asp296, Asp323, ensemble modeling (EOM) approach to predict the likely flex- and Asp328) and one glutamate residue (Glu239). This charged ible movements and multiple conformations in solution by congregation in appropriate proximity suggests a metal binding allowing hinge motions in EF hand domain. We also tested the site. Structural superposition with other known PPases shows bridge helix as a potential source of hinge motion, which could high degree of structural conservation in the arrangement and act as pivot between EF hand domain and PPase domain. position of these residues, all of which have been shown in pre- Indeed, we found that the hinge motion produces better fit to vious studies to coordinate metal ions (38). In contrast, the 2 the SAXS data (␹ ϭ 2.22) (Fig. 4c). Overlay of model into ab positive patch results from tetramerization and causes arrange- initio envelope indicates that EF hand domains bend toward ment of basic residues from discontinuous regions of the PPase each other about the PPase domains, which hold together the domain (Fig. 6b). The underlying subset of residues of this tetramer core preventing it dissociating into lower oligomeric pocket are Lys215, Arg223, Gln309, Lys314, Arg342, Lys345, and state (Fig. 4d). Although a single atomic model suffices to Lys388 of PPase domain that are present on both faces of the explain the experimental SAXS curve, this atomic state may tetramer (Fig. 6b). Lys215, Arg223, Gln309, and Arg342 are buried, represent a true dominant conformation in solution or an aver- whereas Lys314, Lys345, and Lys388 are accessible. All these res- age of an ensemble spanning a subset of conformational spaces. idues show high conservation in VSP homologs from trypano- Either of the above interpretation indicates structural mallea- some family (Fig. 6c). bility of TbbVSP1 in solution in the absence of external forces PPase Activity and Pyrophosphate Binding Site—Studies on such as those exerted by crystal packing. T. brucei gambiense have shown that VSP1 (TbgVSP1) is an Finally, crystal structure derived B-factors display a gradient active PPase (17). Also, the TbbVSP1 PPase domain showed along the body of bridge helix ranging from low (proximal to high structural similarity to yeast PPase. Therefore, we exam-

PPase domain) to high (near the base of EF hand domain, con- ined PPase activity of TbbVSP1 using malachite green assay tributed by Val145, His146 and Ser147). In the tetrameric crystal developed for phosphate estimation in solution (39). At a pro- structure, these residues have limited contacts with other tein tetramer concentration of 0.15 nM, the PPi hydrolysis rate Ϫ1 regions and are least conserved among VSPs. These two attri- had a turnover number (kcat) of 294 s at 37 °C (Fig. 7a, left butes indicate a dynamic tether or flexibility in the hinge region panel). We found that the protein actively hydrolyzes PPi in (Fig. 4e), consistent with the discussion so far. presence of magnesium in an alkaline pH range (7.8–8.5) with ϫ 7 Ϫ1 Ϫ1 TEM Studies—For direct visualization of tetramer complex, kcat/Km of 1.475 10 M s , suggesting that activity is phys- we performed negative staining TEM to produce two-dimen- iological. The main roles attributed to EF hand domain are the sional projections of single particles using highly pure (Ͼ95%) regulation of cellular activity and/or buffering/transporting cal-

TbbVSP1. The gel filtration profile of material used for EM was cium (40). To investigate whether EF hand domain has any role the equivalent tetramer fraction that was used in crystallization in PPi hydrolysis, we also used N-terminal EF hand domain ⌬ and SAXS analyses. For TEM, 80 micrographs of the sample truncated enzyme ( 166 TbbVSP1) that contains the PPase were taken, and 5268 particles were selected with a typical domain only. The resulting kinetic constants for PPi hydrolysis micrograph presented (Fig. 5a). The particles were classified as were similar to full-length protein, suggesting that EF hand was described under “Experimental Procedures,” along with repre- not essential for PPi hydrolysis (Fig. 7a, right panel). sentative classes (Fig. 5b). Different classes were compared with It has been suggested that substitution of Mgϩ2 by transition the two-dimensional projections performed from three-di- metal ions results in efficient hydrolysis of tripolyphosphate mensional crystal structure (Fig. 5, b and c), and a good corre- (polyP3) by class I PPases (41); indeed, we did observe this effect lation between classes and two-dimensional projections was for TbbVSP1 (Table 2). Again, this activity is independent of the observed. Finally, we studied the corresponding three-dimen- EF hand domain. In contrast to PPi hydrolysis, the optimum pH ϩ2 sional view of TbbVSP1 to confirm the orientation of the parti- for poly P3 hydrolysis shifted to a neutral pH of 7.1, when Co cles. Thus, particles observed in TEM by negative staining are and Mnϩ2 were co-factors (Fig. 7b). Interestingly, the same

