Crystal structures reveal a thiol protease-like catalytic triad in the C-terminal region ofPasteurella multocidatoxin

Abstract

Pasteurella multocida toxin (PMT), one of the virulence factors produced by the bacteria, exerts its toxicity by up-regulating various signaling cascades downstream of the heterotrimeric GTPases Gq and G12/13 in an unknown fashion. Here, we present the crystal structure of the C-terminal region (residues 575-1,285) of PMT, which carries an intracellularly active moiety. The overall structure of C-terminal region of PMT displays a Trojan horse-like shape, composed of three domains with a ''feet''-,''body''-, and ''head''-type arrangement, which were designated C1, C2, and C3 from the N to the C terminus, respectively. The C1 domain, showing marked similarity in steric structure to the N-terminal domain of Clostridium difficile toxin B, was found to lead the toxin molecule to the plasma membrane. The C3 domain possesses the Cys-HisAsp catalytic triad that is organized only when the Cys is released from a disulfide bond. The steric alignment of the triad corresponded well to that of papain or other enzymes carrying CysHis-Asp. PMT toxicities on target cells were completely abrogated when one of the amino acids constituting the triad was mutated. Our results indicate that PMT is an enzyme toxin carrying the cysteine protease-like catalytic triad dependent on the redox state and functions on the cytoplasmic face of the plasma membrane of target cells.crystallography ͉ cysteine protease ͉ membrane targeting

Pasteurella multocida toxin activation of heterotrimeric G proteins by deamidation

“…S6C). It has been shown that the positioning of the catalytic triad of PMT is very similar to the catalytic triad of papain and mainly deduced from this similarity PMT has been proposed to act as a protease (19). Our findings indicate that PMT acts as a deamidase to activate G proteins.…”

“…Interestingly, PMT shares similarity with CNFs in the N-terminal binding and translocation domains. However, the C-terminal catalytic domain of CNFs has no obvious structural similarity with PMT, as evidenced by comparison of the crystal structure of the C-terminal C3 domain of PMT (19), harboring the minimal intracellular activity domain (20), with that of the catalytic domain of CNF1 (33) (Fig. S6 A and B).…”

“…A C-terminal fragment of PMT, which consists of 3 domains, covering amino acids 569 to 1285, has been crystallized (19). The structure revealed that the C-terminal C3 domain (residues 1105-1285), which defines the minimal intracellular activity domain responsible for activation of calcium and mitogenic signaling (20), resembles that of a papain-like protease.…”

TIR-only protein RBA1 recognizes a pathogen effector to regulate cell death inArabidopsis

“…This superfamily (Fig. S6) consisted of ChaN, a heme-binding protein from Campylobacter jejuni (32), the EreA-like esterase Bcr136 from Bacillus cereus (33), and a portion of the C2 domain of Pasteurella multocida dermonecrotic toxin (PMT) (34). Modeling of HopBA1-like P. syringae effector proteins revealed that the T3E HopB1 (Fig.…”

Mammalian Target of Rapamycin Complex 1 (mTORC1) Plays a Role in Pasteurella multocida Toxin (PMT)-induced Protein Synthesis and Proliferation in Swiss 3T3 Cells

“…The gene (toxA) encoding P. multocida toxin (PMT), 3 acquired by horizontal transmission (2), has been cloned and sequenced (3). It is a single polypeptide of 146 kDa whose C-terminal activity domain structure has been solved (4). In addition to its mitogenic properties for certain types of cells, including quiescent fibroblast and osteoclast cells, PMT is a strong inducer of anchorageindependent growth (5-7).…”