Ding Haiyuan, Hunt John F, Mukerji Ishita, Oliver Donald
Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459, USA.
Biochemistry. 2003 Jul 29;42(29):8729-38. doi: 10.1021/bi0342057.
SecA ATPase promotes the biogenesis of membrane and secretory proteins into and across the cytoplasmic membrane of Eubacteria. SecA binds to translocon component SecYE and substrate proteins and undergoes ATP-dependent conformational cycles that are coupled to the stepwise translocation of proteins. Our recent crystal structure of B. subtilis SecA [Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., and Deisenhofer, J. (2002) Science 297, 2018-2026] showed two different dimer interactions in the lattice which both buried significant solvent-accessible surface area in their interface and could potentially be responsible for formation of the physiological dimer in solution. In this paper, we utilize fluorescence resonance energy transfer methodology with genetically engineered SecA proteins containing unique pairs of tryptophan and fluorophore-labeled cysteine residues to determine the oligomeric structure of SecA protein in solution. Our results show that of the two dimers interactions observed in the crystal structure, SecA forms an antiparallel dimer in solution that maximizes the buried solvent-accessible surface area and intermolecular contacts. At the submicromolar protein concentrations used in the fluorescence experiments, we saw no evidence for the formation of higher-order oligomers of SecA based on either the alternative dimer or the 3(1) helical fiber observed in the crystal lattice. Our studies are consistent with previous ones demonstrating the existence of a dimerization determinant within the C-domain of SecA as well as those documenting the interaction of N- and C-domains of SecA. Our results also provide a valuable starting point for a determination of whether the subunit status of SecA changes during the protein translocation as well as studies designed to elucidate the conformational dynamics of this multidomain protein during its translocation cycle.
SecA ATP酶促进真细菌的膜蛋白和分泌蛋白进入并穿过细胞质膜的生物合成过程。SecA与转运体组分SecYE及底物蛋白结合，并经历依赖ATP的构象循环，该循环与蛋白的逐步转运相偶联。我们最近解析的枯草芽孢杆菌SecA的晶体结构[亨特，J.F.，温考夫，S.，亨利，L.，法克，J.J.，麦克尼古拉斯，P.，奥利弗，D.B.，和戴森霍费尔，J.（2002年）《科学》297卷，2018 - 2026页]显示，晶格中有两种不同的二聚体相互作用，二者在其界面均掩埋了大量可溶剂接触表面积，并且可能是溶液中生理二聚体形成的原因。在本文中，我们利用荧光共振能量转移方法，使用含有独特的色氨酸对和荧光团标记半胱氨酸残基的基因工程SecA蛋白，来确定溶液中SecA蛋白的寡聚结构。我们的结果表明，在晶体结构中观察到的两种二聚体相互作用中，SecA在溶液中形成反平行二聚体，其掩埋的可溶剂接触表面积和分子间接触最大化。在荧光实验中使用的亚微摩尔蛋白浓度下，我们没有发现基于晶体晶格中观察到的另一种二聚体或3(1)螺旋纤维形成SecA高阶寡聚体的证据。我们的研究与先前的研究一致，先前的研究证明了SecA的C结构域内存在二聚化决定因素，也证明了SecA的N结构域和C结构域之间的相互作用。我们的结果还为确定SecA的亚基状态在蛋白转运过程中是否发生变化以及为阐明该多结构域蛋白在其转运循环中的构象动力学而设计的研究提供了一个有价值的起点。
