The molecular details of WPD-loop movement differ in the protein-tyrosine phosphatases YopH and PTP1B

Abstract

The movement of a conserved protein loop (the WPD-loop) is important in catalysis by protein tyrosine phosphatases (PTPs). Using kinetics, isotope effects, and X-ray crystallography, the different effects arising from mutation of the conserved tryptophan in the WPD-loop were compared in two PTPs, the human PTP1B, and the bacterial YopH from Yersinia. Mutation of the conserved tryptophan in the WPD-loop to phenylalanine has a negligible effect on k(cat) in PTP1B and full loop movement is maintained. In contrast, the corresponding mutation in YopH reduces k(cat) by two orders of magnitude and the WPD loop locks in an intermediate position, disabling general acid catalysis. During loop movement the indole moiety of the WPD-loop tryptophan moves in opposite directions in the two enzymes. Comparisons of mammalian and bacterial PTPs reveal differences in the residues forming the hydrophobic pocket surrounding the conserved tryptophan. Thus, although WPD-loop movement is a conserved feature in PTPs, differences exist in the molecular details, and in the tolerance to mutation, in PTP1B compared to YopH. Despite high structural similarity of the active sites in both WPD-loop open and closed conformations, differences are identified in the molecular details associated with loop movement in PTPs from different organisms.

Figure 1

The general mechanism of the PTP-catalyzed reaction. The WPD-loop assumes a catalytically active closed conformation with the general acid in position to protonate the leaving group during formation of the phosphoenzyme intermediate. In the second step this intermediate is hydrolyzed. After the phosphate product is released the WPD-loop open conformation becomes favored.

Figure 2

Orientation of key residues at the active site of PTP1B (light blue) and YopH (green). (a) Superimposition of the ligand-free structures with the WPD-loop in the open conformation. (b) Superimposition of the vanadate-bound structures with WPD-loop in the closed conformation, using the same orientation as in (a). The lower, italicized residue positions refer to YopH. For the sake of clarity the only backbone carbonyl group is that of W179/W354 shown in (b). Hydrogen bonds in dotted blue lines are shown only for PTP1B. The PDB IDs used to generate each structure were: PTP1B wildtype: 2CM2 (ligand-free form, open WPD-loop)[42] and 3I80 (VO₄ bound, closed WPD-loop)[28]; YopH wildtype: 1YPT (ligand-free form, open WPD-loop)[19] and 2I42 (VO₄ bound, closed WPD-loop).

Figure 3

Orientation of key residues at the active-site of PTP1B W179F in unbound, loop-open (green) and vanadate-bound, loop-closed (light blue) states.

Figure 4

Comparison between PTP1B and YopH structures. Hydrophobic pocket of the conserved tryptophan in the hinge of the WPD-loop, and orientations of the Trp in the native and Phe in the mutant PTPs in open and closed WPD-loop forms. The arrows indicate the movement direction of the Trp or Phe upon WPD-loop closure. These pictures were made from PTP1B and YopH structures superimposed considering the P-loop region, no rotation was applied between each representation. The hydrophobic pockets for all representations are those for the closed or quasi-open (YopH W354F) WPD-loop conformations. The PDB IDs used to generate each structure were: (a) PTP1B wildtype: 2CM2 (ligand-free form, open WPD-loop)[42], 3I80 (VO₄ bound, closed WPD-loop) [28]; (b) PTP1B W179F: 3QKP (ligand-free form, open WPD-loop), 3QKQ (VO₄ bound, closed WPD-loop); (c) YopH wildtype: 1YPT (ligand-free form, open WPD-loop)[19], 2I42 (VO₄ bound, closed WPD-loop); (d) YopH W354F: 3F99 (ligand-free form, quasi-open WPD-loop), 3F9A (WO₄ bound, quasi-open WPD-loop) [24].