Substrate Recognition of MARTX Ras/Rap1-Specific Endopeptidase
Name
nihms905836.pdf
Size
1.54 MB
Format
Adobe PDF
Checksum (MD5)
ee19ab1912b51b12e03cd4f5b401d712
Author(s)
Biancucci, Marco
Satchell, Karla J. F.
Rabideau, Amy
Lu, Zeyu
Loftis, Alexander Robert
Pentelute, Bradley L.
Date Issued
May 2017
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
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.
MIT Department
Massachusetts Institute of Technology. Department of Chemistry
Terms of Use
Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1021/ACS.BIOCHEM.7B00246
