Insights into the molecular basis and mechanism of heme-triggered TLR4 signaling

Introduction Large amounts of labile heme are known to be pro-inflammatory, pro-thrombotic, and cytotoxic by e.g. binding to biomolecules such as proteins [1] [2] [3]. One such example is TLR4, which has been poorly understood with regards to its activation by heme. TLR4 forms a complex with MD2 and the complex then assembles in a dimeric state. In this study, we report in vitro, cellular and in silico analysis of the heme binding to TLR4, MD2, and their complex.

Method Heme binding was investigated on the peptide as well as the protein level by employing prediction of heme-binding motifs by HeMoQuest [4], peptide synthesis and characterization, as well as binding studies by UV/vis spectroscopy experiments. Surface plasmon resonance (SPR) and UV/vis spectroscopic studies were used to analyze heme binding to the proteins. These experiments were further complimented by molecular docking and molecular dynamics simulations of the free and heme-bound proteins TLR4, and MD2, as well as the TLR4-MD2 complex.

Results Our study revealed motifs with high to moderate heme-binding affinity in both proteins, with TLR4 possessing 4 HBMs and MD2 2 HBMs. In contrast to an earlier report [5], two other heme-binding sites were identified in MD2. Cellular experiments analyzing heme-mediated cytokine responses are partially via TLR4 and independently of CD14, which activates TLR4 signaling in the absence of MD2. The further characterization of the most promising and experimentally validated HBMs in TLR4 using mutagenesis studies revealed that heme interacts with TLR4 through distinct binding sites, and individual HBMs are dispensable for full receptor activation.

Conclusion To summarize, a stepstone analysis in the direction of TLR4 signaling activation via transient heme binding was performed. HBMs available in the TLR4-MD2 complex can mediate TLR4 signaling upon heme binding via different mechanisms [6]. Additionally, we also found that TLR4 has higher heme binding affinity as compared to MD2. A detailed exploration of the effects of heme on immune receptors with regard to proinflammatory effects may contribute to a better understanding of hemolytic disorders.

Conflict of Interest

The authors declare no conflict of interest.

• References

• 1 Roumenina LT, Rayes J, Lacroix-Desmazes S, Dimitrov JD.. Heme: Modulator of Plasma Systems in Hemolytic Diseases. Trends Mol Med 2016; 22 (03) 200-213 DOI: 10.1016/j.molmed.2016.01.004.
• 2 Hopp M-T, Imhof D.. Linking Labile Heme with Thrombosis. Journal of Clinical Medicine 2021; 10 (03) 427 DOI: 10.3390/jcm10030427.
• 3 Mubeen S, Domingo-Fernández D, Díaz del Ser S, Solanki DM, Kodamullil AT, Hofmann-Apitius M, Hopp M-T, Imhof D.. Exploring the Complex Network of Heme-Triggered Effects on the Blood Coagulation System. Journal of Clinical Medicine 2022; 11 (19) 5975 DOI: 10.3390/jcm11195975.
• 4 Paul George AA, Lacerda M, Syllwasschy BF, Hopp MT, Wißbrock A, Imhof D.. HeMoQuest: a webserver for qualitative prediction of transient heme binding to protein motifs. BMC Bioinformatics 2020; 21 (01) 124 Published 2020 Mar 27 DOI: 10.1186/s12859-020-3420-2.
• 5 Belcher JD, Zhang P, Nguyen J. et al. Identification of a Heme Activation Site on the MD-2/TLR4 Complex. Front Immunol 2020; 11: 1370 Published 2020 Jun 30 DOI: 10.3389/fimmu.2020.01370.
• 6 Hopp M-T, Holze J, Lauber F, Holtkamp L, Rathod DC, Miteva MA, Prestes EB, Geyer M, Manoury B, Merle NS, Roumenina LT, Bozza MT, Weindl G, Imhof D.. Insights into the molecular basis and mechanism of heme-triggered TLR4 signaling: The role of heme-binding motifs in TLR4 and MD2. Immunology. 2023 (accepted)

Publication History

Article published online:
26 February 2024