Characterization of the membrane-targeting C1 domain in Pasteurella multocida toxin.
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.
Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita-shi, Osaka 565-0871, Japan. skami@biken.osaka-u.ac.jp
Shigeki
S
Kamitani
Kengo
K
Kitadokoro
Masayuki
M
Miyazawa
Hirono
H
Toshima
Aya
A
Fukui
Hiroyuki
H
Abe
Masami
M
Miyake
Yasuhiko
Y
Horiguchi
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
D001426:Bacterial Proteins; D001427:Bacterial Toxins; C062258:Pasteurella multocida toxin; D010743:Phospholipids