Amidzadeh Zahra, Rismani Elham, Shokrgozar Mohammad Ali, Rahimi Hamzeh, Golkar Majid
National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran; Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran.
Department of Molecular Medicine, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
J Mol Graph Model. 2023 Jan;118:108351. doi: 10.1016/j.jmgm.2022.108351. Epub 2022 Oct 12.
Keratinocyte growth factor (KGF) is a potential therapeutic factor in wound healing. However, its applications have been restricted due to its low stability, short half-life, and limited target specificity. We aimed to immobilize KGF on collagen-based biomaterials for long-lasting and targeted therapy by designing fusion forms of KGF with collagen-binding domains (CBD) from natural origins. Twelve fusion proteins were designed consisting of KGF and CBDs with different lengths and amino acid compositions. Three-dimensional (3D) structures of the fusions were predicted by homology modeling. Physiochemical properties and secondary structure of the fusions were evaluated by bioinformatics tools. Moreover, the effect of the CBDs on the 3D structure and dynamic behavior of the fusions was investigated by molecular dynamics (MD) simulation. The binding affinity of the fusions to collagen, KGF receptor, and heparin was assessed using docking tools. Our results demonstrated that fusions with small CBDs like CBD of mammalian collagenase and decapeptide CBD of von Willebrand factor (VWF) were more stable and properly folded than those with larger CBDs. On the other hand, the insertion of bulky CBDs, including Fibronectin CBD and CBD of Clostridium histolyticum collagenase, into KGF resulted in stronger binding to collagen. Therefore, very small or large CBDs are inappropriate for constructing KGF fusions because they suffer from low collagen affinity or poor stability. By comparing the results of MD simulation and docking, this study proposed that CBDs belonging to Vibrio mimicus metalloprotease and A3 domain of VWF would be good candidates to produce stable fusions with proper affinities toward collagen and KGF receptors. Moreover, the secondary structure analysis showed that the overall structure of KGF and CBDs was better preserved when CBDs were inserted at the C-terminal of KGF. This computational information about novel KGF fusions may help find the best constructs for experimental studies.
角质形成细胞生长因子（KGF）是伤口愈合中的一种潜在治疗因子。然而，由于其稳定性低、半衰期短和靶标特异性有限，其应用受到了限制。我们旨在通过设计KGF与天然来源的胶原结合结构域（CBD）的融合形式，将KGF固定在基于胶原蛋白的生物材料上，以实现持久的靶向治疗。设计了12种由KGF和不同长度及氨基酸组成的CBD组成的融合蛋白。通过同源建模预测融合蛋白的三维（3D）结构。利用生物信息学工具评估融合蛋白的理化性质和二级结构。此外，通过分子动力学（MD）模拟研究了CBD对融合蛋白三维结构和动态行为的影响。使用对接工具评估融合蛋白与胶原蛋白、KGF受体和肝素的结合亲和力。我们的结果表明，与较大的CBD相比，具有小CBD（如哺乳动物胶原酶的CBD和血管性血友病因子（VWF）的十肽CBD）的融合蛋白更稳定且折叠正确。另一方面，将包括纤连蛋白CBD和溶组织梭菌胶原酶的CBD在内的大体积CBD插入KGF中，导致与胶原蛋白的结合更强。因此，非常小或非常大的CBD都不适用于构建KGF融合蛋白，因为它们要么胶原蛋白亲和力低，要么稳定性差。通过比较MD模拟和对接的结果，本研究提出，属于拟态弧菌金属蛋白酶的CBD和VWF的A3结构域将是产生对胶原蛋白和KGF受体具有适当亲和力的稳定融合蛋白的良好候选者。此外，二级结构分析表明，当CBD插入KGF的C末端时，KGF和CBD的整体结构得到更好的保留。这些关于新型KGF融合蛋白的计算信息可能有助于找到用于实验研究的最佳构建体。
