Structural insights into the binding of the human receptor for advanced glycation end products (RAGE) by S100B, as revealed by an S100B-RAGE-derived peptide complex

Abstract

S100B is a damage-associated molecular pattern protein that, when released into the extracellular milieu, triggers initiation of the inflammatory response through the receptor for advanced glycation end products (RAGE). Recognition of S100B is accomplished via the amino-terminal variable immunoglobulin domain (V-domain) of RAGE. To gain insights into this interaction, a complex between S100B and a 15-amino-acid peptide derived from residues 54-68 of the V-domain was crystallized. The X-ray crystal structure was solved to 2.55 Å resolution. There are two dimers of S100B and one peptide in the asymmetric unit. The binding interface of this peptide is compared with that found in the complex between S100B and the 12-amino-acid CapZ-derived peptide TRTK-12. This comparison reveals that although the peptides adopt completely different backbone structures, the residues buried at the interface interact with S100B in similar regions to form stable complexes. The binding affinities of S100B for the intact wild-type V-domain and a W61A V-domain mutant were determined to be 2.7 ± 0.5 and 1.3 ± 0.7 µM, respectively, using fluorescence titration experiments. These observations lead to a model whereby conformational flexibility in the RAGE receptor allows the adoption of a binding conformation for interaction with the stable hydrophobic groove on the surface of S100B.

Keywords: RAGE-derived peptide; S100B.

Figure 1

The asymmetric unit contents of the S100B–W61 crystal. (a) Two S100B homodimers and one W61 peptide are visible in the asymmetric unit. Monomers of S100B are shown in shades of green, the W61 peptide is shown in violet and Ca²⁺ ions are shown as black spheres. (b) Final refined 2F _o − F _c electron density (blue mesh) contoured at 1σ above the mean of the composite OMIT map shows the W61 peptide on the surface of S100B chain A in the same orientation as in (a).

Figure 2

The S100B–W61 peptide complex. The surface of S100B is shown coloured according to atom type: carbon, green; oxygen, red; nitrogen, blue; sulfur, yellow. The W61 peptide is shown in stick representation and is coloured according to atom type: carbon, violet; oxygen, red; nitrogen, blue.

Figure 3

Comparison of the S100B–W61 (top) and S100B–TRTK-12 (bottom) crystal structures. (a) Cartoon representation of the peptide backbone (W61, violet; TRTK-12, cyan) on the surface of the hydrophobic groove of S100B. (b) Ala60 in the W61 peptide is positioned in the same pocket as the indole ring of Trp7 in TRTK-12. (c) Val63 of the W61 peptide is positioned in a groove formed by the surface of S100B residues Leu44, Phe76, Met79 and Val80 on the surface of S100B, similar to Ile10 in the TRTK-12 complex.

Figure 4

Fluorescence polarization titration of fluorescein-labeled S100B (1 µM) and the V-domain of RAGE. The titration of the wild-type V-domain is shown by black circles and that of the W61A mutant of the V-domain is shown by half-filled squares. The error bars indicate the standard deviation obtained from three independent experiments. The apparent K _d values obtain from the quadratic fits were 2.7 ± 0.5 µM (solid black line, R = 0.9938) and 1.3 ± 0.7 µM (dashed black line, R = 0.9956) for the wild-type V-domain and the W61A mutant, respectively (Anderson et al., 1988; Leclerc & Vetter, 1998 ▶).