Role of prolyl cis/trans isomers in cyclophilin-assisted Pseudomonas syringae AvrRpt2 protease activation.
In a process contributing to the innate immunity of higher plants, Arabidopsis thaliana cyclophilin ROC1 induces the self-cleavage of Pseudomonas syringae putative cysteine protease AvrRpt2, triggering limited cleavage of A. thaliana RIN4, a negative regulator of plant immunity. We report an increase in AvRpt2 activity in hydrolysis of decapeptide substrates at -GG- sites of more than 5 orders of magnitude, in the presence of cyclophilin-like peptidyl prolyl cis/trans isomerases including ROC1 or hCyp18. Both full-length AvrRpt2 and its 21 kDa self-cleavage product (AvrRpt2(72-255)) were found to be equally active under these conditions. In contrast to classical isomer-specific proteolysis, inertness toward cleavage of a cis/trans prolyl bond isomer at the substrate P4 subsite is not the cause of cyclophilin-mediated activation of the proteolytic reaction. Monitoring single- and double-jump kinetics of proteolytic reactions in the presence of the PPIase inhibitor cyclosporin A revealed that the cis/trans ratio of potentially relevant prolyl bonds of AvrRpt2(72-255) remained the same in the functionally inactive state of AvrRpt2(72-255) and the productive AvrRpt2(72-255)-cyclophilin-substrate complex.
Aumüller
Tobias
T
Max-Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, D-06120 Halle/Saale, Germany.
Jahreis
Günther
G
Fischer
Gunter
G
Schiene-Fischer
Cordelia
C
D017360:Arabidopsis / Q000201:enzymology* / Q000382:microbiology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D021983:Cyclophilins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D006868:Hydrolysis; D019696:Peptidylprolyl Isomerase / Q000737:chemistry / Q000378:metabolism; D011392:Proline / Q000737:chemistry*; D044224:Pseudomonas syringae / Q000201:enzymology* / Q000472:pathogenicity; D013237:Stereoisomerism; D013379:Substrate Specificity / Q000502:physiology; D014774:Virulence
D029681:Arabidopsis Proteins; D001426:Bacterial Proteins; C420919:avrRpt2 protein, Pseudomonas syringae; C437224:cyclophilin hCyp-18; D011392:Proline; D021983:Cyclophilins; C515035:ROC1 protein, Arabidopsis; D019696:Peptidylprolyl Isomerase