Functional dissection of a conserved motif within the pilus retraction protein PilT
Functional dissection of a conserved motif within the pilus retraction protein PilT
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DOI：
10.1128/jb.187.2.611-618.2005
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发表时间：
2005-01-01
影响因子：
3.2
通讯作者：
Forest, KT
中科院分区：
文献类型：
--
作者：
Aukema, KG;Kron, EM;Forest, KT
PilT是革兰氏阴性菌中IV型菌毛收缩所需的六聚体ATP酶。IV型皮利的收缩介导与宿主细胞的紧密附着和宿主细胞中的信号传导、表面运动性、生物膜形成、自然转化和噬菌体敏感性。我们研究了PilT中不同的、高度保守的C-末端AIRNLIRE基序的每个氨基酸在体内和体外的作用。氨基酸A288、I289、L292和I293的取代以及R290和R294的双取代在体内消除了铜绿假单胞菌PilT功能，如通过表面运动性和噬菌体敏感性的损失所测量的。当引入纯化的风产液菌PilT中时，AIRNLIRE基序中的取代不会破坏ATP酶活性或寡聚化。相比之下，广泛保守的核苷酸结合基序中的K136 Q取代阻止了体内和体外PilT功能。我们建议，AIRNLIRE基序形成一个两亲性的α螺旋，它传递信号之间的表面暴露的蛋白质相互作用位点和ATP酶的核心PilT，我们认识到一个潜在的功能同源性在其他II型分泌ATP酶。
PilT is a hexameric ATPase required for type IV pilus retraction in gram-negative bacteria. Retraction of type IV pili mediates intimate attachment to and signaling in host cells, surface motility, biofilm formation, natural transformation, and phage sensitivity. We investigated the in vivo and in vitro roles of each amino acid of the distinct, highly conserved C-terminal AIRNLIRE motif in PilT. Substitution of amino acids A288, I289, L292, and I293 as well as a double substitution of R290 and R294 abolished Pseudomonas aeruginosa PilT function in vivo, as measured by a loss of surface motility and phage sensitivity. When introduced into purified Aquifex aeolicus PilT, substitutions in the AIRNLIRE motif did not disrupt ATPase activity or oligomerization. In contrast, a K136Q substitution in the broadly conserved nucleotide binding motif prevented PilT function in vivo as well as in vitro. We propose that the AIRNLIRE motif forms an amphipathic alpha helix which transmits signals between a surface-exposed protein interaction site and the ATPase core of PilT, and we recognize a potential functional homology in other type II secretion ATPases.