大肠杆菌γ-氨基丁酸转运蛋白GabP(gab通透酶)在其共有两亲性区域(CAR)内含有功能上重要的半胱氨酸残基(Cys-300),一种推定的通道形成结构,从跨膜螺旋8延伸到胺-多胺-胆碱(APC)超家族转运蛋白的相邻细胞质环8-9中。在这里,我们显示了大肠杆菌GabP中的五个半胱氨酸残基(位置158、251、291、300和443),Cys-300是使运输活性对硫醇修饰试剂的抑制作用敏感的一种:而用Ala代替Cys-300模拟了硫醇修饰的抑制作用,在158、251、291或443位取代Ala保留了强大的运输活性,并且不赋予对硫醇失活的抗性;而强大活性的Cys-300单Cys突变体对硫醇修饰完全敏感,其他单Cys突变体(Cys为158、251、291或443)表现出动力学受损的运输活性,可抵抗硫醇试剂的进一步化学失活。本研究还表明,Cys-300表现出(1)对疏水硫醇试剂的敏感性,(2)对大体积(荧光素5-马来酰亚胺)和/或带电荷的{2-磺乙基甲乙磺酸酯或[2-(三甲基铵)乙基]甲乙磺酸酯}硫醇试剂的一般抗性,和(3)对对氯代苯磺酸盐(PCMBS)的特殊敏感性。PCMBS对Cys-300(位于脂质双层的中途)的可及性可能与它与古瓦丁(1,2,3,6-四氢-3-吡啶羧酸)共有的结构相似性有关,运输的GabP底物。这些对硫醇敏感性的结构要求提供了第一个与通道状接近CAR极性表面相一致的化学证据。一种物理配置,可能为理解该区域通常如何影响APC运输机的功能提供基础[Closs,里昂,凯利和坎宁安(1993)J.Biol。Chem.268,20796-20800]和特别是渗透酶[Hu和King(1998)生物化学。J.300,771-776]。
The Escherichia coli gamma-aminobutyric acid transporter GabP (gab permease) contains a functionally significant cysteine residue (Cys-300) within its
consensus amphipathic region (CAR), a putative channel-forming structure that extends out of transmembrane helix 8 and into the adjoining cytoplasmic loop 8-9 of transporters from the amine-polyamine-choline (APC) superfamily. Here we show that of the five cysteine residues (positions 158, 251, 291, 300 and 443) in the E. coli GabP, Cys-300 is the one that renders the transport activity sensitive to inhibition by thiol modification reagents: whereas substituting Ala for Cys-300 mimics the inhibitory effect of thiol modification, substituting Ala at position 158, 251, 291 or 443 preserves robust transport activity and confers no resistance to thiol inactivation; and whereas the robustly active Cys-300 single-Cys mutant is fully sensitive to thiol modification, other single-Cys mutants (Cys at 158, 251, 291 or 443) exhibit kinetically compromised transport activities that resist further chemical inactivation by thiol reagents. The present study reveals additionally that Cys-300 exhibits (1) sensitivity to hydrophobic thiol reagents, (2) general resistance to bulky (fluorescein 5-maleimide) and/or charged {2-sulphonatoethyl methanethiosulphonate or [2-(trimethylammonium)ethyl] methanethiosulphonate} thiol reagents and (3) a peculiar sensitivity to p-chloromercuribenzenesulphonate (PCMBS). The accessibility of PCMBS to Cys-300 (located midway through the lipid bilayer) might be related to the structural similarity that it shares with guvacine (1, 2,3,6-tetrahydro-3-pyridinecarboxylic acid), a transported GabP substrate. These structural requirements for thiol sensitivity provide the first chemical evidence consistent with channel-like access to the polar surface of the CAR, a physical configuration that might provide a basis for understanding how this region impacts the function of APC transporters generally [Closs, Lyons, Kelly and Cunningham (1993) J. Biol. Chem. 268, 20796-20800] and the gab permease particularly [Hu and King (1998) Biochem. J. 300, 771-776].