The crystallization of human ITPA was apparently enhanced and improved by the inclusion of XTP in an equimolar ratio to the enzyme. After we had collected the 1.6 Å X-ray diffraction data from human ITPA crystals using our home source, a similar unpublished structure was deposited in the Protein Data Bank as entry 2car
. Although of high resolution (1.1 Å), this structure resulted from a monoclinic and pseudo-merohedrally twinned crystal. Therefore, we checked our diffraction data using the The Merohedral Crystal Twinning Server and found no evidence of twinning (Yeates, 1997
). Also, the possibility of a monoclinic space group was ruled out by a comparison of parallel refinements. We speculate that the inclusion of XTP in the crystallization experiments may have improved the crystals by eliminating twinning and resulting in the higher symmetry space group.
Crystallographic refinement resulted in a final R
value of 19% with an R
of 24% and the structure refined with reasonable stereochemistry (Table 1). Two residues, both with excellent electron density, have positive ϕ/ψ values (Fig. 2): Gln65 is located in the left-handed helical region of the Ramachandran plot (Ramachandran & Sasisekharan, 1968
) and Lys96 has ϕ = 66° and ψ = 168° and is located just below a generously allowed region as defined by PROCHECK
(Laskowski et al.
). After superposition of Cα
atoms, the r.m.s.d. between 2car
and our coordinates is 0.48 Å, with the largest difference being at loop 124–127, which has differences of up to 5 Å. When the superposition was performed omitting this loop, the r.m.s.d. was only 0.2 Å. The structure has two lobes (Fig. 2
). The top lobe is composed of helices α4, α5, α6, strand β7 and helix α7. The bottom lobe is composed of helices α0, α1 and α2 and strands β1, β2, β3, β4, β5 and β6. The active-site cleft lies between the two lobes. Under physiological conditions, NTPases are most likely to exist as a homodimer (Hwang et al.
; Lin et al.
). A putative homodimer can be extracted from crystallographic symmetry that is very similar to the homodimer found in the 2car
asymmetric unit (Fig. 3
). The active sites are located on opposite sides of the homodimer.
Figure 2 (a) Annotated protein sequence and secondary-structure analysis using JOY software (Mizuguchi et al., 1998 ). The figure is organized as follows: line 1, residue numbers; line 2, an asterisk indicates putative active-site residues that were identified (more ...)
Figure 3 Cross-eyed stereo pairs of the physiological dimer of ITPA. (a) Ribbon diagrams for each monomer are colored in a rainbow with blue at the N-terminus varying to red at the C-terminus. (b) The same drawing rotated 90° about the vertical axis to (more ...)
As noted above, the inclusion of XTP during crystallization was beneficial to the crystals. We were curious to see if we could identify an XTP or possibly XMP bound to ITPA. After refinement of the protein structure was completed, F
electron-density maps were closely examined (Fig. 4
). There was no apparent density for bound ligand. M. jannaschii
NTPase was solved with AMPPNP bound (PDB code 2mjp
). Superposition of Cα
coordinates between ITPA and 2mjp
(r.m.s.d. of 1.3 Å) shows the types of structural movements that can be predicted on ligand binding of human ITPA. Structural movement (towards AMPPNP) that seems to be associated with ligand binding includes helix α1, the loop before α6 and helix α7. These regions include active-site residues (indicated by an asterisk in Fig. 2
). These residues appear to close in and cap off the substrate from the solvent. In order to verify these predictions, future work will include increasing the concentration of XTP or other substrates in the crystallization experiments. With this structure of wild-type human ITPA completed, we are well positioned to perform detailed structure–function studies.
Figure 4 (a) Cross-eyed stereo pair of the electron density surrounding the active site of human ITPA. The REFMAC/σA-weighted 2mF
o − DF
c map is displayed at 1σ. Active-site residues are highlighted (more ...)