Revisiting bone morphogenetic protein-2 knuckle epitope and redesigning the epitope-derived peptides

The bone morphogenetic protein-2 (BMP2) plays a crucial role in bone formation, growth and regeneration, which adopts a conformational wrist epitope and a linear knuckle epitope to interact with its type-I (BRI) and type-II (BRII) receptors, respectively. In this study, we systematically examine the BRII-recognition site of BMP2 at structural, energetic and dynamic levels and accurately locate hotspots of the recognition at BMP2-BRII complex interface. It is revealed that the traditional knuckle epitope (BMP2 residue range 73-92) do fully match the identified hotspots; the BMP2-recognition site includes the C-terminal region of traditional knuckle epitope as well as its flanked β-strands. In addition, the protein context of full-length BMP2 is also responsible for the recognition by addressing conformational constraint on the native epitope segment. Therefore, we herein redefine the knuckle epitope to BMP2 residue range 84-102, which has a similar sequence length but is slid along the protein sequence by ~10 residues as compared to traditional knuckle epitope. The redefined one is also a linear epitope that is natively a double-stranded β-sheet with two asymmetric arms as compared to the natively single β-strand of the traditional version, although their sequences are partially overlapped to each other. It is revealed that the redefined epitope-derived peptide LN^84-102 exhibits an improved affinity by >3-fold relative to the traditional epitope-derived peptide KL^73-92 . Even so, the LN^84-102 peptide still cannot fully represent the BMP2 recognition event by BRII that has been reported to have a nanomolar affinity. We further introduce a disulfide bond across the two arms of double-stranded β-sheet to constrain the free LN^84-102 peptide conformation, which mimics the conformational constraint addressed by protein context. Consequently, several cyclic peptides are redesigned, in which the LN^84-102 (cyc89-101) is determined to exhibit a sub-micromolar affinity; this value is ~5-fold higher than its linear counterpart. Structural analysis also reveals that the cyclic peptide can interact with BRII in a similar binding mode with the redefined knuckle epitope region in full-length BMP2 protein.