Insights into the reaction of protein-tyrosine phosphatase 1B: crystal structures for transition state analogs of both catalytic steps

Abstract

Catalysis by protein-tyrosine phosphatase 1B (PTP1B) occurs through a two-step mechanism involving a phosphocysteine intermediate. We have solved crystal structures for the transition state analogs for both steps. Together with previously reported crystal structures of apo-PTP1B, the Michaelis complex of an inactive mutant, the phosphoenzyme intermediate, and the product complex, a full picture of all catalytic steps can now be depicted. The transition state analog for the first catalytic step comprises a ternary complex between the catalytic cysteine of PTP1B, vanadate, and the peptide DADEYL, a fragment of a physiological substrate. The equatorial vanadate oxygen atoms bind to the P-loop, and the apical positions are occupied by the peptide tyrosine oxygen and by the PTP1B cysteine sulfur atom. The vanadate assumes a trigonal bipyramidal geometry in both transition state analog structures, with very similar apical O-O distances, denoting similar transition states for both phosphoryl transfer steps. Detailed interactions between the flanking peptide and the enzyme are discussed.

FIGURE 1.

The general mechanism of the PTP-catalyzed reaction.

FIGURE 2.

Orientation of key residues at the active site of PTP1B and the binding environment for trigonal bipyramid structures in the transition state analogs. a, first transition state complex (TSA1) between native PTP1B, metavanadate, and the Tyr in the peptide DADEYL; b, second transition state (TSA2) complex between native PTP1B and orthovanadate. The Trp¹⁷⁹ ring was omitted for the sake of clarity, and only the backbone carbonyl group is shown. Hydrogen bond distances (in red) are in Å. See supplemental Fig. S2 for electron density surrounding residues at the active site of TSA1 and TSA2. Stereo images of a and b are included in supplemental Fig. S3.

FIGURE 3.

Crystal structures along the pathway of the reaction catalyzed by PTP1B. Hydrogen bond distances are in Å. The Trp¹⁷⁹ ring was omitted for the sake of clarity, and only the backbone carbonyl group is shown. a, resting state PTP1B apoenzyme (PDB entry 2CM2) (28); b, Michaelis complex between PTP1B C215S and the peptide DADEpYL (PDB entry 1PTU) (19); c, first transition state complex between native PTP1B, metavanadate, and the Tyr in the peptide DADEYL (new structure, PDB entry 3I7Z); d, PTP1B Q262A cysteinyl-phosphate intermediate enzyme (PDB entry 1A5Y) (29); e, second transition state complex, between native PTP1B and orthovanadate (new structure, PDB entry 3I80); f, PTP1B-tungstate product analog complex (PDB entry 2HNQ) (30). These structures are further summarized in supplemental Table S1.

FIGURE 4.

Coordination shell of the water W1 in the active site of PTP1B. a, first transition state; b, second transition state. Stereo images of a and b are included in supplemental Fig. S5.

FIGURE 5.

Close-up of details from crystal structures along the reaction pathway catalyzed by PTP1B. a, initial state, apoenzyme; b, Michaelis complex; c, first transition state; d, phosphoenzyme; e, second transition state; f, product complex. The figure was prepared from superimposed structures, which are presented side by side. In the first step, the phosphorus atom swings from the substrate to the nucleophilic cysteine, whereas the positions of the leaving group oxygen, the nucleophile, and nonbridging oxygens do not change appreciably.