Figure 1.
Two-step synthesis of trypanothione.
For both steps, GSH has first to be activated by phosphorylation. Due to the asymmetry of spermidine the first step of the trypanothione biosynthesis could lead to two different glutathionylspermidines.
Figure 2.
Binding modes of a Gsp homologue.
Two major conformations of the spermidine moiety of the Gsp analogue (C) are observed. In the binding mode A loss of hydrogen bonds (orange dotted lines.) to Glu407 suggests weaker binding than in binding mode B.
Figure 3.
Structure and sequence comparison of the EcGspS crystal structure (pdb: 2ioa; A) and the modeled LmTryS structure (B).
Both structures contain ADP, the Gsp analogue (shown as sticks with carbons in yellow) and two Mg2+ ions (green balls). The yellow circle highlights an additional pocket in the TryS model which results from sequence differences (C). The green surfaces (I and II) indicate flexible loop regions that were not visible in the LmTryS crystal structure (pdb:2vps): In the EcGspS structure (A) region I (Gly 242 to Pro 249) does not offer any additional binding site near the spermidine moiety of the Gsp analogue, while in the LmTryS model (B) the corresponding loop (Gly250 to 262; marked red in C) builds part of the additional binding pocket. II shows the borders of the ATP Grasp fold loop (Pro534 to Gln563 in EcGspS and Val551to Asn579 in LmTryS, respectively). The loop was removed in both structures to demonstrate the positions of ADP and Mg2+.
Figure 4.
Atomic distances within the ‘new’ binding pocket of LmTryS.
Minimum (min) and maximum (max) distances with mean and standard deviations (sd) between Glu614 and Pro626 of the rigid part of the pocket and certain residues of the flexible loop region are given in Å.
Figure 5.
Presumed substrate binding sites of LmTryS as derived from the model with bound ADP/Mg2+ and the Gsp analogue.
ATP is assumed to essentially occupy the position of ADP. The position of the ATP γ-phosphate is mimicked by the phosphoryl group of the Gsp analogue. The red line marks the binding site of GSH, the green line that of spermidine. The empty pocket surrounded by a magenta line can harber the glutathionyl moiety of Gsp and, together with the spermidine binding site, thus could form a Gsp binding site.
Figure 6.
Comparison of N8-Gsp and N1-Gsp binding to LmTryS by trajectory analysis.
2D-rmsd plots of N8-Gsp (left) and N1-Gsp (right) are shown for the respective molecular dynamics simulations of the LmTryS model containing all substrates (Gsp, GSH, ATP and two Mg2+). Each colour point represents the rmsd between the frame conformation on the x-axis and the frame conformation on the y-axis. The analysis reveals that N8-Gsp is essentially moving into a single stable conformation (low rmsd values at the end of the simulation), while N1-Gsp adopts several ones.
Figure 7.
Hydrogen bonding interactions of N8-Gsp with protein residues.
The inset shows a conserved water molecule in a small pocket near the glutathionyl moiety of Gsp. (magenta: N8-Gsp, blue: residues from flexible loop region, green: residues from rigid part of Gsp binding pocket, orange dotted lines: hydrogen bonds). None of the arginine residues directly interacts with the substrate, but both contribute to the stabilisation of the Gsp binding pocket: Arg222 by interacting with Glu614 and Arg613 by interacting with Ser624, Leu274 (not shown) and Glu355.
Figure 8.
LmTryS model with all substrates.
The model represents the last frame of the molecular dynamics simulation including ATP, glutathione and N8-Gsp. The substrate binding regions are shown as green circles. S1: ATP binding pocket; S2: glutathione binding pocket, S3: spermidine binding region, S3+S4: N8-glutathionylspermidine binding pocket. Substrates are shown with carbon atoms in magenta.
Figure 9.
Structural overlay of Gsp binding pocket conformations with bound Gsp.
18 representative conformations are shown (see supporting information II-IV; blue: flexible loop region, green: rigid backbone, grey: rigid residue, magenta: glutathionylspermidine).
Figure 10.
Putative binding mode of TryS inhibitor DDD66604 to LmTryS.
A: DDD66604 (turquoise) bound to the Gsp pocket. B: Chemical formula of DDD66604. C: Comparison of binding mode of DDD66604 (turqouise) and N1-Gsp (purple).in the Gsp binding pocket.