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FIGURE 8. Calcium binding by EF hand domain and interhelical angles. a, representative ITC titrations. The upper panel shows heat flows observed during the experiment; the lower panel shows integrated heats of each Caϩ2 injection; lines show fit of data to the binding model describing ligand interaction with ϩ ϩ a single binding site. Panel i, buffer Ca 2 titrations showing heat of dilutions. Panels ii and iii, EF hand domain Ca 2 titration in 50 mM Na-HEPES and 100 mM NaCl, pH 7.2, at 25 °C (panel ii) and 30 °C (panel iii). b, structural superposition of EF hand domain and calmodulin showing differences in interhelical packing between helices h1 and h2 (left panel) and helices h3 and h4 (right). Left panel shows helix h1 of TbbVSP1 (yellow) and calcium bound calmodulin (orange) are bent by 18° and 42°, respectively, relative to closed state (pink). Whereas, the right panel shows helices h3 are bent by 14° and 40° relative to closed state. PDB codes of calcium bound and unbound forms calmodulin are mentioned in parentheses. Interhelical angles (bottom) are shown in a tabular form.

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TABLE 3 of helices shows that the EF hand domain is in a partially open ؉ Thermodynamic parameters of EF hand-Ca 2 interactions state, deviating from geometry proposed for a closed state (Ref. ⌬ ⌬ Temperature NKa Kd H S 43 and Fig. 8b, bottom panel). Finally, the loop connecting 7 Ϫ1 ϩ2 10 M nM kcal/mol cal/mol °C helices h3 and h4 shows poor overlap with either Ca -bound 25 °C 1.07 Ϯ 0.002 1.55 Ϯ 0.18 64.5 Ϯ 5.5 Ϫ5.25 Ϯ 0.21 14.3 30 °C 1.06 Ϯ 0.005 1.43 Ϯ 0.13 69.9 Ϯ 7.7 Ϫ5.36 Ϯ 0.56 11.9 or unbound forms (Fig. 8b, right panel). Comparison TbbVSP1 Structure with Other PPases—A sequence-based phylogenetic tree construction supports the ϩ2 activity was discernable at acidic pH of 6.2 in presence of Zn notion that TbbVSP1 is a class I PPase because it clusters with (Fig. 7b), and this property of TbbVSP1 is in line with previous animal/fungal class I PPases. However, the branch length of findings on VSP1 from T. brucei gambiense and Leishmania animal/fungal PPase clade tends to be longer than acidocalci- amazonesis (17–18). The kcat values suggest that overall effi- somal VSPs, suggesting a more rapid rate of evolution (Fig. 9a). ciency of polyP3 hydrolysis by TbbVSP1 in presence of transi- A survey of crystal structures of class I PPases in the PDB ϩ2 Ͼ ϩ2 Ͼ ϩ2 tion metal follows the order Zn Co Mn (Table 2). reveals that TbbVSP1 architecture presented in this study rep- Taking these results together, our data indicate that recombi- resents an atypical class I PPase, not only in terms of its domains nant TbbVSP1 hydrolyzes inorganic phosphates over a wide pH but also its oligomer state (Fig. 9a). The PPase domain structure range of 6.2–8.5. of TbbVSP1 is similar to yeast PPase (rmsd 0.6 Å for 202 C␣ We were successful in co-crystallizing TbbVSP1 with mag- atoms). A notable difference is the presence of a longer loop in nesium and IDP (a slow turnover analog of PPi). Omit maps TbbVSP1, which results from insertion of 11 amino acids (res- revealed electron density for IDP and Mg2ϩ ions at the active idues 208–218). This loop contributes to tetramer stability by site of PPase domain (Fig. 7d). The complex comprises of four burying 400 Å2 of accessible area at interface I. Another notable 2ϩ Mg ions (M1, M2, M3, and M4) and one IDP molecule, which difference is an auxiliary space proximal to PPi binding, which is contain two phosphate groups P1 and P2 (Fig. 7e). The active absent in yeast PPase. We find that this structural difference ␤ 374 site consists of residues from loops and -strands that are translates to residue Gly of TbbVSP1 PPase domain. The highly conserved in PPases. In the complex, phosphate group corresponding structurally equivalent residue in yeast PPase is P1 forms hydrogen bonding and ionic interactions with Arg259 Lys198, which points toward the active site and thus occludes 368 369 374 (bidentate interaction), Tyr and Lys , whereas P2 forms the space formation near the PPi binding site. Again, Gly is hydrogen bonding interactions with Lys237 and Tyr270 of the highly conserved among kinetoplastid VSPs. However, the active site (Fig. 7e). M1 and M2 are coordinated exclusively by most striking difference is the absence of C-terminal extension 291 296 protein side chain carboxylates: M1 with Asp , Asp , and from TbbVSP1 PPase domain (Fig. 9c). This C-terminal exten- Asp328 and M2 with Asp296 (Fig. 7e). M3 and M4 are coordi- sion is also absent from bacterial and plant PPases (Fig. 9c). nated with phosphate groups of IDP and side chain carboxy- Superposition of yeast PPase dimer as a whole to dimer com- 239 323 328 lates: M3 with Glu and M4 with Asp and Asp (Fig. 7e). ponent of TbbVSP1 (PPase domains only) gives a very high Coordination geometry for all Mgϩ2 ions in the active is nearly rmsd value of ϳ4 Å, indicating that spatial arrangement of

