Analysis of Shigella flexneri Wzz (RoI) function by mutagenesis and cross-linking:: Wzz is able to oligomerize
Analysis of Shigella flexneri Wzz (RoI) function by mutagenesis and cross-linking:: Wzz is able to oligomerize
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DOI：
10.1046/j.1365-2958.1999.01591.x
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发表时间：
1999-10-01
影响因子：
3.6
通讯作者：
Morona, R
中科院分区：
文献类型：
--
作者：
Daniels, C;Morona, R
志贺氏菌O抗原的模式长度或聚合度(DP)由WZZ/Rol蛋白以一种未知的方式决定。WZZ蛋白通过两个跨膜区(TM1氨基酸32-52和TM2氨基酸295-315)锚定在细胞膜上，其中心环位于周质中。构建了由福氏志贺氏菌WZZ区(WZZ(SF))和鼠伤寒沙门氏菌WZZ区(WZZ(ST))组成的WZZ杂合蛋白编码载体。用定点突变的方法研究了WZZ(SF)氨基/羧基末端高度保守残基的功能意义。一些WZZ(SF)突变体的O抗原模式链长远短于野生型WZZ(SF)，而另一些突变体则完全失活了WZZ(SF)的功能，第三类突变体具有更长的O抗原链长度分布。这些数据表明，WZZ(SF)蛋白全长的氨基酸在确定O-抗原模式链长度时是重要的。通过体内交联实验研究了WZZ蛋白之间的相互作用。实验表明，WZZ(SF)蛋白能够形成至少6个WZZ(SF)蛋白的二聚体和寡聚体。一个氨基酸端194个氨基酸的羧基末端截短的WZZ(SF)蛋白能够齐聚，表明氨基末端区域足以产生WZZ-WZZ相互作用。在氨基末端有内部缺失的缩短的WZZ(SF)蛋白也能够寡聚，这表明59-194残基不是寡聚所必需的。WZZ(SF)蛋白与突变残基的交联表明，WZZ(SF)功能的丧失可能与形成低聚物的能力降低/改变有关，而TM2段甘氨酸残基的突变改变影响了WZZ(SF)-WZZ(SF)在SDS聚丙烯酰胺凝胶中的二聚体迁移率。这些结果为参与O抗原多糖生物合成的蛋白质之间的相互作用提供了第一个证据。
The modal length or degree of polymerization (dp) of the Shigella flexneri O-antigen is determined in an unknown manner by the Wzz/Rol protein. The Wzz protein is anchored into the cytoplasmic membrane by two transmembrane domains (TM1 amino acids 32-52; TM2 amino acids 295-315) with the central loop of the protein located in the periplasm. Plasmids were constructed encoding hybrid Wzz proteins consisting of regions of S. flexneri Wzz (Wzz(SF)) and Salmonella typhimurium Wzz (Wzz(ST)). These imparted O-antigen modal chain lengths that implied that the carboxy-terminal region of Wzz was involved in chain length determination, Site-directed mutagenesis was undertaken to investigate the functional significance of highly conserved residues in amino-/carboxy-terminal domains of Wzz(SF) Some of the Wzz(SF) variants resulted in O-antigen modal chain lengths much shorter than those of wild-type Wzz(SF), whereas other mutants inactivated Wzz(SF) function entirely and a third class had a longer O-antigen chain length distribution. The data indicate that amino acids throughout the length of the Wzz(SF) protein are important in determination of O-antigen modal chain length. In vivo cross-linking experiments were performed to investigate the interactions between Wzz proteins. The experiments indicated that the Wzz(SF) protein is able to form dimers and oligomers of at least six Wzz(SF) proteins. A carboxy-terminal-truncated Wzz(SF) protein having the amino terminal 194 amino acids was able to oligomerize, indicating that the aminoterminal region is sufficient for the Wzz-Wzz interaction observed. Shortened Wzz(SF) proteins having internal deletions in the amino-terminal region were also able to oligomerize, suggesting that residues 59-194 are not essential for oligomerization. Crosslinking of Wzz(SF) proteins with mutationally altered residues showed that loss of Wzz(SF) function may be correlated to a reduced/altered ability to form oligomers, and that mutational alteration of glycine residues in the TM2 segment affects Wzz(SF)-Wzz(SF) dimer mobility in SDS polyacrylamide gels. These results provide the first evidence of protein-protein interactions for proteins involved in O-antigen polysaccharide biosynthesis.