Cell, Molecular, and Structural Biology Program, Miami University, Oxford, OH 45056, USA.
Department of Microbiology, Miami University, Oxford, OH 45056, USA.
Toxins (Basel). 2017 Nov 30;9(12):389. doi: 10.3390/toxins9120389.
Site I inactivation of calmodulin (CaM) was used to examine the importance of aspartic acid 22 at position 3 in CaM calcium binding, protein folding, and activation of the adenylate cyclase toxin domain (CyaA-ACD). NMR calcium titration experiments showed that site I in the CaM mutant (D22A) remained largely unperturbed, while sites II, III, and IV exhibited calcium-induced conformational changes similar to wild-type CaM (CaMWt). Circular dichroism analyses revealed that D22A had comparable -helical content to CaMWt, and only modest differences in -helical composition were detected between CaMWt-CyaA-ACD and D22A-CyaA-ACD complexes. However, the thermal stability of the D22A-CyaA-ACD complex was reduced, as compared to the CaMWt-CyaA-ACD complex. Moreover, CaM-dependent activity of CyaA-ACD decreased 87% in the presence of D22A. Taken together, our findings provide evidence that D22A engages CyaA-ACD, likely through -terminal mediated binding, and that site I inactivation exerts functional effects through the modification of stabilizing interactions that occur between -terminal CaM and CyaA-ACD.
使用钙调蛋白（CaM）的位点 I 失活来研究天冬氨酸 22 在 CaM 钙结合、蛋白质折叠和激活腺苷酸环化酶毒素结构域（CyaA-ACD）中的重要性。NMR 钙滴定实验表明，CaM 突变体（D22A）的位点 I 基本未受影响，而位点 II、III 和 IV 表现出与野生型 CaM（CaMWt）相似的钙诱导构象变化。圆二色性分析表明，D22A 与 CaMWt 具有可比的 α-螺旋含量，并且仅在 CaMWt-CyaA-ACD 和 D22A-CyaA-ACD 复合物之间检测到 α-螺旋组成的微小差异。然而，与 CaMWt-CyaA-ACD 复合物相比，D22A-CyaA-ACD 复合物的热稳定性降低。此外，在存在 D22A 的情况下，CyaA-ACD 的 CaM 依赖性活性降低了 87%。总之，我们的研究结果提供了证据表明，D22A 与 CyaA-ACD 结合，可能通过 C 端介导的结合，而位点 I 失活通过修饰 CaM 和 CyaA-ACD 之间发生的稳定相互作用产生功能效应。
