Division of Biomedical Science and Biochemistry, Research School of Biology, Australian National University, Canberra ACT 0200, Australia.
BMC Biochem. 2011 Dec 22;12:67. doi: 10.1186/1471-2091-12-67.
The three bacteriophage genes gtrA, gtrB and gtr((type)) are responsible for O-antigen glucosylation in Shigella flexneri. Both gtrA and gtrB have been demonstrated to be highly conserved and interchangeable among serotypes while gtr((type)) was found to be specific to each serotype, leading to the hypothesis that the Gtr((type)) proteins are responsible for attaching glucosyl groups to the O-antigen in a site- and serotype- specific manner. Based on the confirmed topologies of GtrI, GtrII and GtrV, such interaction and attachment of the glucosyl groups to the O-antigen has been postulated to occur in the periplasm.
In this study, the topology of GtrIV was experimentally determined by creating different fusions between GtrIV and a dual-reporter protein, PhoA/LacZ. This study shows that GtrIV consists of 8 transmembrane helices, 2 large periplasmic loops, 2 small cytoplasmic N- and C- terminal ends and a re-entrant loop that occurs between transmembrane helices III and IV. Though this topology differs from that of GtrI, GtrII, GtrV and GtrX, it is very similar to that of GtrIc. Furthermore, both the N-terminal periplasmic and the C-terminal periplasmic loops are important for GtrIV function as shown via a series of loop deletion experiments and the creation of chimeric proteins between GtrIV and its closest structural homologue, GtrIc.
The current study provides the basis for elucidating the structure and mechanism of action of this important O-antigen modifying glucosyltransferase.
三种噬菌体基因 gtrA、gtrB 和 gtr((type)) 负责志贺氏菌的 O-抗原葡糖基化。gtrA 和 gtrB 已被证明在血清型之间高度保守且可互换，而 gtr((type)) 则被发现每种血清型都有特异性，这导致了这样一种假设，即 Gtr((type)) 蛋白负责以特定于位点和血清型的方式将葡糖基基团连接到 O-抗原上。基于 GtrI、GtrII 和 GtrV 的已证实拓扑结构，这种相互作用和葡糖基基团与 O-抗原的连接被推测发生在周质中。
在这项研究中，通过在 GtrIV 和双报告蛋白 PhoA/LacZ 之间创建不同的融合，实验确定了 GtrIV 的拓扑结构。这项研究表明，GtrIV 由 8 个跨膜螺旋、2 个大的周质环、2 个小的细胞质 N 和 C 末端以及在跨膜螺旋 III 和 IV 之间发生的再进入环组成。尽管这种拓扑结构与 GtrI、GtrII、GtrV 和 GtrX 不同，但与 GtrIc 非常相似。此外，正如一系列环缺失实验和在 GtrIV 和其最接近的结构同源物 GtrIc 之间创建嵌合蛋白所表明的那样，N 端周质环和 C 端周质环对于 GtrIV 功能都很重要。
本研究为阐明这种重要的 O-抗原修饰葡糖基转移酶的结构和作用机制提供了基础。
