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

Abstract

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 LN84–102 exhibits an improved affinity by >3‐fold relative to the traditional epitope‐derived peptide KL73–92. Even so, the LN84–102 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 LN84–102 peptide conformation, which mimics the conformational constraint addressed by protein context. Consequently, several cyclic peptides are redesigned, in which the LN84–102(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.

A Structure Independent Molecular Fragment Interfuse Model for Mesoscale Dissipative Particle Dynamics Simulation of Peptides

“…18 Therefore, there is a need to redesign peptides that are based on active receptor-binding domains of proteins to address the issues of misfolding, aggregation, enzymatic degradation, and short half-life. 19 Morphogenetic peptide sequences are, in general, 10−30 amino acids in length. As there are 20 different amino acids, there is an enormous number of 10−30 sequences to be studied for identifying possible sequences with morphogenetic activity.…”

“…Furthermore, natural peptides are susceptible to degradation by proteolytic enzymes and their function is limited by short half-life in physiological medium . Therefore, there is a need to redesign peptides that are based on active receptor-binding domains of proteins to address the issues of misfolding, aggregation, enzymatic degradation, and short half-life …”

“… 18 Therefore, there is a need to redesign peptides that are based on active receptor-binding domains of proteins to address the issues of misfolding, aggregation, enzymatic degradation, and short half-life. 19 …”