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Published on: November 11, 2014
Thermosensitive mutants of the MPTP and hPTP1B protein tyrosine phosphatases: isolation and structural analysis
E S Muise1, A Vrielink, M A Ennis
1Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Abstract:
A PCR-based random mutagenesis procedure was employed to identify several thermosensitive mutants of the MPTP enzyme, the murine homologue of the human T-cell PTPase and rat PTP-S enzymes. Four mutants with varying degrees of thermosensitivity were characterized for their thermostability and refolding properties following incubation at the nonpermissive temperature. Structure analysis of these mutations based on the hPTP1B co-ordinate structure demonstrates a clear relationship between the position of each mutated amino acid relative to the catalytic cysteine residue and their thermostability. Introduction of two of these mutations in the related enzyme hPTP1B suggests that the structural defects and the resulting thermosensitivity of these mutations may represent an intrinsic property of all PTPase catalytic domains.
Insights
Researchers identified thermosensitive mutants of the murine MPTP enzyme using random mutagenesis. Mutation position relative to the catalytic site impacts thermostability, suggesting a general property of PTPase domains.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Protein tyrosine phosphatases (PTPases) are crucial enzymes involved in cellular signaling.
- Understanding PTPase structure-function relationships is key to developing targeted therapeutics.
- The murine MPTP enzyme is a homologue of human T-cell PTPase and rat PTP-S.
Purpose of the Study:
- To identify and characterize thermosensitive mutants of the MPTP enzyme.
- To investigate the relationship between mutation location and enzyme thermostability.
- To assess if observed thermosensitivity is an intrinsic property of PTPase catalytic domains.
Main Methods:
- PCR-based random mutagenesis was used to generate mutants.
- Thermostability and refolding properties of mutants were analyzed.
- Structural analysis was performed using the hPTP1B co-ordinate structure.
Main Results:
- Four thermosensitive MPTP enzyme mutants were identified.
- Thermostability correlated with the position of mutated amino acids relative to the catalytic cysteine.
- Introducing two mutations into hPTP1B induced similar thermosensitivity.
Conclusions:
- The position of mutations relative to the catalytic cysteine is critical for PTPase thermostability.
- Thermosensitivity in PTPase catalytic domains may be an intrinsic characteristic.
- This study provides insights into PTPase structure-function and potential therapeutic targeting.
