He M M, Voss J, Hubbell W L, Kaback H R
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90095-1662, USA.
Biochemistry. 1997 Nov 4;36(44):13682-7. doi: 10.1021/bi971531b.
By using a variety of biochemical and biophysical approaches, a helix packing model for the lactose permease of Escherichia coli has been proposed in which the four residues that are irreplaceable with respect to coupling are paired--Glu269 (helix VIII) with His322 (helix X) and Arg302 (helix XI) with Glu325 (helix X). In addition, the substrate translocation pathway is located at the interface between helices V and VIII, which is in close vicinity to the four essential residues. Based on this structural information and functional studies of mutants in the four irreplaceable residues, a molecular mechanism for energy coupling in the permease has been proposed [Kaback, H. R. (1997) Pro.c Natl. Acad. Sci. U.S.A. 94, 5539]. The principle idea of this model is that Arg302 interacts with either Glu325 or Glu269 during turnover. Evidence that Arg302 is in close proximity with Glu325 has been presented [Jung, K., Jung, H., Wu, J., Prive, G. G., & Kaback, H. R. (1993) Biochemistry 32, 12273; He, M. M., Voss, J., Hubbell, W. L., & Kaback, H. R. (1995) Biochemistry 34, 15667]; however, the proximity of Arg302 to Glu269 has not been examined. In this report, it is shown by two methods that Arg302 is also close to Glu269: (i) permease with Glu269-->His, Arg302-->His, and His322-->Phe binds Mn2+ with high affinity at pH 7.5, but not at pH 5.5; and (ii) site-directed spin-labeling of the double Cys mutant Glu269-->Cys/Arg302-->Cys exhibits spin-spin interaction with an interspin distance of about 14-16 A. In addition, the spin-spin interaction is stronger and interspin distance shorter after the permease is reconstituted into proteoliposomes. Taken as a whole, the data are consistent with the idea that Arg302 may interact with either Glu325 or Glu269 during turnover.
通过使用多种生化和生物物理方法，已提出了大肠杆菌乳糖通透酶的螺旋堆积模型，其中在偶联方面不可替代的四个残基形成配对——螺旋VIII的Glu269与螺旋X的His322配对，螺旋XI的Arg302与螺旋X的Glu325配对。此外，底物转运途径位于螺旋V和VIII之间的界面处，该界面紧邻四个必需残基。基于此结构信息以及对四个不可替代残基突变体的功能研究，已提出了通透酶中能量偶联的分子机制[卡巴克，H. R.（1997年）《美国国家科学院院刊》94，5539]。该模型的主要观点是，在周转过程中，Arg302与Glu325或Glu269相互作用。已有证据表明Arg302与Glu325距离很近[荣格，K.，荣格，H.，吴，J.，普里夫，G. G.，&卡巴克，H. R.（1993年）《生物化学》32，12273；何，M. M.，沃斯，J.，哈贝尔，W. L.，&卡巴克，H. R.（1995年）《生物化学》34，15667]；然而，尚未研究Arg302与Glu269的接近程度。在本报告中，通过两种方法表明Arg302也与Glu269接近：（i）将Glu269→His、Arg302→His和His322→Phe的通透酶在pH 7.5时能以高亲和力结合Mn2+，但在pH 5.5时不能；（ii）双半胱氨酸突变体Glu269→Cys/Arg302→Cys的定点自旋标记显示出自旋-自旋相互作用，自旋间距离约为14 - 16埃。此外，将通透酶重组到蛋白脂质体中后，自旋-自旋相互作用更强且自旋间距离更短。总体而言，这些数据与Arg302在周转过程中可能与Glu325或Glu269相互作用的观点一致。
