van der Walle Christopher F, Altroff Harri, Mardon Helen J
Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Women's Centre, Level 3, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK.
Protein Eng. 2002 Dec;15(12):1021-4. doi: 10.1093/protein/15.12.1021.
The ninth and tenth type III domains (FIII9-10) in the central cell binding domain of human fibronectin contain integrin receptor binding sites, including RGD in FIII10 and a synergy site, PHSRN, in FIII9. The specific amino acids that contribute to cell binding have been identified by the use of wild-type and mutant fragments of human fibronectin containing the FIII9-10 domain pair. At high concentrations FIII9-10 mimics, to a large extent, the biological activity of the full-length fibronectin molecule. However, FIII9 is conformationally unstable, even in the context of the FIII9-10 pair. Here we report the construction of a series of hybrid mouse-human FIII9-10 pairs that confer varying degrees of conformational stability to FIII9. The conformational stability of the human FIII9 module was increased 2-3-fold by substitution of Leu1408 with Pro. We demonstrate that the biological activity of this mutant is enhanced. The resulting FIII9-10 mutant has good solution properties and will provide a template into which further mutations can be incorporated in order to probe the structure-function relationship of the cell binding module of fibronectin.
人纤连蛋白中央细胞结合域中的第九和第十个III型结构域（FIII9 - 10）含有整合素受体结合位点，包括FIII10中的RGD和FIII9中的协同位点PHSRN。通过使用包含FIII9 - 10结构域对的人纤连蛋白野生型和突变片段，已确定了有助于细胞结合的特定氨基酸。在高浓度下，FIII9 - 10在很大程度上模拟了全长纤连蛋白分子的生物活性。然而，即使在FIII9 - 10对的情况下，FIII9在构象上也是不稳定的。在此，我们报告了一系列杂交小鼠 - 人FIII9 - 10对的构建，这些对赋予FIII9不同程度的构象稳定性。通过将Leu1408替换为Pro，人FIII9模块的构象稳定性提高了2 - 3倍。我们证明该突变体的生物活性增强。所得的FIII9 - 10突变体具有良好的溶液性质，并将提供一个模板，可在其中引入进一步的突变以探究纤连蛋白细胞结合模块的结构 - 功能关系。
