br Perspective and conclusion Collagen Toolkits

Perspective and conclusion Collagen Toolkits II and III have been used to determine the Z-VDVAD-FMK for DDR1 and DDR2, and the main binding site is the GVM-GFO motif [103,108]. The co-crystal structure of the DS domain of human DDR2 bound to a synthetic collagen-like peptide containing the GVM-GFO motif further elucidated the exact DDR2–collagen interactions [109]. Recombinant collagen has been used to provide further validation of the important GVM-GFO binding motif [120]. However, regions other than the GVM-GFO motif were found to exhibit DDR binding as well and further investigation is needed to understand their roles in DDR function [103,108]. Furthermore, it remains unclear how collagen binding leads to DDR activation. In collagen III sequence, at least two triplets N-terminal to the essential GVM-GFO binding motif were required for DDR2 activation by synthetic collagen-like peptides, and even with these additional triplets, a very high concentration of peptides was needed [103]. Another direction that can further expand our knowledge of these interactions is the development of Toolkit libraries that span the collagen domains of the other types of collagen. For example, the ability to synthesize well-defined, stable, and biologically active collagen-like heterotrimers (see Section 3.1 for active research areas) will help elucidate how DDR1 and DDR2 interact with type I collagen. However, owing to the interruptions in their triple-helical domains, it could be challenging to apply the Toolkit philosophy to non-fibrillar collagens such as collagen IV and X [57]. Scl2 recombinant collagen construct has been used to study DDR–collagen interactions. However, these recombinant proteins are typically expressed in E. coli, which lacks prolyl hydroxylases, and thus do not contain Hyp. This difference of Hyp vs Pro has been cited as the cause of decreased (but not eliminated) binding affinity in multiple cases [[116], [117], [118], [119]], including in the case of DDRs [120]. An active area of research has been in developing strategies to introduce the Pro to Hyp post-translational modifications in E. coli [[137], [138], [139]]. These strategies have shown varying levels of effectiveness to hydroxylate larger sequences and accuracy in hydroxylating the Yaa proline. Advances in this area will help develop recombinant proteins that better mimic animal collagen. Understanding the nature of protein–collagen interactions is also crucial in developing biomaterials that replicate the structural and biological characteristics of collagen [140]. These biomaterials can then be applied to develop scaffolds or tissues that can be valuable in surgeries or medicine [7,140]. These biomaterials can also incorporate synthetic peptides and recombinant collagen, highlighting the adaptability of these tools. For example, chemically crosslinked collagen has been studied as a biomaterial in tissue engineering [140]. While this chemical treatment is important to create mechanically stable collagen film, it unfortunately disrupts the film's cell reactivity [141,142]. To restore reactivity to DDR2, a synthetic collagen-like peptide including the important DDR2-binding motif, (GPP)5-GPR-GQO-GVNle-GFO-(GPP)5, and a linker have been incorporated to crosslinked collagen films [143]. These crosslinked films coupled with the collagen-like peptide were then able to bind to DDR2 and induce DDR2 activation [143]. The recombinant Scl2 collagen system has shown capability as a biomaterial as well because of its adaptability and scalability. Scl2 was functionalized to crosslink into a hydrogel without disrupting its triple helix [130]. The Scl2–hydrogel crosslinking also did not disrupt cell adhesion and integrin binding when the α1 and α2 integrin binding motifs of collagen were inserted into the Scl2 [130]. This Scl2–hydrogel has been incorporated in the development of a vascular graft with suitable biomechanical properties [144] and in the development of an injectable medicine to stimulate chondrogenesis [145]. Additionally, a high-throughput batch purification methodology for Scl2 recombinant collagen has been developed [146]. This methodology has shown to be very scalable and produce a high percentage (>95%) of pure protein [146,147]. As our understanding of DDR–collagen interactions advances, it shall be possible to engineer biomaterials with DDR-activating or inhibiting properties.