octahedral, which we validated using the Check My Metal tool monomers forming the primary dimer interface in TbbVSP1 is (42). different from yeast PPase (Fig. 9d). This difference can also be Caϩ2 Binding Affinity of EF Hand Domain—We investigated judged by examining the orientation of catalytic center in each ϩ2 whether EF hand domain is a functional Ca binder. We monomer of yeast and TbbVSP1; in the former, they are ϩ2 deployed ITC to determine Ca binding and measure binding arranged on opposite faces, whereas in TbbVSP1, they consort affinities along with thermodynamic parameters. Because in a sideways manner (Fig. 9d). Overlay of aforementioned PPase domains have divalent cation binding capabilities, we dimers also reveals a notable difference in their packing angles,

generated a protein encompassing 1–160 residues that contains which entails a rotational shift of 20° in TbbVSP1 monomer the EF hand domain. Initial titration of Caϩ2 in HEPES buffer with respect to yeast PPase monomer; this rotational shift is against 50 ␮M protein showed a sharp transition in ITC curves, accompanied by translation of 101 Å. The dimer interface of yeast indicating high affinity interaction. The results presented in PPase (total buried area, ϳ1920 Å2) is also more substantial than ϳ 2 Fig. 8a show good fit to a simple one-site model, describing TbbVSP1 ( 1650 Å ). This may be explained by the fact that specific binding driven by both favorable enthalpy and entropy C-terminal extension of yeast PPase contributes significantly changes. As in Table 3, binding affinity of EF hand domain is in (ϳ43% total buried surface) to dimerization interface of yeast ϭϳ ° nanomolar range (Kd 65 nM at 25 C), and this affinity PPase. Further, theoretical free energy of dissociation from PISA decreases slightly upon increase in temperature, which is again indicates that C-terminal is important for stability of the yeast accompanied by favorable changes in heat and entropy. Note PPase dimer. Therefore, C-terminal extensions of yeast/animal that the structure of EF hand domain presented in this work is PPases might act as clamps holding monomers, and their absence ϩ2 in Ca -free form. Comparison of EF hand domain with an in TbbVSP1 might increase the fluidity of dimer interface region, archetypal Caϩ2 binding protein calmodulin (PDB ID 1EXR; resulting in different spatial arrangement. Caϩ2-bound form and 1CFD in unbound form) revealed that the putative Caϩ2 binding loop which connects the helices h1 Discussion

and h2 in EF hand domain adopts a conformation similar to TbbVSP1 has a bidomain architecture consisting of the EF Caϩ2-bound form of calmodulin (Fig. 8b, left panel). Further, hand domain and the PPase domain. Although most known measurement of interhelical angles and angular displacement class I PPases are single domain proteins that form hexameric/

