Nayak Ashok Ranjan, Karade Sharanbasappa Shrimant, Srivastava Vijay Kumar, Rana Ajay Kumar, Gupta C M, Sahasrabuddhe Amogh A, Pratap J Venkatesh
Division of Molecular and Structural Biology, CSIR - Central Drug Research Institute, Jankipuram Extension, Sitapur Road, Lucknow 226031, India.
Division of Molecular and Structural Biology, CSIR - Central Drug Research Institute, Jankipuram Extension, Sitapur Road, Lucknow 226031, India.
J Struct Biol. 2016 Jul;195(1):129-38. doi: 10.1016/j.jsb.2016.02.020. Epub 2016 Mar 2.
Coiled coils are ubiquitous structural motifs that serve as a platform for protein-protein interactions and play a central role in myriad physiological processes. Though the formation of a coiled coil requires only the presence of suitably spaced hydrophobic residues, sequence specificities have also been associated with specific oligomeric states. RhXXhE is one such sequence motif, associated with parallel trimers, found in coronins and other proteins. Coronin, present in all eukaryotes, is an actin-associated protein involved in regulating actin turnover. Most eukaryotic coronins possess the RhXXhE trimerization motif. However, a unique feature of parasitic kinetoplastid coronin is that the positions of R and E are swapped within their coiled coil domain, but were still expected to form trimers. To understand the role of swapped motif in oligomeric specificity, we determined the X-ray crystal structure of Leishmania donovani coronin coiled coil domain (LdCoroCC) at 2.2Å, which surprisingly, reveals an anti-parallel tetramer assembly. Small angle X-ray scattering studies and chemical crosslinking confirm the tetramer in solution and is consistent with the oligomerization observed in the full length protein. Structural analyses reveal that LdCoroCC possesses an inherent asymmetry, in that one of the helices of the bundle is axially shifted with respect to the other three. The analysis also identifies steric reasons that cause this asymmetry. The bundle adapts an extended a-d-e core packing, the e residue being polar (with an exception) which results in a thermostable bundle with polar and apolar interfaces, unlike the existing a-d-e core antiparallel homotetramers with apolar core. Functional implications of the anti-parallel association in kinetoplastids are discussed.
卷曲螺旋是普遍存在的结构基序，作为蛋白质 - 蛋白质相互作用的平台，在众多生理过程中发挥核心作用。虽然卷曲螺旋的形成仅需要适当间隔的疏水残基的存在，但序列特异性也与特定的寡聚状态相关。RhXXhE就是这样一种序列基序，与平行三聚体相关，存在于冠蛋白和其他蛋白质中。冠蛋白存在于所有真核生物中，是一种与肌动蛋白相关的蛋白质，参与调节肌动蛋白的周转。大多数真核生物的冠蛋白都具有RhXXhE三聚化基序。然而，寄生性动基体冠蛋白的一个独特特征是，R和E在其卷曲螺旋结构域内的位置互换，但仍预期形成三聚体。为了理解互换基序在寡聚特异性中的作用，我们确定了杜氏利什曼原虫冠蛋白卷曲螺旋结构域（LdCoroCC）在2.2Å分辨率下的X射线晶体结构，令人惊讶的是，它揭示了一种反平行四聚体组装。小角X射线散射研究和化学交联证实了溶液中的四聚体，并且与全长蛋白中观察到的寡聚化一致。结构分析表明，LdCoroCC具有内在的不对称性，即束中的一个螺旋相对于其他三个螺旋在轴向上发生了位移。分析还确定了导致这种不对称性的空间原因。该束采用扩展的a - d - e核心堆积，e残基是极性的（有一个例外），这导致了一个具有极性和非极性界面的热稳定束，这与现有的具有非极性核心的a - d - e核心反平行同型四聚体不同。讨论了动基体中反平行缔合的功能意义。
