Kim Ju Yaen, Kinoshita Misaki, Kume Satoshi, Gt Hanke, Sugiki Toshihiko, Ladbury John E, Kojima Chojiro, Ikegami Takahisa, Kurisu Genji, Goto Yuji, Hase Toshiharu, Lee Young-Ho
Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
Cellular Function Imaging Team, Division of Bio-function Dynamics Imaging, RIKEN Center for Life Science Technologies, Kobe, Hyogo 650-0047, Japan Multi-Modal Microstructure Analysis Unit, RIKEN CLST-JEOL Collaboration Center, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
Biochem J. 2016 Nov 1;473(21):3837-3854. doi: 10.1042/BCJ20160658. Epub 2016 Aug 22.
Although electrostatic interactions between negatively charged ferredoxin (Fd) and positively charged sulfite reductase (SiR) have been predominantly highlighted to characterize complex formation, the detailed nature of intermolecular forces remains to be fully elucidated. We investigated interprotein forces for the formation of an electron transfer complex between Fd and SiR and their relationship to SiR activity using various approaches over NaCl concentrations between 0 and 400 mM. Fd-dependent SiR activity assays revealed a bell-shaped activity curve with a maximum ∼40-70 mM NaCl and a reverse bell-shaped dependence of interprotein affinity. Meanwhile, intrinsic SiR activity, as measured in a methyl viologen-dependent assay, exhibited saturation above 100 mM NaCl. Thus, two assays suggested that interprotein interaction is crucial in controlling Fd-dependent SiR activity. Calorimetric analyses showed the monotonic decrease in interprotein affinity on increasing NaCl concentrations, distinguished from a reverse bell-shaped interprotein affinity observed from Fd-dependent SiR activity assay. Furthermore, Fd:SiR complex formation and interprotein affinity were thermodynamically adjusted by both enthalpy and entropy through electrostatic and non-electrostatic interactions. A residue-based NMR investigation on the addition of SiR to N-labeled Fd at the various NaCl concentrations also demonstrated that a combination of electrostatic and non-electrostatic forces stabilized the complex with similar interfaces and modulated the binding affinity and mode. Our findings elucidate that non-electrostatic forces are also essential for the formation and modulation of the Fd:SiR complex. We suggest that a complex configuration optimized for maximum enzymatic activity near physiological salt conditions is achieved by structural rearrangement through controlled non-covalent interprotein interactions.
尽管带负电荷的铁氧化还原蛋白（Fd）与带正电荷的亚硫酸盐还原酶（SiR）之间的静电相互作用在表征复合物形成方面得到了主要强调，但分子间力的详细性质仍有待充分阐明。我们使用多种方法研究了在0至400 mM NaCl浓度范围内Fd和SiR之间形成电子转移复合物的蛋白间作用力及其与SiR活性的关系。Fd依赖性SiR活性测定显示出钟形活性曲线，在约40 - 70 mM NaCl时达到最大值，且蛋白间亲和力呈反向钟形依赖性。同时，在甲基紫精依赖性测定中测得的内在SiR活性在NaCl浓度高于100 mM时表现出饱和。因此，两种测定方法都表明蛋白间相互作用对于控制Fd依赖性SiR活性至关重要。量热分析表明，随着NaCl浓度增加，蛋白间亲和力单调下降，这与Fd依赖性SiR活性测定中观察到的反向钟形蛋白间亲和力不同。此外，Fd:SiR复合物的形成和蛋白间亲和力通过静电和非静电相互作用在热力学上由焓和熵共同调节。在不同NaCl浓度下对N标记的Fd添加SiR进行的基于残基的核磁共振研究也表明，静电和非静电力的组合稳定了具有相似界面的复合物，并调节了结合亲和力和模式。我们的研究结果阐明，非静电力对于Fd:SiR复合物的形成和调节也至关重要。我们认为，通过受控的非共价蛋白间相互作用进行结构重排，可在生理盐条件下实现针对最大酶活性优化的复合物构型。
