Rapid loop dynamics of yersinia protein tyrosine phosphatases

Laura J. Juszczak, Zhong-Yin Zhang, Li Wu, David S. Gottfried, Daniel D. Eads

Research output: Contribution to journalArticle

64 Citations (Scopus)

Abstract

The Yersinia protein tyrosine phosphatases (PTPase) contain a single and invariant tryptophan (W354) located at one of the hinge positions of the flexible loop (WpD loop), which is essential for catalysis. The wild-type Yersinia PTPase and an active site mutant in which the esential Cys 403 has been replaced by serine (C403S) have been examined using both time-resolved fluorescence anisotropy and steady-state UV resonance Raman (UVRR) spectroscopies. Both enzymes were examined with and without the bound inhibitor arsenate. The UVRR spectra indicate that in solution the ligand- free, wild-type PTPase exists as an equilibrium mixture of two tryptophan rotamer structures with χ2,1 dihedral angles of -4°and -90°. The two rotamers have been attributed to the presence of both 'closed' and 'open' WpD loop conformers of the ligand-free enzyme. Conversely, the UVRR spectra of the arsenate-ligated, wild-type PTPase and of ligand-free and arsenate- ligated C403S PTPase contain a single W3 hand which is correlated to the - 4°rotamer of W354, indicating a predominance of the closed WpD loop conformer. The tryptophan fluorescence anisotropy decay measurements of the ligand-bound, wild-type Yersinia PTPase and of both ligation states of the C403S PTPase reveal a single correlation time of 30-48 ns due to the rotational motion of the protein, while the ligand-free, wild-type PTPase is found to have two correlation times of 31 and 3.8 ns. The 3.8 ns correlation time of the ligand-free enzyme is attributed to the hinged movement of the WpD loop which contains W354. These results indicate that under physiological conditions, the nonligated, wild-type Yersinia PTPase alternates between an open WpD loop and a closed loop form with a rate constant of ~42.6 x 108 s-1. We conclude that the rate of WpD loop closure of the wild-type Yersinia PTPase is thus independent of the presence of ligand, whereas in the presence of ligand the rate of opening is dramatically reduced resulting in a closed conformation on ligand binding. In contrast, the ligand-free and ligated C403S PTPase remain in the loop closed configuration over the time course of our dynamic measurements. The lack of WpD loop motion in the C403S PTPase is believed to be due to either a loss of repulsive potential between the anionic thiolate and Asp 356 of the WpD loop and/or the formation of a hydrogen bond or water bridged hydrogen bond between Ser 403 and Asp 356.

Original languageEnglish (US)
Pages (from-to)2227-2236
Number of pages10
JournalBiochemistry
Volume36
Issue number8
DOIs
StatePublished - Feb 25 1997
Externally publishedYes

Fingerprint

Yersinia
Protein Tyrosine Phosphatases
Ligands
Tryptophan
Fluorescence Polarization
Raman scattering
Hydrogen
Hydrogen bonds
Anisotropy
Enzymes
Fluorescence
Raman Spectrum Analysis
Dihedral angle
Hinges
Catalysis
Serine
Ligation
Raman spectroscopy
Conformations
Rate constants

ASJC Scopus subject areas

  • Biochemistry

Cite this

Juszczak, L. J., Zhang, Z-Y., Wu, L., Gottfried, D. S., & Eads, D. D. (1997). Rapid loop dynamics of yersinia protein tyrosine phosphatases. Biochemistry, 36(8), 2227-2236. https://doi.org/10.1021/bi9622130

Rapid loop dynamics of yersinia protein tyrosine phosphatases. / Juszczak, Laura J.; Zhang, Zhong-Yin; Wu, Li; Gottfried, David S.; Eads, Daniel D.

In: Biochemistry, Vol. 36, No. 8, 25.02.1997, p. 2227-2236.

Research output: Contribution to journalArticle

Juszczak, LJ, Zhang, Z-Y, Wu, L, Gottfried, DS & Eads, DD 1997, 'Rapid loop dynamics of yersinia protein tyrosine phosphatases', Biochemistry, vol. 36, no. 8, pp. 2227-2236. https://doi.org/10.1021/bi9622130
Juszczak, Laura J. ; Zhang, Zhong-Yin ; Wu, Li ; Gottfried, David S. ; Eads, Daniel D. / Rapid loop dynamics of yersinia protein tyrosine phosphatases. In: Biochemistry. 1997 ; Vol. 36, No. 8. pp. 2227-2236.
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AU - Juszczak, Laura J.

AU - Zhang, Zhong-Yin

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AU - Eads, Daniel D.

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N2 - The Yersinia protein tyrosine phosphatases (PTPase) contain a single and invariant tryptophan (W354) located at one of the hinge positions of the flexible loop (WpD loop), which is essential for catalysis. The wild-type Yersinia PTPase and an active site mutant in which the esential Cys 403 has been replaced by serine (C403S) have been examined using both time-resolved fluorescence anisotropy and steady-state UV resonance Raman (UVRR) spectroscopies. Both enzymes were examined with and without the bound inhibitor arsenate. The UVRR spectra indicate that in solution the ligand- free, wild-type PTPase exists as an equilibrium mixture of two tryptophan rotamer structures with χ2,1 dihedral angles of -4°and -90°. The two rotamers have been attributed to the presence of both 'closed' and 'open' WpD loop conformers of the ligand-free enzyme. Conversely, the UVRR spectra of the arsenate-ligated, wild-type PTPase and of ligand-free and arsenate- ligated C403S PTPase contain a single W3 hand which is correlated to the - 4°rotamer of W354, indicating a predominance of the closed WpD loop conformer. The tryptophan fluorescence anisotropy decay measurements of the ligand-bound, wild-type Yersinia PTPase and of both ligation states of the C403S PTPase reveal a single correlation time of 30-48 ns due to the rotational motion of the protein, while the ligand-free, wild-type PTPase is found to have two correlation times of 31 and 3.8 ns. The 3.8 ns correlation time of the ligand-free enzyme is attributed to the hinged movement of the WpD loop which contains W354. These results indicate that under physiological conditions, the nonligated, wild-type Yersinia PTPase alternates between an open WpD loop and a closed loop form with a rate constant of ~42.6 x 108 s-1. We conclude that the rate of WpD loop closure of the wild-type Yersinia PTPase is thus independent of the presence of ligand, whereas in the presence of ligand the rate of opening is dramatically reduced resulting in a closed conformation on ligand binding. In contrast, the ligand-free and ligated C403S PTPase remain in the loop closed configuration over the time course of our dynamic measurements. The lack of WpD loop motion in the C403S PTPase is believed to be due to either a loss of repulsive potential between the anionic thiolate and Asp 356 of the WpD loop and/or the formation of a hydrogen bond or water bridged hydrogen bond between Ser 403 and Asp 356.

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