RRSP Exhibits Novel Proteolytic Activity
Authors: Lam Matthew , Biancucci Marco , Satchell Karla JF 
Published: November 2023

Rat sarcoma protein (Ras) is a protein involved in the transduction of signals necessary for cell survival and proliferation. Mutations in Ras can inhibit its enzymatic function and leave it constitutively active, resulting in uncontrollable cell proliferation, culminating in tumor growth. In addition, many bacterial protein toxins and effector domains target Ras GTPases to destroy eukaryotic cells and reduce cellular response against bacterial infection. The Multifunctional‐Autoprocessing‐Repeats‐in‐Toxins (MARTX) toxin is the primary virulence factor of Vibrio vulnificus, a bacterium that causes sepsis and death from wound infections or contaminated seafood. The domain in the 5th position of MARTX toxins produced by V. vulnificus has been shown to cleave between Y32 and D33 residues within the Switch I region of all Ras isoforms, inhibiting the Ras‐MAPK pathway and subsequently cell proliferation.

Identification of the catalytic site of this Ras/Rap1‐specific endopeptidase (RRSP) was directed by bioinformatics suggesting that the C2B subdomain of RRSP is similar to the active sites of other enzymes such as bacterial erythromycin esterases EreA and EreB, and mammalian protein Tiki2. Despite these enzymes having different substrates, it was revealed that RRSP‐C2B shares highly conserved residues that form the active sites of the otherwise distinct proteins. Consequently, a putative active site of RRSP composed of two conserved glutamate and three histidine residues could be modeled.

Point mutations targeting these suspected catalytic residues were generated to assess the enzymatic activity of RRSP mutants. The purified mutants were then incubated with recombinant KRas. Alanine substitutions in three of the five conserved residues prevented in vitro processing of KRas only partially, suggesting that these residues play a more supportive role, such as substrate binding. However, mutations in the other two residues inhibited in vitro KRas processing entirely, and they were deemed as catalytic residues. Fluorescence thermal shift (FTS) was conducted to confirm that the structure of the RRSP mutant variants did not differ significantly from that of the wild type, indicating that the amino acid substitutions impacted enzyme activity and not the overall structural fold of the protein. Thus, the active site of RRSP is shown to be comprised of a pair of Glu/His residues and is most similar to that of the erythromycin esterase EreB. Unlike the metalloproteins EreA and Tiki2, treating RRSP with metal ions chelators such as EDTA and phenanthroline had no effect on substrate processing. Overall, these findings suggest that RRSP conserves a specific set of catalytic residues representative of a proposed erythromycin esterase‐Tiki family, within which it has novel protease activity divergent from the metalloproteases of this family.

Support or Funding Information

This work was supported by a Northwestern Undergraduate Research Grant (to M.R.L.), the Pancreatic Cancer Action Network KRas Post‐doctoral Fellowship (to M.B.) and grants from the National Institute of Allergy and Infectious Diseases R01AI092825 and R01AI098369 (to K.J.F.S.)