PREDICTED STRUCTURE AND BINDING MOTIFS OF COLLAGEN a1(XI)

The amino propeptide of collagen a1(XI) (NPP)has been shown to bind glycosaminoglycans and to form adimer. While these are independent biochemical events, it islikely that dimerization facilitates the interaction with glycosaminoglycansor alternatively, that glycosaminoglycan interactionfacilitates the formation of an NPP:NPP dimer. Thecomputer program MODELLER was used to generate a homologymodel of the collagen a1(XI) NPP monomer using thecrystal structure of the closely related noncollagenous-4 (NC4)domain of collagen a1(IX) (PDB:2UUR) as the template. Additionally,a dimer model of collagen a1(XI) NPP domain wascreated based upon the thrombospondin dimer template(PDB:1Z78). The structure of the dimer created inMODELLER was validated by comparison to a dimer modelgenerated by docking two monomers of PDB:2UUR usingClusPro. Calculations of relative binding energy for the interactionbetween each collagen 1(XI) NPP model and glycosaminoglycansas ligands was performed using AutoDock4.Computational results support a higher affinity between heparansulfate and a dimer compared to a monomer. These findingsare supported by affinity chromatography experiments inwhich distinct monomer and dimer peaks were observed. Sequentialpoint mutation studies of the putative binding site(147-KKKITK-152) indicated the importance of the basiclysine residue for binding to heparan sulfate. Two orders ofmagnitude change in binding affinity was predicted whencomparing wild type to the mutation K152A. Experimentaldata supports the predicted change in affinity