Crystal structure of the TRANCE/RANKL cytokine reveals determinants of receptor-ligand specificity

Abstract

RANK, the receptor activator of NF-kappaB, and its ligand RANKL (initially termed TRANCE, also termed ODF and OPGL), are a TNF superfamily receptor-ligand pair that govern the development and function of osteoclasts, lymphoid tissue, and mammary epithelium. While TNF family cytokines share a common structural scaffold, individual receptor-ligand pairs associate with high specificity. Given the low level of amino acid conservation among members of the TNF superfamily, the means by which these molecules achieve specificity cannot be completely understood without knowledge of their three-dimensional structures. To determine the elements of RANKL that mediate RANK activation, we have crystallized the ectodomain of murine RANKL and solved its structure to a resolution of 2.6 A. RANKL self-associates as a homotrimer with four unique surface loops that distinguish it from other TNF family cytokines. Mutagenesis of selected residues in these loops significantly modulates RANK activation, as evidenced by in vitro osteoclastogenesis, thereby establishing their necessity in mediating the biological activities of RANKL. Such structural determinants of RANKL-RANK specificity may be of relevance in the pharmacologic design of compounds to ameliorate osteopenic disorders of bone.

Figure 1

Crystal structure of RANKL. (a) Ribbon diagram of the RANKL trimer, shown with the β-strands (green) and connecting loops (orange) of one RANKL monomer labeled according to standard TNF-β–sandwich nomenclature. The other two RANKL monomers are cyan and magenta, respectively. (b) In this view, oriented identically to a, the RANKL transmembrane stalk projects to the top of the image, while the membrane-distal region is toward the bottom. The homotrimer exhibits the shape of a truncated pyramid, being slightly wider at the membrane-proximal end. (c) Ribbon diagram of the RANKL trimer viewed down the axis of threefold symmetry, oriented with the membrane-distal face forward. The secondary structure of monomer X is labeled as in a. (d) The RANKL trimer, shown with the molecular surfaces of monomers X, Y, and Z colored in green, cyan, and magenta, respectively. The orientation of the molecule is identical to that in c. (e) Comparison of a single RANKL monomer with those of TNF and TRAIL. The β-strands of all three structures are colored green, while the connecting loops are colored orange for RANKL, gray for TNF (PDB code 1tnr), and blue for TRAIL (PDB code 1d4v). When the structures of these proteins are superimposed, it is apparent that the β-strands of RANKL superimpose almost identically with those of TNF and TRAIL. In contrast, the AA′′, CD, EF, and DE loops of RANKL exhibit unique topology when compared with those of other TNF family cytokines. (f) Electron density of the E-D-G β-strands. The structure is viewed from the solvent-accessible surface of the RANKL monomer, with an orientation similar to (e). Displayed in magenta is a 2.6-Å composite omit map (contoured at 1.2 ς) with the RANKL structure depicted in green (carbon), red (oxygen), blue (nitrogen), and yellow (sulfur).

Figure 2

Structure-based alignment of TNF family cytokines with RANKL. The sequence of the extracellular core domains of the TNF family cytokines TRAIL, CD40L, TNF-α, TNF-β, and ACRP30 are shown aligned to that of murine RANKL. Structural alignments to RANKL are based upon pairwise topological residue superposition of the crystal structure of RANKL with those of TRAIL (1d4v), CD40L (1aly), TNF-α (2tnf), TNF-β (1tnr), and ACRP30 (1c28). Residue numbers and secondary structure assignments for RANKL are depicted above the sequences. The ten β-strands that constitute the TNF family β-sandwich are drawn as green arrows, with standardized nomenclature. Solvent-accessible loops and coil regions connecting the β-strands are illustrated as brown lines. The AA′′ loop (orange line), connecting β-strands A and A′′, encompasses the A′ β-strand. Brown triangles above the residues indicate those involved in the trimeric interface of RANKL. The degree of homology at structurally equivalent positions among the family members (excluding ACRP30) is shown by colored circles below the sequences, 0–50% conservation (no circles), 50–90% (tan), >90% (brown). TRAIL residues boxed in green denote DR5 receptor contact sites on TRAIL. TNF-β residues boxed in magenta identify TNFR contact sites on TNF-β. By substituting RANKL in place of the ligands in the TRAIL:DR5 and TNF:TNFR co-crystal structures, we calculated potential contact sites for both of these receptors on RANKL. Boxed residues of RANKL indicate those calculated to physically contact DR5 (green), TNFR (magenta), or both (yellow) receptors during the docking analysis. Structural elements that are unique to RANKL are clustered in the solvent-accessible AA′′, CD, DE, and EF loops.

Figure 3

Receptor contact regions of RANKL. (a) Schematic depiction of a RANKL trimer (green, cyan, and magenta monomers) docked with its receptor RANK (drawn here as four CRDs, yellow). (b) Surface representation of a RANKL trimer, oriented as in a, docked with a monomer of DR5, the TRAIL receptor (yellow Cα backbone worm). Areas on the surface of RANKL calculated to engage DR5 are shaded green, as is the sequence in Figure 2. (c) Surface representation of a RANKL trimer docked with a monomer of the TNFR (yellow Cα backbone worm). Areas on the surface of RANKL calculated to engage the TNFR are shaded magenta. (d) Comparison of DR5 (green shading) and TNFR (magenta shading) contact sites on the surface of RANKL. Overlapping areas that contact both receptors are shown in yellow. (e) Surface representation of a RANKL trimer with degrees of conservation mapped to the surface. The degree of conservation is directly proportional to intensity of colorization (white, no conservation; brown, conserved). Areas that contact both receptors show little to no sequence conservation across this family of cytokines (light tan to white shading). These unique regions provide specificity for receptor-ligand recognition, underlying the lack of cross-reactivity among receptors and ligands in this cytokine family. (f) Potential receptor contact areas of RANKL that were targeted for structure-based mutagenesis are shown in red.

Figure 4

Structure-based mutagenesis of RANKL. (a) Ribbon view of AA′′ loop illustrating the AA′′ loop deletion and AA′′ loop-swap mutants. For the AA′′ loop deletion, SGSHKVTLSS was mutated to SSS by removal of the segment depicted in red (GSHKVTL). For the AA′′ loop-swap mutant, SGSHKVTLSS was mutated to SLL by swapping the segments depicted in blue and red with the analogous segments from TNF. (b) Dose-response curves for native and mutant forms of RANKL (filled square, native RANKL; filled circle, I248D point substitution; filled triangle, AA′′ loop deletion; open triangle, AA′′ loop swap) are plotted for in vitro osteoclastogenesis. ED₅₀ of native RANKL is ∼12 ng/ml, compared with ∼100 ng/ml for I248D RANKL. AA′′ loop deletion and AA′′ loop-swap mutants failed to induce detectable osteoclastogenesis at any dosage. (c) Osteoclastogenic cultures are depicted at identical magnification after 7 days of exposure to the various forms of RANKL. Cultures were stained for osteoclast-specific TRAP activity (red reaction product). Native RANKL induces the formation of large, multinucleated, TRAP-expressing osteoclasts. I248D RANKL generates TRAP-positive cells that lack the characteristic morphology of osteoclasts, with the majority of cells exhibiting mononuclearity. AA′′ loop deletion and loop-swap mutants fail to induce differentiation along the osteoclastogenic pathway.