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Leishmania major (cytosolic) 0.3 96 Trypanosoma brucei (cytosolic) 0.2 0.2 Trypanosoma cruzi (cytosolic) 0.2 69 Trypanosoma brucei brucei (acidocalcisomal) 100 Trypanosoma cruzi (acidocalcisomal) 0.2 Leishmania major (acidocalcisomal) 0.1 98 Komagataella pastoris 82 83 Debaryomyces hansenii 90 0.2 Kluyveromyces marxianus 99 Zygosaccharomyces bailii 0.2 96 Rhizopus microsporus 0.3 Caenorhabditis elegans 69 0.2 Xenopus laevis 96 Homo sapiens 52 0.1 99 Rattus norvegicus 97 0.2 Mus musculus 0.2 99 Pyrococcus horikoshii 0.3 Thermococcus thioreducens 54 Mycobacterium tuberculosis 0.2 0.5 Helicobacter pylori 67 61 0.4 Ricktessia prowazekii 0.2 0.2 97 0.3 59 Enterobacter aerogenes 36 0.2 100 100 Salmonella bongori 0.3 1.0 Escherichia coli Thermus thermophilus 0.5 48 Glycine max 96 Arabidopsis thaliana 98 0.1 Zea mays 0.3 Chlamydomonas reinhardtii 0.2

0.2

FIGURE 9. Comparison of crystal structures/biological assemblies and phylogeny. a, maximum likelihood tree based on protein sequences of soluble PPase domains. Boot strap values from 500 iterations are shown in red font, and branch lengths are shown in black font. The 0.2 bar represents amino acid substitution per site. Putative cytosolic PPases of kinetoplastid are used as the out group. b, biological assemblies of TbbVSP1 and PPases from E. coli and S. cerevisiae. c, schematic shows the C-terminal extension present in animal/fungal PPases. Residues that contribute to dimer interface of yeast PPase are conserved and highlighted in purple/magenta in the sequence alignment. d, ribbon cartoon of PPase domain of yeast and TbbVSP1 showing different spatial arrangement of monomers, which is also indicated by topology of active site in each dimer. The C-terminal extension of ScPPase colored in magenta.

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dimeric assemblies, TbbVSP1 is distinct in being tetrameric. binding site (in the PPase domain). This hence explains the Aside from TbbVSP1 crystal structure, SAXS and EM confirm inertness of the EF hand domain toward TbbVSP1 enzyme the unprecedented tetrameric assembly of TbbVSP1. Although activity, and this explanation may also hold true for TbgVSP1, crystal structure analysis indicates a static tetrameric arrange- although spatial arrangement of EF hand and PPase domains in ment, our SAXS data hint at a rather dynamic assembly in solu- this assembly will vary, because the protein is reportedly hexa- tion. Together, the SAXS modeling and crystal structure sug- meric (17). gest that the flexibility manifests itself via hinge motion in EF A comparison between class I eukaryotic (animal/fungal)

hand domains, although the functional implication of flexibility PPase family and TbbVSP1 suggests an intriguing evolutionary remains unclear. This is also a nice example where “in solution” variety from dimeric to tetrameric states. On the basis of struc- and “in crystallo” structural techniques together are more tural and phylogenetic data, a possible scenario emerges in informative than either alone. which C-terminal extension has been acquired by animal/fun- Although structurally intriguing, biochemically the gal PPases, independently after divergence from a protein that

TbbVSP1 is similar to TbgVSP1. Both substrate specificity pro- formed the last common ancestor between animal/fungal and file and active site structure strongly indicate that PPi is a phys- VSPs. From atomic resolution crystal structures of yeast PPase iological substrate for TbbVSP1; however, PPase activity is opti- and TbbVSP1, it is apparent that the C-terminal extension mal at alkaline pH range only, indicating that PPi may not holds PPase domains like a clamp, thus resulting in a stable hydrolyzed constitutively by TbbVSP1 in acidocalcisomes. dimeric state. From the structural data so far, it seems that However, depending on identity of the co-factor, catalytic capa- prokaryotic/archaeal and plant class I PPases also lack this typ- bilities of TbbVSP1 expand beyond PPi, resulting in hydrolysis ical C-terminal extension and interestingly show higher or non of poyP3 over a range of pH, both acidic and neutral. This sug- dimeric oligomerization state (trimers and hexamers). It might gests a regulatory role for TbbVSP1 in acidocalcisome, which is also be intriguing to suggest that the absence of such an exten- rich in polyphosphates. In this context, Lemercier et al. (17) sion in TbbVSP1 leads to a different packing arrangement of have previously suggested that to prevent polyP3 accumulation PPase domains in primary dimers, which is amenable to in the cell compartment where polyP hydrolysis is occurring, tetramer formation. Indeed, studies in past have shown that ϩ2 polyP3 is further hydrolyzed by VSP1 in the presence of Zn at loss or addition of certain sequences or structural elements can an acidic pH. That followed by an increase in pH and release of result in spatial reorientation of subunits that can induce dif- Caϩ2,Hϩ ions by VSP1 and exopolyphosphatase could drive ferent oligomeric states (47, 48). the reaction forward producing Pi and PPi within acidcocalci- In conclusion, our study presents an unprecedented struc- some (17). Furthermore, acidocalcisomes seem active in several tural assembly of the EF hand and the PPase domain within one

