Melchers K, Weitzenegger T, Buhmann A, Steinhilber W, Sachs G, Schäfer K P
Byk Gulden Pharmaceuticals, Department of Molecular Biology, Konstanz, Germany.
J Biol Chem. 1996 Jan 5;271(1):446-57. doi: 10.1074/jbc.271.1.446.
Southern blot screening of a genomic Helicobacter pylori library was employed to find a P type ATPase using a mixture of 16 DNA oligonucleotides coding for the DKTGT(I/L)T consensus sequence specific for the phosphorylation site of this family of ATPases. A positive clone, pRH439, was isolated and sequenced. The inserted 3.4-kb H. pylori DNA contained an intact open reading frame encoding a protein of 686 amino acids carrying the consensus sites for phosphorylation and ATP binding. The amino acid sequence exhibits a 25-30% identity with bacterial Cd2+ and Cu2+ ATPases. Genomic Southern blot analysis showed that this ATPase was present in all H. pylori strains examined, whereas it was not detectable in Campylobacter jejuni and other bacteria. The membrane topology of this ATPase was investigated using in vitro transcription/translation of fusion vectors to find signal anchor and/or stop transfer sequences. Eight regions of the H. pylori ATPase acted as signal anchor and/or stop transfer sequences and were ordered pairwise along the polypeptide chain placing the N and C-terminal amino acids in the cytoplasm. These transmembrane segments are contained between positions 73 and 92 (H1), 98 and 125 (H2), 128 and 148 (H3), 149 and 176 (H4), 309 and 327 (H5), 337 and 371 (H6), 637 and 658 (H7), and 659 and 685 (H8). The membrane domain of the ATPase, therefore, consists of at least four pairs of transmembrane segments with the phosphorylation site and ATP binding domain located in the large cytoplasmic loop between H6 and H7. The cytoplasmic domain contains several histidines and cysteines, perhaps indicative of divalent cation binding sites. There are several charged amino acids (3 Lys, 2 Glu, 2 Asp), predicted to be in the membrane domain mainly in H2, H3, and H4 and a Cys-Pro-Cys putative metal ion site in H6. The extracytoplasmic domain also has several charged amino acids (5 Glu, 1 Asp, 1 Lys, 1 Arg). It is likely that this novel protein is a heavy metal cation transporting ATPase and belongs to a family of P type ATPases containing eight transmembrane segments.
利用编码DKTGT(I/L)T共有序列的16种DNA寡核苷酸混合物，对幽门螺杆菌基因组文库进行Southern杂交筛选，以寻找P型ATP酶。筛选出一个阳性克隆pRH439并进行测序。插入的3.4kb幽门螺杆菌DNA包含一个完整的开放阅读框，编码一个686个氨基酸的蛋白质，该蛋白质带有磷酸化和ATP结合的共有位点。氨基酸序列与细菌的Cd2+和Cu2+ATP酶有25%-30%的同源性。基因组Southern杂交分析表明，这种ATP酶存在于所有检测的幽门螺杆菌菌株中，而在空肠弯曲菌和其他细菌中未检测到。利用融合载体的体外转录/翻译研究了这种ATP酶的膜拓扑结构，以寻找信号锚定和/或终止转移序列。幽门螺杆菌ATP酶的8个区域作为信号锚定和/或终止转移序列，沿多肽链成对排列，使N端和C端氨基酸位于细胞质中。这些跨膜区段位于第73至92位（H1）、98至125位（H2）、128至148位（H3）、149至176位（H4）、309至327位（H5）、337至371位（H6）、637至658位（H7）和659至685位（H8）之间。因此，该ATP酶的膜结构域至少由四对跨膜区段组成，磷酸化位点和ATP结合结构域位于H6和H7之间的大细胞质环中。细胞质结构域含有几个组氨酸和半胱氨酸，可能指示二价阳离子结合位点。有几个带电荷的氨基酸（3个赖氨酸、2个谷氨酸、2个天冬氨酸），预计主要在H2、H3和H4的膜结构域中，以及H6中的一个半胱氨酸-脯氨酸-半胱氨酸假定金属离子位点。胞外结构域也有几个带电荷的氨基酸（5个谷氨酸、1个天冬氨酸、1个赖氨酸、1个精氨酸）。这种新蛋白质可能是一种重金属阳离子转运ATP酶，属于含有八个跨膜区段的P型ATP酶家族。
