Vermeulen Wim, Van Troys Marleen, Bourry David, Dewitte Daisy, Rossenu Stefaan, Goethals Marc, Borremans Frans A M, Vandekerckhove Joël, Martins José C, Ampe Christophe
NMR and Structure Analysis Unit, Department of Organic Chemistry, Faculty of Sciences, Ghent University, Belgium.
J Mol Biol. 2006 Jun 23;359(5):1277-92. doi: 10.1016/j.jmb.2006.04.042. Epub 2006 May 6.
The HeadPiece (HP) domain, present in several F-actin-binding multi-domain proteins, features a well-conserved, solvent-exposed PXWK motif in its C-terminal subdomain. The latter is an autonomously folding subunit comprised of three alpha-helices organised around a hydrophobic core, with the sequence motif preceding the last helix. We report the contributions of each conserved residue in the PXWK motif to human villin HP function and structure, as well as the structural implications of the naturally occurring Pro to Ala mutation in dematin HP. NMR shift perturbation mapping reveals that substitution of each residue by Ala induces only minor, local perturbations in the full villin HP structure. CD spectroscopic thermal analysis, however, shows that the Pro and Trp residues in the PXWK motif afford stabilising interactions. This indicates that, in addition to the residues in the hydrophobic core, the Trp-Pro stacking within the motif contributes to HP stability. This is reinforced by our data on isolated C-terminal HP subdomains where the Pro is also essential for structure formation, since the villin, but not the dematin, C-terminal subdomain is structured. Proper folding can be induced in the dematin C-terminal subdomain by exchanging the Ala for Pro. Conversely, the reverse substitution in the villin C-terminal subdomain leads to loss of structure. Thus, we demonstrate a crucial role for this proline residue in structural stability and folding potential of HP (sub)domains consistent with Pro-Trp stacking as a more general determinant of protein stability.
头部结构域（HP）存在于多种F-肌动蛋白结合多结构域蛋白中，其C末端亚结构域具有一个保守性良好、暴露于溶剂中的PXWK基序。后者是一个自主折叠的亚基，由围绕疏水核心排列的三个α螺旋组成，最后一个螺旋之前有序列基序。我们报告了PXWK基序中每个保守残基对人绒毛蛋白HP功能和结构的贡献，以及去膜蛋白HP中天然存在的脯氨酸到丙氨酸突变的结构影响。核磁共振位移扰动图谱显示，每个残基被丙氨酸取代仅在完整的绒毛蛋白HP结构中引起轻微的局部扰动。然而，圆二色光谱热分析表明，PXWK基序中的脯氨酸和色氨酸残基提供了稳定的相互作用。这表明，除了疏水核心中的残基外，基序内的色氨酸-脯氨酸堆积有助于HP的稳定性。我们关于分离的C末端HP亚结构域的数据进一步证实了这一点，其中脯氨酸对于结构形成也是必不可少的，因为绒毛蛋白而非去膜蛋白的C末端亚结构域是有结构的。通过将丙氨酸换成脯氨酸，可以在去膜蛋白C末端亚结构域中诱导正确折叠。相反，在绒毛蛋白C末端亚结构域中进行反向取代会导致结构丧失。因此，我们证明了这个脯氨酸残基在HP（亚）结构域的结构稳定性和折叠潜力中起着关键作用，这与脯氨酸-色氨酸堆积作为蛋白质稳定性的更普遍决定因素是一致的。
