o-Vanillin binds covalently to MAL/TIRAP Lys-210 but independently inhibits TLR2

Abstract

Toll-like receptor (TLR) innate immunity signalling protects against pathogens, but excessive or prolonged signalling contributes to a range of inflammatory conditions. Structural information on the TLR cytoplasmic TIR (Toll/interleukin-1 receptor) domains and the downstream adaptor proteins can help us develop inhibitors targeting this pathway. The small molecule o-vanillin has previously been reported as an inhibitor of TLR2 signalling. To study its mechanism of action, we tested its binding to the TIR domain of the TLR adaptor MAL/TIRAP (MAL^TIR). We show that o-vanillin binds to MAL^TIR and inhibits its higher-order assembly in vitro. Using NMR approaches, we show that o-vanillin forms a covalent bond with lysine 210 of MAL. We confirm in mouse and human cells that o-vanillin inhibits TLR2 but not TLR4 signalling, independently of MAL, suggesting it may covalently modify TLR2 signalling complexes directly. Reactive aldehyde-containing small molecules such as o-vanillin may target multiple proteins in the cell.

Keywords:

• Covalent modification
• o-vanillin
• MyD88 adaptor-like (MAL)
• nuclear magnetic resonance (NMR)
• Toll-like receptor (TLR)
• Toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP)

Acknowledgements

We would like to thank Ashley Mansell and Paul Herzog (Hudson Institute of Medical Research, Clayton, Australia) for MAL-KO mice.

Authors’ contributions

Conception and design: M.H.R., S.J.T., M.J.M., J.D.N., X.J., P.R.V., P.M., A.H., I.V., T.H., A.A.B.R., B.D., T.V., M.M., K.J.S., B.K.; analysis and interpretation of the data: M.H.R., S.J.T., M.J.M., H.K., I.V., J.D.N., M.M., K.J.S., B.K.; the drafting of the paper: M.H.R., S.J.T., M.J.M., M.M., K.J.S., B.K.; revising it critically for intellectual content: all authors; final approval of the version to be published: all authors. All authors agree to be accountable for all aspects of the work.

Disclosure statement

The authors report no conflicts of interest.

Data availability statement

The NMR structure and chemical shifts of MAL were deposited in the Protein Data Bank, with ID 8JZM, and the Biological Magnetic Resonance Bank, with accession number 36579, respectively.

Additional information

Funding

The work was supported by the National Health and Medical Research Council (grants 1196590 and 1108859 to T.V., 1160570 to B.K., K.J.S, M.M. and T.V., 2003688 to K.J.S., and 2025931 to B.K.) and the Australian Research Council Future Fellowship (FT200100572) to T.V. and Laureate Fellowship (FL180100109) to B.K.