Conformational and dynamic changes of Yersinia protein tyrosine phosphatase induced by ligand binding and active site mutation and revealed by H/D exchange and electrospray ionization fourier transform ion cyclotron resonance mass spectrometry

Fang Wang, Weiqun Li, Mark Emmett, Christopher L. Hendrickson, Alan G. Marshall, Yan Ling Zhang, Li Wu, Zhong Yin Zhang

Research output: Contribution to journalArticle

67 Citations (Scopus)

Abstract

Protein tyrosine phosphatases (PTPase) play important roles in the intracellular signal transduction pathways that regulate cell transformation, growth, and proliferation. Here, solvent accessibility is determined for backbone amide protons from various segments of wild-type Yersinia PTPase in the presence or absence of 220 μM vanadate, a competitive inhibitor, as well as an active site mutant in which the essential cysteine 403 has been replaced by serine (C403S). The method consists of solution-phase H/D exchange, followed by pepsin digestion, high-performance liquid chromatography, and electrospray ionization high-field (9.4 T) Fourier transform ion cyclotron resonance mass spectrometry. Proteolytic segments spanning ~93.5% of the primary sequence are analyzed. Binding of vanadate reduces the H/D exchange rate throughout the protein, both for the WpD loop and for numerous other residues that are shielded when that loop is pulled down over the active site on binding of the inhibitor. The single active site C403S mutation reduces solvent access to the WpD loop itself, but opens up the structure in several other segments. Although the 3D structure of the ligand-bound C403S mutant is similar to that of the wild-type PTPase, and the C403S mutant and the wild-type enzyme display similar affinities for vanadate, the thermodynamics for binding of vanadate is different for the two proteins. Collectively, these results establish the flexibility of the WpD loop (previously inferred by comparing PTPase X-ray single-crystal diffraction structures in the presence and absence of a tungstate inhibitor), as well as several other significant changes in segment exposure and/or flexibility that are not evident from X-ray structures.

Original languageEnglish (US)
Pages (from-to)15289-15299
Number of pages11
JournalBiochemistry
Volume37
Issue number44
DOIs
StatePublished - Nov 3 1998
Externally publishedYes

Fingerprint

Cyclotrons
Yersinia
Cyclotron resonance
Electrospray ionization
Protein Tyrosine Phosphatases
Vanadates
Fourier Analysis
Mass spectrometry
Mass Spectrometry
Catalytic Domain
Fourier transforms
Binding Sites
Ions
Ligands
Mutation
X-Rays
X rays
Signal transduction
Pepsin A
High performance liquid chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Conformational and dynamic changes of Yersinia protein tyrosine phosphatase induced by ligand binding and active site mutation and revealed by H/D exchange and electrospray ionization fourier transform ion cyclotron resonance mass spectrometry. / Wang, Fang; Li, Weiqun; Emmett, Mark; Hendrickson, Christopher L.; Marshall, Alan G.; Zhang, Yan Ling; Wu, Li; Zhang, Zhong Yin.

In: Biochemistry, Vol. 37, No. 44, 03.11.1998, p. 15289-15299.

Research output: Contribution to journalArticle

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title = "Conformational and dynamic changes of Yersinia protein tyrosine phosphatase induced by ligand binding and active site mutation and revealed by H/D exchange and electrospray ionization fourier transform ion cyclotron resonance mass spectrometry",
abstract = "Protein tyrosine phosphatases (PTPase) play important roles in the intracellular signal transduction pathways that regulate cell transformation, growth, and proliferation. Here, solvent accessibility is determined for backbone amide protons from various segments of wild-type Yersinia PTPase in the presence or absence of 220 μM vanadate, a competitive inhibitor, as well as an active site mutant in which the essential cysteine 403 has been replaced by serine (C403S). The method consists of solution-phase H/D exchange, followed by pepsin digestion, high-performance liquid chromatography, and electrospray ionization high-field (9.4 T) Fourier transform ion cyclotron resonance mass spectrometry. Proteolytic segments spanning ~93.5{\%} of the primary sequence are analyzed. Binding of vanadate reduces the H/D exchange rate throughout the protein, both for the WpD loop and for numerous other residues that are shielded when that loop is pulled down over the active site on binding of the inhibitor. The single active site C403S mutation reduces solvent access to the WpD loop itself, but opens up the structure in several other segments. Although the 3D structure of the ligand-bound C403S mutant is similar to that of the wild-type PTPase, and the C403S mutant and the wild-type enzyme display similar affinities for vanadate, the thermodynamics for binding of vanadate is different for the two proteins. Collectively, these results establish the flexibility of the WpD loop (previously inferred by comparing PTPase X-ray single-crystal diffraction structures in the presence and absence of a tungstate inhibitor), as well as several other significant changes in segment exposure and/or flexibility that are not evident from X-ray structures.",
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AU - Emmett, Mark

AU - Hendrickson, Christopher L.

AU - Marshall, Alan G.

AU - Zhang, Yan Ling

AU - Wu, Li

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N2 - Protein tyrosine phosphatases (PTPase) play important roles in the intracellular signal transduction pathways that regulate cell transformation, growth, and proliferation. Here, solvent accessibility is determined for backbone amide protons from various segments of wild-type Yersinia PTPase in the presence or absence of 220 μM vanadate, a competitive inhibitor, as well as an active site mutant in which the essential cysteine 403 has been replaced by serine (C403S). The method consists of solution-phase H/D exchange, followed by pepsin digestion, high-performance liquid chromatography, and electrospray ionization high-field (9.4 T) Fourier transform ion cyclotron resonance mass spectrometry. Proteolytic segments spanning ~93.5% of the primary sequence are analyzed. Binding of vanadate reduces the H/D exchange rate throughout the protein, both for the WpD loop and for numerous other residues that are shielded when that loop is pulled down over the active site on binding of the inhibitor. The single active site C403S mutation reduces solvent access to the WpD loop itself, but opens up the structure in several other segments. Although the 3D structure of the ligand-bound C403S mutant is similar to that of the wild-type PTPase, and the C403S mutant and the wild-type enzyme display similar affinities for vanadate, the thermodynamics for binding of vanadate is different for the two proteins. Collectively, these results establish the flexibility of the WpD loop (previously inferred by comparing PTPase X-ray single-crystal diffraction structures in the presence and absence of a tungstate inhibitor), as well as several other significant changes in segment exposure and/or flexibility that are not evident from X-ray structures.

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