Structural Biology Laboratory, Council of Scientific and Industrial Research-Institute of Microbial Technology, G. N. Ramachandran Protein Centre, Chandigarh, India.
Council of Scientific and Industrial Research-Institute of Microbial Technology, G. N. Ramachandran Protein Centre, Chandigarh, India.
J Biol Chem. 2021 Jan-Jun;296:100308. doi: 10.1016/j.jbc.2021.100308. Epub 2021 Jan 22.
The δ-proteobacteria Myxococcus xanthus displays social (S) and adventurous (A) motilities, which require pole-to-pole reversal of the motility regulator proteins. Mutual gliding motility protein C (MglC), a paralog of GTPase-activating protein Mutual gliding motility protein B (MglB), is a member of the polarity module involved in regulating motility. However, little is known about the structure and function of MglC. Here, we determined ∼1.85 Å resolution crystal structure of MglC using Selenomethionine Single-wavelength anomalous diffraction. The crystal structure revealed that, despite sharing <9% sequence identity, both MglB and MglC adopt a Regulatory Light Chain 7 family fold. However, MglC has a distinct ∼30° to 40° shift in the orientation of the functionally important α2 helix compared with other structural homologs. Using isothermal titration calorimetry and size-exclusion chromatography, we show that MglC binds MglB in 2:4 stoichiometry with submicromolar range dissociation constant. Using small-angle X-ray scattering and molecular docking studies, we show that the MglBC complex consists of a MglC homodimer sandwiched between two homodimers of MglB. A combination of size-exclusion chromatography and site-directed mutagenesis studies confirmed the MglBC interacting interface obtained by molecular docking studies. Finally, we show that the C-terminal region of MglB, crucial for binding its established partner MglA, is not required for binding MglC. These studies suggest that the MglB uses distinct interfaces to bind MglA and MglC. Based on these data, we propose a model suggesting a new role for MglC in polarity reversal in M. xanthus.
δ-变形菌黏球菌呈现出社会（S）和冒险（A）两种运动性，这两种运动性都需要运动调节蛋白进行极到极的反转。互滑运动蛋白 C（MglC）是 GTP 酶激活蛋白互滑运动蛋白 B（MglB）的同源物，是参与调节运动的极性模块的成员。然而，关于 MglC 的结构和功能知之甚少。在这里，我们使用硒代蛋氨酸单波长反常散射法测定了 MglC 的约 1.85Å分辨率晶体结构。晶体结构表明，尽管 MglB 和 MglC 仅共享<9%的序列同一性，但它们都采用 Regulatory Light Chain 7 家族折叠。然而，与其他结构同源物相比，MglC 中功能重要的α2 螺旋的取向有明显的30°至40°的偏移。通过等温滴定量热法和尺寸排阻色谱法，我们表明 MglC 以亚毫摩尔范围的解离常数与 MglB 以 2:4 的比例结合。通过小角 X 射线散射和分子对接研究，我们表明 MglBC 复合物由夹在两个 MglB 同源二聚体之间的 MglC 同源二聚体组成。大小排阻色谱和定点突变研究的组合证实了通过分子对接研究获得的 MglBC 相互作用界面。最后，我们表明，MglB 与其已建立的伴侣 MglA 结合所必需的 C 末端区域对于与 MglC 结合并非必需。这些研究表明，MglB 使用不同的界面来结合 MglA 和 MglC。基于这些数据，我们提出了一个模型，表明 MglC 在黏球菌极性反转中具有新的作用。
