Szmelcman S, Sassoon N, Hofnung M
Unité de Programmation Moléculaire & Toxicologie Génétique-CNRS Ura 1444, Institut Pasteur, Paris, France.
Protein Sci. 1997 Mar;6(3):628-36. doi: 10.1002/pro.5560060312.
The periplasmic maltose binding protein, MalE, is a major element in maltose transport and in chemotaxis towards this sugar. Previous genetic analysis of the MalE protein revealed functional domains involved in transport and chemotactic functions. Among them the surface located alpha helix 7, which is part of the C-lobe, one of the two lobes forming the three dimensional structure of MalE. Small deletions in this region abolished maltose transport, although maintaining wild-type affinity and specificity as well as a normal chemoreceptor function. It was suggested that alpha helix 7 may be implicated in interactions between the maltose binding protein and the membrane-bound protein complex (Duplay P, Szmelcman S. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. II. Chemotaxis towards maltose. J Mol Biol 194:675-678: Duplay P, Szmelcman S, Bedouelle H, Hofnung M. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. I: Transport of maltose. J Mol Biol 194:663-673). In this study, we submitted a region of 14 residues--Asp 207 to Gly 220--encompassing alpha helix 7, to genetic analysis by oligonucleotide mediated random mutagenesis. Out of 127 identified mutations, twelve single and five double mutants with normal affinities towards maltose were selected for further investigation. Two types of mutations were characterized, silent mutations that did not affect maltose transport and mutations that heavily impaired transport kinetics, even thought the maltose binding capacity of the mutant proteins remained normal. Three substitutions at Tyr 210 (Y210S, Y210L, Y210N) drastically reduced maltose transport. One substitution at Ala 213 (A213I) and one substitution at Glu 214 (E214K) also impaired transport. These three identified residues, Tyr 210, Ala 213, and Glu 214, which are constituents of alpha helix 7, therefore seem to play some important role in maltose transport, most probably in a productive interaction between the MalE protein and the membrane bound MalFGK2 complex.
周质麦芽糖结合蛋白MalE是麦芽糖转运以及对该糖趋化作用的主要元件。先前对MalE蛋白的遗传学分析揭示了参与转运和趋化功能的功能结构域。其中位于表面的α螺旋7是C叶的一部分，C叶是构成MalE三维结构的两个叶之一。该区域的小缺失消除了麦芽糖转运，尽管保持了野生型亲和力和特异性以及正常的化学感受器功能。有人提出α螺旋7可能参与麦芽糖结合蛋白与膜结合蛋白复合物之间的相互作用（Duplay P，Szmelcman S. 1987.大肠杆菌K12周质麦芽糖结合蛋白的沉默和功能变化。II.对麦芽糖的趋化作用。《分子生物学杂志》194:675 - 678；Duplay P，Szmelcman S，Bedouelle H，Hofnung M. 1987.大肠杆菌K12周质麦芽糖结合蛋白的沉默和功能变化。I：麦芽糖的转运。《分子生物学杂志》194:663 - 673）。在本研究中，我们通过寡核苷酸介导的随机诱变对包含α螺旋7的14个残基区域（Asp 207至Gly 220）进行了遗传分析。在127个鉴定出的突变中，选择了12个对麦芽糖具有正常亲和力的单突变体和5个双突变体进行进一步研究。鉴定出了两种类型的突变，即不影响麦芽糖转运的沉默突变和严重损害转运动力学的突变，尽管突变蛋白的麦芽糖结合能力保持正常。Tyr 210处的三个取代（Y210S、Y210L、Y210N）极大地降低了麦芽糖转运。Ala 213处的一个取代（A213I）和Glu 214处的一个取代（E214K）也损害了转运。因此，这三个已鉴定的残基Tyr 210、Ala 213和Glu 214作为α螺旋7的组成部分，似乎在麦芽糖转运中发挥了一些重要作用，很可能在MalE蛋白与膜结合的MalFGK2复合物之间的有效相互作用中发挥作用。
