Thermodynamic study of ligand binding to protein-tyrosine phosphatase 1B and its substrate-trapping mutants

Yan Ling Zhang, Zhu Jun Yao, Mauro Sarmiento, Li Wu, Terrence R. Burke, Zhong-Yin Zhang

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

The binding of several phosphonodifluoromethyl phenylalanine (F2Pmp)-containing peptides to protein-tyrosine phosphatase 1B (PTP1B) and its substrate-trapping mutants (C215S and D181A) has been studied using isothermal titration calorimetry. The binding of a high affinity ligand, Ac-Asp-Ala-Asp-Glu-F2Pmp-Leu-NH2, to PTP1B (K(d) = 0.24 μM) is favored by both enthalpic and entropic contributions. Disruption of ionic interactions between the side chain of Arg-47 and the N-terminal acidic residues reduces the binding affinity primarily through the reduction of the TΔS term. The role of Arg-47 may be to maximize surface contact between PTPIB and the peptide, which contributes to high affinity binding. The active site Cys-215 → Ser mutant PTP1B binds ligands with the same affinity as the wild-type enzyme. However, unlike wild-type PTP1B, peptide binding to C2158 is predominately driven by enthalpy change, which likely results from the elimination of the electrostatic repulsion between the thiolate anion and the phosphonate group. The increased enthalpic contribution is offset by reduction in the binding entropy, which may be the result of increased entropy of the unbound protein caused by this mutation. The general acid-deficient mutant D181A binds the peptide 5-fold tighter than the C215S mutant, consistent with the observation that the Asp to Ala mutant is a better 'substrate-trapping' reagent than C215S. The increased binding affinity for D181A as compared with the wild-type PTP1B results primarily from an increase in the ΔH of binding in the mutant, which may be related to decreased electrostatic repulsion between the phosphate moiety and PTP1B. These results have important implications for the design of high affinity PTP1B inhibitors.

Original languageEnglish (US)
Pages (from-to)34205-34212
Number of pages8
JournalJournal of Biological Chemistry
Volume275
Issue number44
StatePublished - Nov 3 2000
Externally publishedYes

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Non-Receptor Type 1 Protein Tyrosine Phosphatase
Thermodynamics
Carrier Proteins
Ligands
Substrates
Peptides
Entropy
Static Electricity
Electrostatics
Organophosphonates
Calorimetry
Mutant Proteins
Phenylalanine
Titration
Anions
Enthalpy
Catalytic Domain
Phosphates
Mutation
Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Thermodynamic study of ligand binding to protein-tyrosine phosphatase 1B and its substrate-trapping mutants. / Zhang, Yan Ling; Yao, Zhu Jun; Sarmiento, Mauro; Wu, Li; Burke, Terrence R.; Zhang, Zhong-Yin.

In: Journal of Biological Chemistry, Vol. 275, No. 44, 03.11.2000, p. 34205-34212.

Research output: Contribution to journalArticle

Zhang, Yan Ling ; Yao, Zhu Jun ; Sarmiento, Mauro ; Wu, Li ; Burke, Terrence R. ; Zhang, Zhong-Yin. / Thermodynamic study of ligand binding to protein-tyrosine phosphatase 1B and its substrate-trapping mutants. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 44. pp. 34205-34212.
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