Structural definition of the discrete hotspot sites of BMP-2 conformational wrist epitope and rational design of the hotspot-derived osteogenic peptides against chondrocyte senescence

Abstract

The bone morphogenetic protein-2 (BMP-2) is an essential regulator of bone formation and remodeling, which has also been implicated in the pathogenesis of osteoarthritis and its closely related chondrocyte senescence. The BMP-2 uses a conformational wrist epitope and a linear knuckle epitope to interact with type-I (BMPR-I) and type-II (BMPR-II) receptors, respectively. Previously, the knuckle epitope has been intensely studied, but the wrist epitope still remains largely unexplored due to its discontinuous nature. In the present work, the intermolecular interaction of BMP-2 with BMPR-I was investigated systematically at structural, energetic and dynamic levels. Three discrete hotspots that represent the key BMPR-I recognition sites of BMP-2 were identified; they are spatially dispersed over the two monomers of BMP-2 dimer and totally account for 83.5 % binding potency of BMP-2 to BMPR-I (hotspot 1: residues 49–70 in monomer 1; hotspot 2: residues 24–31 in monomer 2; hotspot 3: residues 88–107 in monomer 2). Therefore, we defined the three discrete hotspot sites as the core region of wrist epitope; their contribution to the binding increases in the order: hotspot 2 < hotspot 3 < hotspot 1. We demonstrated that the primary hotspot 1 site has a native U-shaped conformation in the full-length BMP-2 protein context, but it cannot maintain in the native conformation when split from the context to obtain a free hotspot-1 peptide, thus largely impairing its binding potency to BMPR-I. We further employed disulfide-bonded cyclization and head-to-tail cyclization to constrain the peptide conformation, and found that only the former can effectively constrain the peptide into native conformation, thus considerably improving its binding affinity to BMPR-I, whereas the latter totally disorders the native conformation, thus rendering the peptide as a full nonbinder of BMPR-I.