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Substrate Recognition of MARTX Ras/Rap1-Specific Endopeptidase

Author(s)
Biancucci, Marco; Satchell, Karla J. F.; Rabideau, Amy; Lu, Zeyu; Loftis, Alexander Robert; Pentelute, Bradley L.; ... Show more Show less
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Abstract
Ras/Rap1-specific endopeptidase (RRSP) is a cytotoxic effector domain of the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin of highly virulent strains of Vibrio vulnificus. RRSP blocks RAS-MAPK kinase signaling by cleaving Ras and Rap1 within the switch I region between Y32 and D33. Although the RRSP processing site is highly conserved among small GTPases, only Ras and Rap1 have been identified as proteolytic substrates. Here we report that residues Y32 and D33 at the scissile bond play an important role in RRSP substrate recognition, while the nucleotide state of Ras has an only minimal effect. In addition, substrate specificity is generated by residues across the entire switch I region. Indeed, swapping the Ras switch I region into either RalA or RhoA, GTPases that are not recognized by RRSP, generated chimeras that are substrates of RRSP. However, a difference in the processing efficiency of Ras switch I in the context of Ras, RalA, or RhoA indicates that protein regions outside Ras switch I also contribute to efficient RRSP substrate recognition. Moreover, we show that synthetic peptides corresponding to the Ras and Rap1, but not RalA, switch I regions are cleaved by RRSP, demonstrating sequence-specific substrate recognition. In conclusion, this work demonstrates that the GTPase recognition of RRSP is independent of the nucleotide state and is mainly driven by the Ras and Rap1 switch I loop and also influenced by additional protein-protein interactions, increasing the substrate specificity of RRSP.
Date issued
2017-05
URI
http://hdl.handle.net/1721.1/116350
Department
Massachusetts Institute of Technology. Department of Chemistry
Journal
Biochemistry
Publisher
American Chemical Society (ACS)
Citation
Biancucci, Marco et al. “Substrate Recognition of MARTX Ras/Rap1-Specific Endopeptidase.” Biochemistry 56, 21 (May 2017): 2747–2757 © 2017 American Chemical Society
Version: Author's final manuscript
ISSN
0006-2960
1520-4995

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