Figure 1.
(A) StPurL structure is depicted in cartoon with N-terminal domain in green, linker domain in yellow, glutaminase domain in red, and FGAM synthetase domain with structural sub-domains 1 and 2 shown in blue and cyan colors respectively. The two active sites and the auxillary ADP are shown in sticks and Xenon atoms are depicted as orange spheres. In the side panel (B, C and D), mFo-DFc densitiy maps of the three Xenon atoms at 5.0 σ are shown in black mesh and residues forming the cavities around them are shown in sticks.
Table 1.
Data processing and refinement statistics.
Figure 2.
Unfolding and stability of StPurL.
(A) Unfolding of StPurL: Thermal denaturation profile of the StPurL protein as observed by measuring ellipticity at 222 nm. (B) Bar graph showing percentage activity (FGAM synthetase) at two different temperatures for StPurL (before and after the first transition) is depicted (C) B-factors: StPurL crystal structure depicted as a function of B-factors. Red, orange and yellow colors represent high B-factors. Blue, cyan and green colors depict low B-factors. (D) Secondary structure: percentage of residues with α helical and β sheet ramachandran angles is pictorially depicted with green color representing N-terminal domain, yellow color depicting linker domain, blue color depicting FGAM synthetase domain and red color depicting glutaminase domain. (E) Conservation: Average relative entropy values representing extent of evolutionary conservation of each domain are plotted. (F) Unfolding mechanism: Proposed unfolding mechanism of StPurL is depicted. The N-terminal domain (green) and linker domain (yellow) unfold in step 1. The FGAM synthetase domain (blue) and glutaminase domain (red) unfold in step 2.
Figure 3.
Characterization of StPurLMutants.
(A) Secondary structure analysis: Wavelength scan using circular dichroism spectroscopy performed for all variants at the same molar concentration. (B) Stability: Thermal denaturation monitored using CD signal at 222 nm. (C) Activity (FGAM synthetase): Percentage activity of all mutants at 37°C with respect to that of native StPurL protein.
Figure 4.
Crystal Structure of F209W mutant.
(A) mFo-DFc electron density map for Trp209 at 3.0 σ is shown in black mesh (B) Xe3 cavity region of F209W structure is shown in magenta cartoons and StPurL-Xenon complex is shown color coded with FGAM synthetase domain in blue and linker domain in yellow color. Location of xenon atom is depicted as an orange sphere. Select residues with rmsd of more than 1 Å are shown in sticks.
Figure 5.
Statistical Coupling Analysis.
Sector residues of blue (A), red (B), and magenta (C) are shown in surface representation with darker color in each figure representing higher SCA scores and lighter colors representing lower scores. Xenon atoms are depicted as orange spheres. In (A) locations of the two active sites and auxiliary ADP are shown in sticks. In (B) and (C), residues of the oxyanion hole and the thioester intermediate are depicted in yellow and red sticks respectively.
Figure 6.
Structural alignment of glutaminase domains.
StPurL (PDB file 1T3T) in green color, TmPurQ (PDB file 3D54) in cyan color, IGP synthase (PDB file 1GPW) in salmon color, CPS (PDB file 1C30) in magenta color and GMP synthase (PDB file 1GPM) in yellow color. Secondary structure elements are labeled as per StPurL structure. Active site is demarcated by Cys-glutamine intermediate of StPurL and TmPurQ and inhibitor trapped in IGP synthase shown in sticks. The location of the xenon atom in the StPurL-Xenon complex is shown in an orange sphere and residues blocking the xenon binding pocket in CPS, IGP synthase and GMP synthase are shown in sticks.
Figure 7.
(A) Glutaminase domain is shown in surface representation in purple color with active site residues, helix α27 and α30 highlighted in cartoon and stick representations. A2 domain of the FGAM synthetase is shown in pink cartoons with the long and short loops in green color. The structurally conserved long loop region of TmPurL is shown in yellow cartoon. ADP is shown in magenta sticks. The β-sheet regions of A1 domain involved in making the β-barrel core of the FGAM synthetase domain are shown in marine blue cartoon representation. Locations of the xenon atoms trapped in the structure are depicted as orange spheres. (B) View of the interface between the FGAM synthetase loop regions and the glutaminase domain showing various hydrogen bonding and van der waals interactions.