Integrated in Silico–in Vitro Rational Design of Osteogenic Peptides derived from the Armpit Epitope of Human Bone Morphogenetic Proteins

Bone morphogenetic proteins (BMPs) are multi-functional growth factors that initiate, promote and maintain cartilage and bone morphogenesis, differentiation and regeneration in both the developing embryo and adult. The proteins have a conformational wrist epitope and a linear knuckle epitope responsible for, respectively, type-I and type-II receptor binding, as well as a hybrid armpit epitope targeted by natural BMP antagonists. In this study, the recognition and interaction between human BMPs and their pan-antagonist Crossveinless was investigated systematically at molecular level. It is revealed that the armpit epitope shares a roughly common region over different BMPs, which consists of a loop segment and a turn segment that are sequentially discontinuous but spatially vicinal on these BMP protein surfaces. Turn segment is the primary binding site that can be bound effectively by Crossveinless using a tightly packed mode. The segment was further extended at its two termini to cover a complete double-stranded sheet of BMPs, which was then split from the interfacial context of BMP–Crossveinless complexes to derive a series of osteogenic peptides; they exhibit moderate intrinsic disorder in free state, but can be constrained into a native-like conformation by stapling a disulfide bridge across two strands of the sheet. The disulfide bridge was rationally designed and optimized to avoid disrupting the native interaction of BMP sheet peptides with the active pocket of Crossveinless. Biophysical assays substantiated that the binding affinities of resulting cyclic peptides were improved by 2–6-fold relative to their linear counterpart upon the stapling, in which the cyclic peptide Bmp7-sb1 (S[CLYFDDNSNVILC]K) derived from the double-stranded sheet region of BMP7 armpit epitope was determined to have the highest affinity to Crossveinless in all tested samples. These rationally designed epitope-derived peptides can be used as osteogenic agents to activate the human BMP signaling by competitively targeting their natural antagonist.