Characterization of the Membrane-targeting C1 Domain in Pasteurella multocida Toxin
Characterization of the Membrane-targeting C1 Domain in Pasteurella multocida Toxin
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DOI：
10.1074/jbc.m110.102285
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发表时间：
2010-08-13
影响因子：
4.8
通讯作者：
Horiguchi, Yasuhiko
中科院分区：
文献类型：
--
作者：
Kamitani, Shigeki;Kitadokoro, Kengo;Horiguchi, Yasuhiko
多杀性巴氏杆菌毒素（PMT）是导致某些形式巴氏杆菌病发病的毒力因子。毒素通过异三聚体gtp酶α亚基的谷氨酰胺残基脱酰胺激活G(q)-和G（12/13）依赖途径。我们最近报道了PMT （C-PMT）的C端（残基575-1285）的晶体结构，它由三个结构域（C1， C2和C3）组成，并且C1结构域参与C-PMT定位到质膜，C3结构域具有半胱氨酸蛋白酶样催化三联体。在本研究中，我们详细分析了C1结构域的膜靶向功能。C1结构域由7个螺旋组成，其中前4个（残基590670）与艰难梭菌毒素B的N端结构相似，参与了C-PMT向质膜的募集。缺乏这些螺旋的C-PMT （C-PMT δ C1(4H)）既不定位于质膜，也不刺激G（q/12/13）依赖的信号通路。当膜靶向特性被带有n -肉豆蔻酰基序的肽标签补充时，C-PMT δ C1（4H）恢复了PMT活性。表面等离子体共振分析证实了C1结构域与含磷脂脂质体之间的直接结合。这些结果表明，C-PMT的C1结构域是质膜的靶向信号。
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 590670), 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 Delta C1(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 Delta C1(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.