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Identification of Kinase-substrate Pairs Using High Throughput Screening
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Substrate Recognition of MARTX Ras/Rap1-Specific Endopeptidase
Marco Biancucci1, Amy E Rabideau2, Zeyu Lu2
1Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine , Chicago, Illinois 60611, United States.
Biochemistry
|May 2, 2017
Summary
Ras/Rap1-specific endopeptidase (RRSP) from Vibrio vulnificus targets Ras and Rap1 GTPases. Substrate specificity is primarily determined by the switch I loop, not nucleotide state, with other protein interactions refining recognition.
Area of Science:
- Molecular Biology
- Biochemistry
- Microbiology
Background:
- Ras/Rap1-specific endopeptidase (RRSP) is a key component of the Vibrio vulnificus MARTX toxin.
- RRSP inhibits RAS-MAPK signaling by cleaving Ras and Rap1 GTPases at a conserved site.
Purpose of the Study:
- To investigate the molecular determinants of RRSP substrate recognition.
- To understand the role of the switch I region and nucleotide state in RRSP activity.
Main Methods:
- Site-directed mutagenesis to create chimeric GTPases.
- In vitro cleavage assays using synthetic peptides.
- Analysis of GTPase processing efficiency.
Main Results:
- Residues Y32 and D33 at the scissile bond are crucial for RRSP substrate recognition.
- The switch I region of Ras, when transferred to other GTPases, confers RRSP substrate activity.
- Peptide cleavage assays confirm sequence-specific recognition of Ras and Rap1 switch I regions.
Conclusions:
- RRSP substrate specificity is mainly driven by the switch I loop of Ras and Rap1.
- Nucleotide-bound state has minimal impact on RRSP cleavage.
- Additional protein-protein interactions contribute to RRSP's precise substrate targeting.
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