Solution NMR structure of putidaredoxin-cytochrome P450cam complex via a combined residual dipolar coupling-spin labeling approach suggests a role for Trp106 of putidaredoxin in complex formation.
Solution NMR structure of putidaredoxin-cytochrome P450cam complex via a combined residual dipolar coupling-spin labeling approach suggests a role for Trp106 of putidaredoxin in complex formation.
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DOI：
10.1016/j.jmb.2008.09.037
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发表时间：
2008-12-12
影响因子：
5.6
通讯作者：
Jain NU
中科院分区：
文献类型：
--
作者：
Zhang W;Pochapsky SS;Pochapsky TC;Jain NU
用高分辨溶液核磁共振研究了恶臭假单胞菌细胞色素P450 cam与铁氧还蛋白(2Fe-2S)、恶臭还蛋白(PDX)形成的58 kDa复合体。在底物樟脑转化为5-外羟基樟脑的酶促反应中，PDX既是CYP101的生理还原剂，又是其效应剂。为了获得氧化后的PDX-CyP101络合物的实验结构，采用了一种组合方法，该方法利用了来自残留偶极偶联的两个蛋白质的取向数据和来自PDX特定位置的自旋标记的距离限制。利用15N和13C核磁共振谱首次绘制了PDX与CYP101络合物中顺磁金属簇区及其附近残基的光谱变化，从而直接鉴定了受CYP101结合强烈影响的残基。PDX-CYP101复合体的新的核磁共振结构与先前的突变和涉及结合界面残基的生物物理研究的结果很好地吻合，例如在PDX的Asp38和CYP101的Arg112之间形成盐桥，同时确定了不同于早期建模研究的关键特征。对该复合体结合界面的分析表明，Trp106的侧链是PDX的C端残基，对与CYP101结合至关重要，它位于CyP101的血红素结合环的对面，并与CYP101上血红素基团附近的几个残基形成非极性接触，表明它在复合体的形成中具有潜在的重要作用。
The 58 kDa complex formed between the [2Fe-2S] ferredoxin, putidaredoxin (Pdx), and cytochrome P450cam (CYP101) from the bacterium Pseudomonas putida has been investigated by high-resolution solution NMR spectroscopy. Pdx serves as both the physiological reductant and effector for CYP101 in the enzymatic reaction involving conversion of substrate camphor to 5-exo-hydroxy-camphor. In order to obtain an experimental structure for the oxidized Pdx-CYP101 complex, a combined approach using orientational data on the two proteins derived from residual dipolar couplings and distance restraints from site-specific spin labeling of Pdx has been applied. Spectral changes for residues in and near the paramagnetic metal cluster region of Pdx in complex with CYP101 have also been mapped for the first time using 15N and 13C NMR spectroscopy, leading to direct identification of the residues strongly affected by CYP101 binding. The new NMR structure of the Pdx-CYP101 complex agrees well with results from previous mutagenesis and biophysical studies involving residues at the binding interface such as formation of salt bridge between Asp38 of Pdx and Arg112 of CYP101, while at the same time identifying key features different from earlier modeling studies. Analysis of the binding interface of the complex reveals that the side-chain of Trp106, the C-terminal residue of Pdx and critical for binding to CYP101, is located across from the heme-binding loop of CYP101 and forms non-polar contacts with several residues in the vicinity of heme group on CYP101, pointing to a potentially important role in complex formation.