Structure-based development of a receptor activator of nuclear factor-κB ligand (RANKL) inhibitor peptide and molecular basis for osteopetrosis

Abstract

The receptor activator of nuclear factor-κB (RANK) and its ligand RANKL, which belong to the tumor necrosis factor (TNF) receptor-ligand family, mediate osteoclastogenesis. The crystal structure of the RANKL ectodomain (eRANKL) in complex with the RANK ectodomain (eRANK) combined with biochemical assays of RANK mutants indicated that three RANK loops (Loop1, Loop2, and Loop3) bind to the interface of a trimeric eRANKL. Loop3 is particularly notable in that it is structurally distinctive from other TNF-family receptors and forms extensive contacts with RANKL. The disulfide bond (C125-C127) at the tip of Loop3 is important for determining the unique topology of Loop3, and docking E126 close to RANKL, which was supported by the inability of C127A or E126A mutants of RANK to bind to RANKL. Inhibitory activity of RANK mutants, which contain loops of osteoprotegerin (OPG), a soluble decoy receptor to RANKL, confirmed that OPG shares the similar binding mode with RANK and OPG. Loop3 plays a key role in RANKL binding. Peptide inhibitors designed to mimic Loop3 blocked the RANKL-induced differentiation of osteoclast precursors, suggesting that they could be developed as therapeutic agents for the treatment of osteoporosis and bone-related diseases. Furthermore, some of the RANK mutations associated with autosomal recessive osteopetrosis (ARO) resulted in reduced RANKL-binding activity and failure to induce osteoclastogenesis. These results, together with structural interpretation of eRANK-eRANKL interaction, provided molecular understanding for pathogenesis of ARO.

Crystal Structure of Human RANKL Complexed with Its Decoy Receptor Osteoprotegerin

“…4C, 4D). Similarly, it has also been shown that mutation of RANK residue Asp 85 at site I did not significantly affect the RANKL binding and inhibition of osteoclast differentiation by soluble RANK (26). At site II, the general binding mode of an inserted loop from receptor into RANKL trimer is also conserved in the RANKL/OPG-CRD and RANKL/RANK complexes.…”

“…Mouse RANKL Lys 256 has interaction with RANK Lys 97 at the interface. Mutation of either Glu 126 or Lys 97 at RANK completely abrogated the RANKL binding and inhibition of osteoclast differentiation by soluble RANK (26). We also performed a GST pull-down experiment using human GST-RANKL and its mutants with human OPG-CRD or human RANK.…”

“…The crystal structure of the mouse RANKL/RANK complex was recently solved independently by two groups, providing precise molecular details of RANKL/RANK interaction (26,27). In this article, we report the complex structure of human RANKL ectodomain with N-terminal four CRD domains of human OPG (OPG-CRD).…”

The skeleton: a multi-functional complex organ. The role of key signalling pathways in osteoclast differentiation and in bone resorption

“…RANK is a type I transmembrane receptor belonging to the TNF receptor superfamily. The crystal structure of the extracellular domain of RANK has recently been described in the presence and absence of RANKL (Ta et al 2010). These studies showed tight binding between RANK and RANKL and illustrate how single base changes to the receptor-binding domain in RANKL drastically reduce interaction with RANKL and prevent osteoclast formation.…”

Dimerization of LTβR by LTα1β2is necessary and sufficient for signal transduction

“…The extracellular domain (ECD) of LTβR comprises four CRDs and is expected to have similar overall architecture to other multidomain TNFRSF members such as TNFR1. Crystal structures of several ligand-receptor complexes in this superfamily (15)(16)(17)(18)(19)(20)(21)(22)(23) revealed that receptors bind in a symmetrical Significance Cytokines are proteins that modulate the activity of target cells via activation of cell-surface receptors. The trimeric cytokines of the tumor necrosis factor superfamily typically signal by inducing homotrimerization of their cognate receptors.…”