Center for Human Immunology, Siebens-Drake Research Institute, Departments of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada.
J Bacteriol. 2010 Dec;192(23):6160-71. doi: 10.1128/JB.00141-10. Epub 2010 Sep 24.
Wzx belongs to a family of membrane proteins involved in the translocation of isoprenoid lipid-linked glycans, which is loosely related to members of the major facilitator superfamily. Despite Wzx homologs performing a conserved function, it has been difficult to pinpoint specific motifs of functional significance in their amino acid sequences. Here, we elucidate the topology of the Escherichia coli O157 Wzx (Wzx(EcO157)) by a combination of bioinformatics and substituted cysteine scanning mutagenesis, as well as targeted deletion-fusions to green fluorescent protein and alkaline phosphatase. We conclude that Wzx(EcO157) consists of 12 transmembrane (TM) helices and six periplasmic and five cytosolic loops, with N and C termini facing the cytoplasm. Four TM helices (II, IV, X, and XI) contain polar residues (aspartic acid or lysine), and they may form part of a relatively hydrophilic core. Thirty-five amino acid replacements to alanine or serine were targeted to five native cysteines and most of the aspartic acid, arginine, and lysine residues. From these, only replacements of aspartic acid-85, aspartic acid-326, arginine-298, and lysine-419 resulted in a protein unable to support O-antigen production. Aspartic acid-85 and lysine-419 are located in TM helices II and XI, while arginine-298 and aspartic acid-326 are located in periplasmic and cytosolic loops 4, respectively. Further analysis revealed that the charge at these positions is required for Wzx function since conservative substitutions maintaining the same charge polarity resulted in a functional protein, whereas those reversing or eliminating polarity abolished function. We propose that the functional requirement of charged residues at both sides of the membrane and in two TM helices could be important to allow the passage of the Und-PP-linked saccharide substrate across the membrane.
Wzx 属于参与异戊烯脂质连接聚糖易位的膜蛋白家族，与主要易化剂超家族的成员关系松散。尽管 Wzx 同源物执行保守功能，但很难确定其氨基酸序列中具有功能意义的特定模体。在这里，我们通过生物信息学和取代半胱氨酸扫描诱变以及针对绿色荧光蛋白和碱性磷酸酶的靶向缺失融合，阐明了大肠杆菌 O157 Wzx（Wzx(EcO157)）的拓扑结构。我们得出的结论是，Wzx(EcO157)由 12 个跨膜 (TM) 螺旋和 6 个周质和 5 个细胞质环组成，N 和 C 末端朝向细胞质。四个 TM 螺旋 (II、IV、X 和 XI) 包含极性残基（天冬氨酸或赖氨酸），它们可能构成相对亲水核心的一部分。针对五个天然半胱氨酸和大部分天冬氨酸、精氨酸和赖氨酸残基，靶向 35 个氨基酸替换为丙氨酸或丝氨酸。其中，只有天冬氨酸-85、天冬氨酸-326、精氨酸-298 和赖氨酸-419 的替换导致无法支持 O-抗原产生的蛋白质。天冬氨酸-85 和赖氨酸-419 位于 TM 螺旋 II 和 XI 中，而精氨酸-298 和天冬氨酸-326 分别位于周质和细胞质环 4 中。进一步分析表明，这些位置的电荷对于 Wzx 功能是必需的，因为保持相同电荷极性的保守取代导致功能蛋白，而那些反转或消除极性的取代则使功能丧失。我们提出，跨膜两侧和两个 TM 螺旋中带电荷残基的功能要求对于允许 Und-PP 连接的糖底物穿过膜可能很重要。
