Smith Dustin, Lloyd Laura, Wei Elaine, Radmanesh Paria, Wei Chin-Chuan
Department of Chemistry, College of Arts and Sciences, Southern Illinois University Edwardsville, Edwardsville, IL, 62026, USA.
Department of Biochemistry, School of Molecular and Cellular Biology, University of Illinois, Urbana Champaign, IL, 61801, USA.
Biochem Biophys Rep. 2022 Jan 15;29:101198. doi: 10.1016/j.bbrep.2021.101198. eCollection 2022 Mar.
Superoxide generated by NADPH Oxidase 5 (Nox5) is regulated by Ca through the interaction of its self-contained Ca binding domain and dehydrogenase domain (DH). Recently, calmodulin (CaM) has been reported to enhance the Ca sensitivity of Nox5 by binding to the CaM-binding domain sequence (CMBD), in which the interaction between CaM and Nox5 is largely unclear. Here, we used the CMBD peptide and truncated DH constructs, and separately studied their interaction with CaM by fluorescence, calorimetry, and dynamic light scattering. Our results revealed that each half-domain of CaM binds one CMBD peptide with a binding constant near 10 M and a binding enthalpy change of -3.81 kcal/mol, consistent with an extended 1:2 CaM:CMBD structure. However, the recombinant truncated DH proteins exist as oligomers, possibly trimer and tetramer. The oligomeric states are concentration and salt dependent. CaM binding appears to stabilize the DH dimer complexed with CaM. The thermodynamics of CaM binding to the DH is comparable to the peptide-based study except that the near unity binding stoichiometry and a large conformational change were observed. Our result suggests that the oligomeric states of Nox5, mediated by its DH domain and CaM, may be important for its superoxide-generating activity.
烟酰胺腺嘌呤二核苷酸磷酸氧化酶5（Nox5）产生的超氧化物通过其自身包含的钙结合结构域与脱氢酶结构域（DH）的相互作用受钙调节。最近，有报道称钙调蛋白（CaM）通过与钙调蛋白结合结构域序列（CMBD）结合来增强Nox5对钙的敏感性，而CaM与Nox5之间的相互作用在很大程度上尚不清楚。在此，我们使用CMBD肽和截短的DH构建体，分别通过荧光、量热法和动态光散射研究它们与CaM的相互作用。我们的结果表明，CaM的每个半结构域结合一个CMBD肽，结合常数接近10⁶ M，结合焓变-3.81 kcal/mol，这与扩展的1:2 CaM:CMBD结构一致。然而，重组截短的DH蛋白以寡聚体形式存在，可能是三聚体和四聚体。寡聚状态取决于浓度和盐浓度。CaM结合似乎稳定了与CaM复合的DH二聚体。CaM与DH结合的热力学与基于肽的研究相当，但观察到接近1的结合化学计量和大的构象变化。我们的结果表明，由其DH结构域和CaM介导的Nox5寡聚状态可能对其超氧化物生成活性很重要。
