Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Lucknow 226031, India; Academy of Scientific and Innovative Research, New Delhi 110025, India.
Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Lucknow 226031, India.
Biochim Biophys Acta Proteins Proteom. 2018 Oct;1866(10):1008-1020. doi: 10.1016/j.bbapap.2018.06.007. Epub 2018 Jul 6.
The GMF class of the ADF-H domain family proteins regulate actin dynamics by binding to the Arp2/3 complex and F-actin through their Site-1 and Site-2, respectively. CeGMF of C. elegans is analogous to GMFγ of human and mouse and is 138 amino acids in length.
We have characterized the solution structure and dynamics of CeGMF by solution NMR spectroscopy and its thermal stability by DSC.
The solution structure of CeGMF shows canonical ADF-H fold with two additional β-strands in the β4-β5 loop region. The Site-1 of CeGMF is well formed and residues of all three regions of Site-1 show dynamic flexibility. However, the β4-β5 loop of Site-2 is less inclined towards the C-terminal, as the latter is truncated by four residues in comparison to GMF isoforms of human and mouse. Regions of Site-2 show motions on ns-ps timescale, but dynamic flexibility of β4-β5 loop is low in comparison to corresponding F-loop region of ADF/cofilin UNC-60B. A general difference in packing of α3 and α1 between GMF and ADF/cofilins was noticed. Additionally, thermal stability of CeGMF was significantly higher than its ADF/cofilin homologs.
We have presented the first solution structure of GMF from C. elegans, which highlights the structural differences between the Site-2 of CeGMF and mammalian GMF isoforms. Further, we have seen the differences in structure, dynamics, and thermal stability of GMF and ADF/cofilin.
This study provides a useful insight to structural and dynamics factors that define the specificity of GMF towards Arp2/3 complex.
ADF-H 结构域家族蛋白的 GMF 类通过分别与 Arp2/3 复合物和 F-肌动蛋白结合其 Site-1 和 Site-2 来调节肌动蛋白动力学。秀丽隐杆线虫的 CeGMF 类似于人类和小鼠的 GMFγ，长度为 138 个氨基酸。
我们通过溶液 NMR 光谱法和 DSC 法分别对 CeGMF 的溶液结构和动力学及其热稳定性进行了表征。
CeGMF 的溶液结构显示出典型的 ADF-H 折叠，在β4-β5 环区域有两个额外的β-链。CeGMF 的 Site-1 形成良好，所有三个区域的 Site-1 残基均表现出动态灵活性。然而，Site-2 的β4-β5 环较不倾向于 C 末端，因为与人类和小鼠的 GMF 同工型相比，后者被截断了四个残基。Site-2 区域的运动发生在 ns-ps 时间尺度上，但与 ADF/ 原肌球蛋白 UNC-60B 的相应 F 环区域相比，β4-β5 环的动态灵活性较低。GMF 和 ADF/ 原肌球蛋白之间注意到 α3 和 α1 之间的一般包装差异。此外，CeGMF 的热稳定性明显高于其 ADF/ 原肌球蛋白同源物。
我们展示了秀丽隐杆线虫 GMF 的首个溶液结构，突出了 CeGMF 的 Site-2 与哺乳动物 GMF 同工型之间的结构差异。此外，我们还看到了 GMF 和 ADF/ 原肌球蛋白在结构、动力学和热稳定性方面的差异。
这项研究为 GMF 对 Arp2/3 复合物的特异性定义的结构和动力学因素提供了有用的见解。
