Glaenzer Janin, Peter Martin F, Thomas Gavin H, Hagelueken Gregor
Institute for Physical & Theoretical Chemistry, University of Bonn, Bonn, Germany.
Department of Biology, University of York, York, UK.
Biophys J. 2017 Jan 10;112(1):109-120. doi: 10.1016/j.bpj.2016.12.010.
The tripartite ATP-independent periplasmic (TRAP) transporters are a widespread class of membrane transporters in bacteria and archaea. Typical substrates for TRAP transporters are organic acids including the sialic acid N-acetylneuraminic acid. The substrate binding proteins (SBP) of TRAP transporters are the best studied component and are responsible for initial high-affinity substrate binding. To better understand the dynamics of the ligand binding process, pulsed electron-electron double resonance (PELDOR, also known as DEER) spectroscopy was applied to study the conformational changes in the N-acetylneuraminic acid-specific SBP VcSiaP. The protein is the SBP of VcSiaPQM, a sialic acid TRAP transporter from Vibrio cholerae. Spin-labeled double-cysteine mutants of VcSiaP were analyzed in the substrate-bound and -free state and the measured distances were compared to available crystal structures. The data were compatible with two clear states only, which are consistent with the open and closed forms seen in TRAP SBP crystal structures. Substrate titration experiments demonstrated the transition of the population from one state to the other with no other observed forms. Mutants of key residues involved in ligand binding and/or proposed to be involved in domain closure were produced and the corresponding PELDOR experiments reveal important insights into the open-closed transition. The results are in excellent agreement with previous in vivo sialylation experiments. The structure of the spin-labeled Q54R1/L173R1 R125A mutant was solved at 2.1 Å resolution, revealing no significant changes in the protein structure. Thus, the loss of domain closure appears to be solely due to loss of binding. In conclusion, these data are consistent with TRAP SBPs undergoing a simple two-state transition from an open-unliganded to closed-liganded state during the transport cycle.
三方ATP非依赖型周质（TRAP）转运蛋白是细菌和古细菌中广泛存在的一类膜转运蛋白。TRAP转运蛋白的典型底物是有机酸，包括唾液酸N - 乙酰神经氨酸。TRAP转运蛋白的底物结合蛋白（SBP）是研究得最为透彻的组分，负责最初的高亲和力底物结合。为了更好地理解配体结合过程的动力学，采用脉冲电子 - 电子双共振（PELDOR，也称为DEER）光谱来研究N - 乙酰神经氨酸特异性SBP VcSiaP的构象变化。该蛋白是VcSiaPQM的SBP，VcSiaPQM是一种来自霍乱弧菌的唾液酸TRAP转运蛋白。对VcSiaP的自旋标记双半胱氨酸突变体在底物结合态和游离态下进行了分析，并将测量距离与现有的晶体结构进行了比较。数据仅与两种清晰的状态相符，这与TRAP SBP晶体结构中所见的开放和闭合形式一致。底物滴定实验表明群体从一种状态转变为另一种状态，未观察到其他形式。产生了参与配体结合和/或被认为参与结构域闭合的关键残基的突变体，相应的PELDOR实验揭示了对开放 - 闭合转变的重要见解。结果与先前的体内唾液酸化实验非常吻合。自旋标记的Q54R1/L173R1 R125A突变体的结构在2.1 Å分辨率下得到解析，显示蛋白质结构没有显著变化。因此，结构域闭合的丧失似乎仅仅是由于结合的丧失。总之，这些数据与TRAP SBP在转运循环中经历从开放 - 未结合到闭合 - 结合状态的简单双态转变一致。
