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. 2016 Jan 27:10:455-63.
doi: 10.2147/DDDT.S98466. eCollection 2016.

Determination of the binding mode for anti-inflammatory natural product xanthohumol with myeloid differentiation protein 2

Weitao Fu  1 , Lingfeng Chen  1 , Zhe Wang  1 , Chengwei Zhao  1 , Gaozhi Chen  1 , Xing Liu  1 , Yuanrong Dai  2 , Yuepiao Cai  1 , Chenglong Li  3 , Jianmin Zhou  1 , Guang Liang  1
Affiliations

Determination of the binding mode for anti-inflammatory natural product xanthohumol with myeloid differentiation protein 2

Weitao Fu et al. Drug Des Devel Ther. .

Abstract

It is recognized that myeloid differentiation protein 2 (MD-2), a coreceptor of toll-like receptor 4 (TLR4) for innate immunity, plays an essential role in activation of the lipopolysaccharide signaling pathway. MD-2 is known as a neoteric and suitable therapeutical target. Therefore, there is great interest in the development of a potent MD-2 inhibitor for anti-inflammatory therapeutics. Several studies have reported that xanthohumol (XN), an anti-inflammatory natural product from hops and beer, can block the TLR4 signaling by binding to MD-2 directly. However, the interaction between MD-2 and XN remains unknown. Herein, our work aims at characterizing interactions between MD-2 and XN. Using a combination of experimental and theoretical modeling analysis, we found that XN can embed into the hydrophobic pocket of MD-2 and form two stable hydrogen bonds with residues ARG-90 and TYR-102 of MD-2. Moreover, we confirmed that ARG-90 and TYR-102 were two necessary residues during the recognition process of XN binding to MD-2. Results from this study identified the atomic interactions between the MD-2 and XN, which will contribute to future structural design of novel MD-2-targeting molecules for the treatment of inflammatory diseases.

Keywords: binding mode; inflammation; molecular dynamics simulation; myeloid differentiation 2; xanthohumol.

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Figures

Figure 1
Figure 1
XN’s binding activity to MD-2 protein. Notes: (A) The structure of XN. (B) SPR shows direct interactions between XN and the wild type of MD-2 protein. (C) ELISA assay showing XN inhibition of biotin-LPS binding to wild-type MD-2 protein. Data are mean values (± SEM) of at least three separate repeated experiments (**P<0.01). Abbreviations: ELISA, enzyme-linked immunosorbent assay; LPS, lipopolysaccharide; MD-2, myeloid differentiation protein 2; SEM, standard error of the mean; SPR, surface plasmon resonance; XN, xanthohumol; Ka, association (‘on rate’); Kd, dissociation rates (‘off rate’); KD, equilibrium dissociation constant (‘binding constant’); WT, wild type.
Figure 2
Figure 2
Molecular docking analysis of XN to the activity cavity of MD-2. Notes: (A) XN overlapped with LPS in the binding site of MD-2 together with TLR4. TLR4 (cyan), MD-2 (wheat), LPS (green sticks), XN (blue spheres). (B) Overlapped region of XN (blue sticks) and LPS (green sticks). (C) Molecular docking analysis of three-dimensional binding pose between XN and MD-2. MD-2 (wheat cartoon), XN (cyan sticks), hydrogen bonds (black dotted lines). Abbreviations: LPS, lipopolysaccharide; MD-2, myeloid differentiation protein 2; TLR4, toll-like receptor 4; XN, xanthohumol.
Figure 3
Figure 3
Backbone RMSDs are shown as a function of time for apo MD-2 and XN/MD-2 complex structures at 50 ns. Notes: (A) Time evolution of the RMSD of MD-2 and XN are shown with black lines and red lines, respectively. (B) The time evolution of the RMSD of apo MD-2. (C) The XN/MD-2 complex system. The alignments of starting (wheat) and final (blue) structures. (D) Apo MD-2 system. The alignments of starting (wheat) and final (blue) structures. Abbreviations: MD-2, myeloid differentiation protein 2; RMSD, root-mean square deviation; XN, xanthohumol.
Figure 4
Figure 4
The “openness” of apo MD-2 and XN/MD-2 complex. Notes: (A) Total distance between center of mass of apo MD-2 and every center of mass of β strand. (B) Total distance between center of mass of XN/MD-2 complex and every center of mass of β strand. Abbreviations: MD-2, myeloid differentiation protein 2; XN, xanthohumol.
Figure 5
Figure 5
Key hydrogen interactions between XN and residues ARG-90 and TYR-102 of MD-2. Notes: (A) XN (yellow sticks), residues AGR-90 and TYR-102 (white sticks covered with transparent surface), hydrogen bonds (black dotted lines); hydrogen atoms are omitted. (B) The distance for the hydrogen bonds between XN and the residue TYR-102 as a function of time. (C) The distances for the hydrogen bonds between XN and residue ARG-90 as a function of time. Abbreviations: MD-2, myeloid differentiation protein 2; XN, xanthohumol.
Figure 6
Figure 6
XN shows low binding affinity to the MD-2R90A/Y102A mutant. Notes: (A) SPR assay indicating loss of high affinity binding of XN with the R90A and Y102A mutants of MD-2. (B) ELISA assay of XN competition with biotin-LPS binding to the MD-2R90A/Y102A mutant. Abbreviations: ELISA, enzyme-linked immunosorbent assay; LPS, lipopolysaccharide; MD-2, myeloid differentiation protein 2; SPR, surface plasmon resonance; XN, xanthohumol; Ka, association (‘on rate’); Kd, dissociation rates (‘off rate’); KD, equilibrium dissociation constant (‘binding constant’).
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