Grabarczyk Daniel B, Chappell Paul E, Johnson Steven, Stelzl Lukas S, Lea Susan M, Berks Ben C
Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom;
Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.
Proc Natl Acad Sci U S A. 2015 Dec 29;112(52):E7166-75. doi: 10.1073/pnas.1506386112. Epub 2015 Dec 11.
The bacterial Sox (sulfur oxidation) pathway is an important route for the oxidation of inorganic sulfur compounds. Intermediates in the Sox pathway are covalently attached to the heterodimeric carrier protein SoxYZ through conjugation to a cysteine on a protein swinging arm. We have investigated how the carrier protein shuttles intermediates between the enzymes of the Sox pathway using the interaction between SoxYZ and the enzyme SoxB as our model. The carrier protein and enzyme interact only weakly, but we have trapped their complex by using a "suicide enzyme" strategy in which an engineered cysteine in the SoxB active site forms a disulfide bond with the incoming carrier arm cysteine. The structure of this trapped complex, together with calorimetric data, identifies sites of protein-protein interaction both at the entrance to the enzyme active site tunnel and at a second, distal, site. We find that the enzyme distinguishes between the substrate and product forms of the carrier protein through differences in their interaction kinetics and deduce that this behavior arises from substrate-specific stabilization of a conformational change in the enzyme active site. Our analysis also suggests how the carrier arm-bound substrate group is able to outcompete the adjacent C-terminal carboxylate of the carrier arm for binding to the active site metal ions. We infer that similar principles underlie carrier protein interactions with other enzymes of the Sox pathway.
细菌的Sox（硫氧化）途径是无机硫化合物氧化的重要途径。Sox途径中的中间体通过与蛋白质摆动臂上的半胱氨酸结合，共价连接到异二聚体载体蛋白SoxYZ上。我们以SoxYZ与酶SoxB之间的相互作用为模型，研究了载体蛋白如何在Sox途径的酶之间穿梭中间体。载体蛋白与酶的相互作用很弱，但我们通过“自杀酶”策略捕获了它们的复合物，即在SoxB活性位点中设计一个半胱氨酸，使其与进入的载体臂半胱氨酸形成二硫键。这种捕获复合物的结构以及量热数据，确定了在酶活性位点通道入口处和第二个远端位点的蛋白质-蛋白质相互作用位点。我们发现，酶通过载体蛋白底物和产物形式相互作用动力学的差异来区分它们，并推断这种行为源于酶活性位点构象变化的底物特异性稳定。我们的分析还表明，与载体臂结合的底物基团如何能够在与活性位点金属离子结合时，胜过载体臂相邻的C末端羧酸盐。我们推断，类似的原理是载体蛋白与Sox途径其他酶相互作用的基础。
