S100B as an Antagonist To Interfere with the Interface Area Flanked by S100A11 and RAGE V Domain

Abstract

The Ca²⁺-sensing protein S100A11 of the S100 family is an important mediator of numerous biological functions and pathological conditions including cancer. The receptor for advanced glycation end products (RAGE) has been well accepted as the major receptor for several S100 family members. Here, we take the S100B protein as an antagonist to interfere with the interaction flanked by S100A11 and the RAGE V domain. We employed NMR spectroscopy to describe the interactions between the S100A11 and S100B proteins. ¹H-¹⁵N heteronuclear single-quantum correlation-NMR titrations showed the potential binding dynamics of S100A11 and S100B interactions. In the HADDOCK program, we constructed the S100A11-S100B heterodimer complex that was then superimposed with the S100A11-S100B complex structure in the same orientation as the S100A11-RAGE V domain complex. This overlay analysis showed that S100B could interfere in the binding section of S100A11 and the RAGE V domain. Additionally, water-soluble tetrazolium-1 assay provided a functional read-out of the effects of these proteins in an in vitro cancer model. Our study establishes that the development of an S100B antagonist could perform a vital part in the treatment of S100- and RAGE-dependent human diseases.

Figure 1

Analysis of the binding interface (S100A11/S100B) region in S100A11. (a) ¹H–¹⁵N HSQC spectra overlay of free ¹⁵N S100A11 (red) and ¹⁵N S100A11 binding to unlabeled S100B (blue). Cross-peaks illustrating changes in intensities are shown in boxes. Horizontal lines connect the NH₂ side chains. (b) The cross-peak intensity plot (I/I₀) where (I) denotes the cross-peak intensity of S100A11–S100B complex and (I₀) indicates the early intensity of free S100A11 versus a number of amino-acid residues (1–105) illustrated as a bar diagram. (c) A picture illustration of the S100A11 monomer with residues exhibiting decreases in cross-peak signals distinguished by the cyan color.

Figure 2

Analysis of the binding interface (S100A11/S100B) region in S100B. (a) ¹H–¹⁵N HSQC spectra overlay of free ¹⁵N S100B (red) and ¹⁵N S100B binding to unlabeled S100A11 (blue). Cross-peaks showing changes in intensities are shown in boxes. (b) Cartoon representation of the S100B monomer with residues exhibiting a decrease in cross-peak signals are shown in yellow.

Figure 3

Cartoon representing S100A11–S100B complex created with the HADDOCK program. S100A11 is depicted in red and S100B in blue color. Residues near the interaction sites are illustrated in cyan (from the S100A11 side) and yellow (from S100B side) respectively.

Figure 4

¹H–¹⁵N HSQC spectra of free ¹⁵N S100A11 (red).

Figure 5

¹H–¹⁵N HSQC spectra overlay of free ¹⁵N S100A11 (red) and ¹⁵N S100A11 homodimer + S100B homodimer mixture (blue).

Figure 6

¹H–¹⁵N HSQC spectra overlay of free ¹⁵N S100A11 (red) and ¹⁵N S100A11–S100B heterodimers (green).

Figure 7

Dissociation constant. K_d calculated by the designated residues detected in ¹⁵N S100A11 HSQC titrations with S100B (yellow) and vice-versa (brown). The overall average K_d is about 2.65 μM.

Figure 8

WST-1 assay analysis. The SW-480 cells were treated with 100 nM of S100A11 (dark blue), 100 nM of S100B (light blue), and 100 nM of S100A11–S100B heterodimers (green). The proportional cell counts after subsequent treatment with S100A11 and S100B are depicted as fold inductions with serum-free media alone as the control (red).

Figure 9

S100B interfering S100A11–RAGE V domain. (a) The complex model of the S100A11–RAGE V domain constructed via HADDOCK. (b) Superimposition of the complex between S100A11 (red) and the RAGE V domain (green), with the complex amid S100A11 (red) and S100B (blue). (c) The magnified depiction of the clear and significant hindrance to S100A11–RAGE V domain associated with the S100B protein.