Wilken S, Schmees G, Schneider E
Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, Germany.
Mol Microbiol. 1996 Nov;22(4):655-66. doi: 10.1046/j.1365-2958.1996.d01-1724.x.
The ATP-binding-cassette (ABC) protein LacK of Agrobacterium radiobacter displays high sequence similarity to the MalK subunit of the Salmonella typhimurium maltose-transport system (MalFGK2). We have used LacK as a tool to identify sites of interaction of MalK with the membrane-integral components MalF and MalG. Small amounts of LacK, resulting from the expression of the plasmid-borne lacK gene, proved to be sufficient for partial restoration of growth of a malK strain of S. typhimurium on maltose. LacK failed to substitute for MalK in regulating the expression of maltose-inducible genes but the hybrid complex MalFGLacK2 was sensitive to inducer exclusion. The lacK gene also complemented a ugpC mutant of Escherichia coli to growth on sn-glycerol-3-phosphate as the phosphate source. Partially purified LacK exhibited a spontaneous ATPase activity comparable to that of MalK. A MalK"-'LacK chimeric protein was isolated (by in vivo recombination) in which the N-terminal 140 amino acids of MalK are fused to residues 141-363 of LacK. The protein substituted for MalK in maltose transport considerably better than LacK. Furthermore, random mutagenesis of the plasmid-borne lacK gene yielded three clones that were superior to wild-type lacK in complementing a malK mutation. Single mutations (V114M or L123F) substantially improved the growth of a malK strain on maltose, whereas a double mutation (L123F, S295N) resulted in growth and transport rates that were indistinguishable from those obtained with MalK. In contrast, the introduction of the single change S295N into LacK had no effect but combination with the V114M mutation led to a further twofold increase in transport activity. These results indicate that a putative helical domain in MalK, encompassing residues 89-140, is crucial for a functional, high-affinity interaction with MalF and MalG.
放射土壤杆菌的ATP结合盒（ABC）蛋白LacK与鼠伤寒沙门氏菌麦芽糖转运系统（MalFGK2）的MalK亚基具有高度的序列相似性。我们利用LacK作为工具来确定MalK与膜整合成分MalF和MalG的相互作用位点。由质粒携带的lacK基因表达产生的少量LacK，被证明足以部分恢复鼠伤寒沙门氏菌malK菌株在麦芽糖上的生长。LacK在调节麦芽糖诱导基因的表达方面无法替代MalK，但杂交复合物MalFGLacK2对诱导剂排斥敏感。lacK基因也能互补大肠杆菌的ugpC突变体，使其能够以sn-甘油-3-磷酸作为磷源生长。部分纯化的LacK表现出与MalK相当的自发ATP酶活性。通过体内重组分离出一种MalK"-'LacK嵌合蛋白，其中MalK的N端140个氨基酸与LacK的141-363位残基融合。该蛋白在麦芽糖转运中替代MalK的效果比LacK好得多。此外，对质粒携带的lacK基因进行随机诱变产生了三个克隆，它们在互补malK突变方面优于野生型lacK。单突变（V114M或L123F）显著改善了malK菌株在麦芽糖上的生长，而双突变（L123F，S295N）导致的生长和转运速率与MalK的情况难以区分。相比之下，将单一变化S295N引入LacK没有效果，但与V114M突变结合会使转运活性进一步提高两倍。这些结果表明，MalK中一个假定的螺旋结构域，包含89-140位残基，对于与MalF和MalG进行功能性的高亲和力相互作用至关重要。