biological processes after alkalization, which also involves hy- TbbVSP1. This work provides a structural foundation both for drolysis of polyphosphates to short chain phosphates and pyro- mechanistic exploration of TbbVSP1 and for scoring feasibility phosphates (44, 45). This may well provide the basis for biolog- of small molecule inhibitor development in future.

ical activity of TbbVSP1. Our ITC data show that the EF hand domain of TbbVSP1 is a Author Contributions—A. S. and A. J. conceived the study. A. J. per- ϩ2 functional and specific Ca ion binder. Surprisingly, TbbVSP1 formed purification of the enzyme, biochemical assays, ITC, deter- structure reveals a conformation similar to an open or Caϩ2- mined and analyzed the x-ray structure. M. Y. and H. B. collected bound state, despite absence of metal bound in the putative x-ray data. A. R. R. performed and analyzed SAXS experiments. L. B. and C. V.-B. conducted TEM studies. The manuscript was written binding site (Fig. 8b). Inspection of the TbbVSP1 tetramer structure reveals that conformation of calcium binding loops is primarily by A. S. and A. J., but all authors contributed. stabilized by electrostatic and hydrogen bonding interactions with a symmetry-related molecule (Interface II; Fig. 3c). Thus, Acknowledgment—We express deep gratitude to V. K. Aves for con- crystal forces might partially mimic a Caϩ2 atom, stabilizing an stant encouragement. apparent “open” conformation. However, this remains to be

verified using crystals in which TbbVSP1 is packed in alternate References forms. 1. Brun, R., Blum, J., Chappuis, F., and Burri, C. (2010) Human African ϩ2 We also observed that Ca is able to inhibit both full-length trypanosomiasis. Lancet 375, 148–159 ⌬ ϭϳ ␮ TbbVSP1 and 166 TbbVSP1 alike (IC50 50 M in presence 2. Hotez, P. J., Molyneux, D. H., Fenwick, A., Kumaresan, J., Sachs, S. E., ϩ2 ϩ2 3mM Mg ) (Fig. 7c), suggesting that EF hand binding to Ca Sachs, J. D., and Savioli, L. (2007) Control of neglected tropical diseases. New Engl. J. Med. 357, 1018–1027 does not play a role in inhibition of PPi hydrolysis; this was also ϩ2 3. Keita, M., Bouteille, B., Enanga, B., Vallat, J. M., and Dumas, M. (1997) observed previously for TbgVSP1 (17). Instead, the Ca -PPi Trypanosoma brucei brucei: a long-term model of human African ϩ2 complex acts a competitive inhibitor of Mg -PPi complex, trypanosomiasis in mice, meningo-encephalitis, astrocytosis, and neuro- inhibiting PPase activity by a mechanism suggested previously logical disorders. Exp. Parasitol. 85, 183–192 (46). Furthermore, EF hand domain deletion also has no effect 4. Kirchhoff, L. V., Bacchi, C. J., Wittner, M., and Tanowitz, H. B. (2000) African trypanosomiasis (sleeping sickness). Curr. Treat. Options Infect. on either PPi or polyP3 hydrolysis by TbbVSP1. In this context, the crystal structure of Tb VSP1 presented in this study indi- Dis. 2, 66–69 b 5. Kennedy, P. G. (2004) Human African trypanosomiasis of the CNS: cur- cates that relative positions of EF hand domain in TbbVSP1 rent issues and challenges. J. Clin. Invest. 113, 496–504 monomer, dimeric, or tetrameric states disallow role for EF 6. Docampo, R., and Moreno, S. N. (2003) Current chemotherapy of human hand in direct interactions with substrate or co-factor metal African trypanosomiasis. Parasitol. Res. 90, S10–S13

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