Structural and Functional Highlights of Vacuolar Soluble Protein 1 from Pathogen Trypanosoma brucei brucei

Abstract

Trypanosoma brucei (T. brucei) is responsible for the fatal human disease called African trypanosomiasis, or sleeping sickness. The causative parasite, Trypanosoma, encodes soluble versions of inorganic pyrophosphatases (PPase), also called vacuolar soluble proteins (VSPs), which are localized to its acidocalcisomes. The latter are acidic membrane-enclosed organelles rich in polyphosphate chains and divalent cations whose significance in these parasites remains unclear. We here report the crystal structure of T. brucei brucei acidocalcisomal PPases in a ternary complex with Mg(2+) and imidodiphosphate. The crystal structure reveals a novel structural architecture distinct from known class I PPases in its tetrameric oligomeric state in which a fused EF hand domain arranges around the catalytic PPase domain. This unprecedented assembly evident from TbbVSP1 crystal structure is further confirmed by SAXS and TEM data. SAXS data suggest structural flexibility in EF hand domains indicative of conformational plasticity within TbbVSP1.

Keywords: Trypanosoma brucei; crystal structure; electron microscopy (EM); isothermal titration calorimetry (ITC); small-angle x-ray scattering (SAXS).

FIGURE 1.

Domain organization and crystal structure of Tb_bVSP1. 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 Tb_bVSP1, monitored by absorbance at 280 nm. Peaks are color-coded according to divalent cations mixed with the protein: red, mixed with Ca⁺²; brown, mixed with Mg⁺²; 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”). K_av values for full-length Tb_bVSP1 (red) and its EF hand deletion mutant (blue) are indicated. The bottom panel depicts size exclusion chromatography profile of EF hand deletion Tb_bVSP1. 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 Tb_bVSP1 monomer with individual domains colored same as in the schematic. Secondary structures elements are labeled: h for helix and β for strand. The lower panel shows conformational difference in the bridge helix, revealed upon superposition of individual subunits in the asymmetric unit.

FIGURE 2.

Dimer of dimers assembly of Tb_bVSP1. 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 Tb_bVSP1 assembly containing two dimers: A-B and A′-B′. A-B is highlighted domain-wise as in panel a, and similarly A′-B′ 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.

FIGURE 3.

Intermolecular interactions within Tb_bVSP1 tetramer subunits. a, schematic of the tetramer of Tb_bVSP1 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/π … π edge to face, face to face interactions and N-H … π interactions were calculated/measured from centroids of aromatic rings that are shown as green spheres.

FIGURE 4.

SAXS analysis of Tb_bVSP1 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 = 4π sin (θ) λ⁻¹, where θ is the scattering angle, and λ is the x-ray wavelength. d, two views of Tb_bVSP1 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 Tb_bVSP1 crystal structure colored from low to high values (40–105 Ų) 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.

FIGURE 5.

TEM studies. a, typical micrograph as observed in TEM (negative staining) with nominal magnification of 39,600× where the scale bar represents 50 nm. b, class averages of Tb_bVSP1. 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.

FIGURE 6.

Electrostatic surface potential. a, two views of Tb_bVSP1 tetramer surface potential where blue indicates positive and red indicates negative charge foci, displaying range of ± 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 (Tb_bVSP1), T. brucei gambiense (Tb_gVSP1), 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.

FIGURE 7.

Substrate specificity and pyrophosphate binding pocket of Tb_bVSP1. a, Michaelis-Menten plot of initial velocity versus substrate concentration for Tb_bVSP1 (left panel) and Δ166 Tb_bVSP1 (right panel). K_m and k_cat values are indicated along with adjusted regression co-efficient (R²) for the curves. b, optimum pH measurement with polyP₃. The curves show variation in tripolyphosphatase activity of Tb_bVSP1 over a pH range of 5.2–9.0 and in the presence of 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 6.0–9.0) was used in these experiments. c, inhibition of PP_i hydrolysis by Ca⁺². PPase activity was determined with increasing concentrations of CaCl₂ with substrate (PP_i) concentration near K_m value (25 μm) and co-factor (3 mm of Mg⁺²). d, stereo view of F_o − F_c simulated annealing OMIT electron density map contoured at 6σ showing IDP (blue mesh) and Mg²⁺ (orange mesh) in the active site. e, important interactions between IDP, Mg⁺² 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 Mg⁺² ions (chartreuse) and water (pale green) are shown as spheres.

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 Ca⁺² injection; lines show fit of data to the binding model describing ligand interaction with 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 Tb_bVSP1 (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.

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 Tb_bVSP1 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 Tb_bVSP1 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.