Gupta Sakshi, Tiwari Neha, Verma Jyoti, Waseem Mohd, Subbarao Naidu, Munde Manoj
School of Physical Sciences, Jawaharlal Nehru University New Delhi-110067 India
School of Computational and Integrative Sciences, Jawaharlal Nehru University New Delhi-110067 India.
RSC Adv. 2020 May 27;10(34):20288-20301. doi: 10.1039/d0ra01773f. eCollection 2020 May 26.
The HEP II (Heparin-binding site II) region of fibronectin (FN) containing domain III plays a crucial role in cell adhesion and migration through heparin-binding on the cell surface. There are two such fibronectin heparin interacting peptide (FHIP I and FHIP II) sequences present in HEP II. However, the molecular principles by which these sites orchestrate heparin-binding processes are poorly understood. Such knowledge would have great implications in the therapeutic targeting of FN. With this aim, we have explored the binding studies of FHIP I and FHIP II with heparin using various biophysical methods. A fluorescence melting study specifically revealed the preference of heparin for domain III in FN, indicating the key contribution of FHIP I and FHIP II in heparin binding. In isothermal titration calorimetry (ITC), the higher binding affinity observed for FHIP II (∼10 mol) compared to FHIP I (∼10 mol) is expected due to the presence of a superior cluster of Arg and Lys residues in FHIP II, which can facilitate specific H-bonding interactions with heparin. Based on heat capacity changes, the key role of H-bonding, electrostatic and hydrophobic interactions was demonstrated in binding. Finally, the molecular docking and MD simulation results reinforced that the interaction of heparin (dodecasaccharide) is stronger and stable with the FHIP II peptide. The results described here suggest that these peptides provide all the structural and thermodynamic elements necessary for heparin-binding of HEP II of FN. Subsequently, it can be concluded that FHIP II could be a better location for therapeutic intervention in cell adhesion activity by FN.
纤连蛋白（FN）含结构域III的HEP II（肝素结合位点II）区域通过与细胞表面的肝素结合，在细胞黏附和迁移中起关键作用。HEP II中有两个这样的纤连蛋白肝素相互作用肽（FHIP I和FHIP II）序列。然而，这些位点协调肝素结合过程的分子原理尚不清楚。此类知识对FN的治疗靶向具有重要意义。为此，我们使用各种生物物理方法探索了FHIP I和FHIP II与肝素的结合研究。荧光熔解研究特别揭示了肝素对FN中结构域III的偏好，表明FHIP I和FHIP II在肝素结合中起关键作用。在等温滴定量热法（ITC）中，由于FHIP II中存在更优质的精氨酸和赖氨酸残基簇，可促进与肝素的特异性氢键相互作用，因此预计FHIP II（10 mol）比FHIP I（10 mol）具有更高的结合亲和力。基于热容变化，证明了氢键、静电和疏水相互作用在结合中的关键作用。最后，分子对接和分子动力学模拟结果强化了肝素（十二糖）与FHIP II肽的相互作用更强且更稳定。此处描述的结果表明，这些肽提供了FN的HEP II肝素结合所需的所有结构和热力学元素。随后，可以得出结论，FHIP II可能是FN细胞黏附活性治疗干预的更好靶点。
