Abstract
Pasteurella multocida toxin (PMT) is a virulence factor responsible for the pathogenesis of some forms of pasteurellosis. The toxin activates G(q)- and G(12/13)-dependent pathways through the deamidation of a glutamine residue in the alpha-subunit of heterotrimeric GTPases. We recently reported the crystal structure of the C terminus (residues 575-1285) of PMT (C-PMT), which is composed of three domains (C1, C2, and C3), and that the C1 domain is involved in the localization of C-PMT to the plasma membrane, and the C3 domain possesses a cysteine protease-like catalytic triad. In this study, we analyzed the membrane-targeting function of the C1 domain in detail. The C1 domain consists of seven helices of which the first four (residues 590-670), showing structural similarity to the N terminus of Clostridium difficile toxin B, were found to be involved in the recruitment of C-PMT to the plasma membrane. C-PMT lacking these helices (C-PMT DeltaC1(4H)) neither localized to the plasma membrane nor stimulated the G(q/12/13)-dependent signaling pathways. When the membrane-targeting property was complemented by a peptide tag with an N-myristoylation motif, C-PMT DeltaC1(4H) recovered the PMT activity. Direct binding between the C1 domain and liposomes containing phospholipids was evidenced by surface plasmon resonance analyses. These results indicate that the C1 domain of C-PMT functions as a targeting signal for the plasma membrane.
Highlights
Pasteurella multocida toxin (PMT)2 is a highly potent mitogen for various types of cultured cells including fibroblasts and osteoblastic cells [1,2,3]
We recently reported the crystal structure of the C terminus of PMT (C-PMT), which is composed of three domains (C1, C2, and C3), and that the C1 domain is involved in the localization of C-PMT to the plasma membrane, and the C3 domain possesses a cysteine proteaselike catalytic triad
C1 Domain of C-PMT Functions as Targeting Signal for Plasma Membrane—We have recently determined the crystal structure of C-PMT, which exhibits intracellular toxicity [23], and found that it was composed of three distinct domains, 3 W
Summary
Pasteurella multocida toxin (PMT) is a highly potent mitogen for various types of cultured cells including fibroblasts and osteoblastic cells [1,2,3]. Previous studies implied that the toxin is internalized by endocytosis after binding to a putative receptor on target cells and escapes from endosomes into the cytoplasm [7, 16] where it activates heterotrimeric GTPases through deamidation. In the C-terminal region of PMT, there is neither a known catalytic motif nor a region homologous with any other toxin or enzyme. The results revealed that the C1 domain, essential to the toxicity of PMT, was exchangeable with a different signal motif for membrane targeting. The direct binding of the C1 domain with liposomes including phospholipids was demonstrated From these results, we conclude that the C1 domain functions to recruit PMT to the plasma membrane where the heterotrimeric GTPases, the substrates for the toxin, reside
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