Biomimetic design of affinity ligands for collagen based on Glycoprotein VI-collagen interface

Collagen is a vital structural protein with applications including tissue engineering, drug delivery systems, cosmetics, etc. Purification of collagen is still a challenge in consideration of the structural integrity, biological activity, selectivity, and purity. This highlights the urgent need for efficient affinity ligands of collagen to facilitate its separation and purification via affinity chromatography. Glycoprotein VI (GPVI) has been reported as a native counterpart capable of binding on collagen, providing a foundation for the development of collagen-specific ligands. Therefore, peptide ligands targeting collagen were developed based on the molecular interactions involved in the binding of GPVI on collagen. Molecular dynamics (MD) simulations and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) analysis were used to investigate molecular interactions. Affinity ligand libraries DXXWXK and DXXWXEK (where X represented arbitrary residue of 20 standard amino acids) targeting collagen were then constructed, in which DYHWHEK and DYMWHEK were screened by molecular docking, MD simulations, etc. Furthermore, the binding of the ligands to collagen was experimentally verified by a significant higher OD₄₅₀ of 1.9600 ± 0.1677 (DYHWHEK) and 2.5801 ± 0.1696 (DYMWHEK) as compared to control in enzyme-linked immunosorbent assay, and an increased melting temperature of 5.241 °C (DYHWHEK) and 2.642 °C (DYMWHEK) for the complex of ligand-collagen in thermal shift assay. Subsequently, DYHWHEK was coupled to NHS-activated Sepharose 4 Fast Flow to obtain DYHWHEK-4FF. Successful adsorption and elution of collagen using DYHWHEK-4FF confirmed DYHWHEK as a high-affinity ligand for collagen, demonstrating its potential for efficient collagen purification.