Identification of a second aryl phosphate-binding site in protein-tyrosine phosphatase 1B: a paradigm for inhibitor design - PubMed (original) (raw)

Identification of a second aryl phosphate-binding site in protein-tyrosine phosphatase 1B: a paradigm for inhibitor design

Y A Puius et al. Proc Natl Acad Sci U S A. 1997.

Abstract

The structure of the catalytically inactive mutant (C215S) of the human protein-tyrosine phosphatase 1B (PTP1B) has been solved to high resolution in two complexes. In the first, crystals were grown in the presence of bis-(para-phosphophenyl) methane (BPPM), a synthetic high-affinity low-molecular weight nonpeptidic substrate (Km = 16 microM), and the structure was refined to an R-factor of 18. 2% at 1.9 A resolution. In the second, crystals were grown in a saturating concentration of phosphotyrosine (pTyr), and the structure was refined to an R-factor of 18.1% at 1.85 A. Difference Fourier maps showed that BPPM binds PTP1B in two mutually exclusive modes, one in which it occupies the canonical pTyr-binding site (the active site), and another in which a phosphophenyl moiety interacts with a set of residues not previously observed to bind aryl phosphates. The identification of a second pTyr molecule at the same site in the PTP1B/C215S-pTyr complex confirms that these residues constitute a low-affinity noncatalytic aryl phosphate-binding site. Identification of a second aryl phosphate binding site adjacent to the active site provides a paradigm for the design of tight-binding, highly specific PTP1B inhibitors that can span both the active site and the adjacent noncatalytic site. This design can be achieved by tethering together two small ligands that are individually targeted to the active site and the proximal noncatalytic site.

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Figures

Figure 1

Figure 1

Structures of PTP1B substrates BPPM (a) and pTyr (b).

Figure 2

Figure 2

(a and b) Simulated annealing omit maps showing unbiased electron density for bound BPPM (a) and phoshphotyrosine (b) molecules. The density shown is an _F_o − _F_c map contoured at 2.0 σ, with the refined models superimposed. Molecules bound to the canonical pTyr binding site are colored green, and ligands bound to the second site are colored yellow. (c) Superposition of BPPM and pTyr ligands. pTyr A is drawn in black, and pTyr B is gray. [Diagrams were generated with program

o

(16)].

Figure 3

Figure 3

Stereo representations of the binding modes of BPPM A (a), BPPM B (b), and pTyr B (c). Contacts represented by dashed lines are distances less than 3.6 Å, except for certain interactions with aromatic rings. Interactions between the amide nitrogens of residues 216–221 and the phosphate groups of ligand A are too numerous to represent. [Diagrams were generated with the program

o

(16)].

Figure 4

Figure 4

Schematic representations of the interactions between PTP1B/C215S and BPPM A (a), BPPM B (b), and pTyr B (c). A distance cutoff of 3.6 Å was used, except for certain interactions with aromatic rings.

Figure 5

Figure 5

A strategy for creating selective and high-affinity PTP1B inhibitors. Based on the principle of additivity of free energy of binding, high-affinity ligands can be designed by linking two functional groups (each with modest affinity to the target protein) identified experimentally. The added specificity arises from the fact that the tethered ligand has to bind both sites simultaneously.

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