Solution structure of the TLR adaptor MAL/TIRAP reveals an intact BB loop and supports MAL Cys91 glutathionylation for signaling

Hughes, Mark M., Lavrencic, Peter, Coll, Rebecca C., Ve, Thomas, Ryan, Dylan G., Williams, Niamh C., Menon, Deepthi, Mansell, Ashley, Board, Philip G., Mobli, Mehdi, Kobe, Bostjan and O'Neill, Luke A. J. (2017) Solution structure of the TLR adaptor MAL/TIRAP reveals an intact BB loop and supports MAL Cys91 glutathionylation for signaling. Proceedings of the National Academy of Sciences, 114 32: E6480-E6489. doi:10.1073/pnas.1701868114


Author Hughes, Mark M.
Lavrencic, Peter
Coll, Rebecca C.
Ve, Thomas
Ryan, Dylan G.
Williams, Niamh C.
Menon, Deepthi
Mansell, Ashley
Board, Philip G.
Mobli, Mehdi
Kobe, Bostjan
O'Neill, Luke A. J.
Title Solution structure of the TLR adaptor MAL/TIRAP reveals an intact BB loop and supports MAL Cys91 glutathionylation for signaling
Journal name Proceedings of the National Academy of Sciences   Check publisher's open access policy
ISSN 1091-6490
0027-8424
Publication date 2017-08-08
Year available 2017
Sub-type Article (original research)
DOI 10.1073/pnas.1701868114
Open Access Status Not yet assessed
Volume 114
Issue 32
Start page E6480
End page E6489
Total pages 10
Place of publication Washington, DC, United States
Publisher National Academy of Sciences
Language eng
Abstract MyD88 adaptor-like (MAL) is a critical protein in innate immunity, involved in signaling by several Toll-like receptors (TLRs), key pattern recognition receptors (PRRs). Crystal structures of MAL revealed a nontypical Toll/interleukin-1 receptor (TIR)-domain fold stabilized by two disulfide bridges. We therefore undertook a structural and functional analysis of the role of reactive cysteine residues in the protein. Under reducing conditions, the cysteines do not form disulfides, but under oxidizing conditions they are highly amenable to modification. The solution structure of the reduced form of the MAL TIR domain, determined by NMR spectroscopy, reveals a remarkable structural rearrangement compared with the disulfide-bonded structure, which includes the relocation of a beta-strand and repositioning of the functionally important "BB-loop" region to a location more typical for TIR domains. Redox measurements by NMR further reveal that C91 has the highest redox potential of all cysteines in MAL. Indeed, mass spectrometry revealed that C91 undergoes glutathionylation in macrophages activated with the TLR4 ligand lipopolysaccharide (LPS). The C91A mutation limits MAL glutathionylation and acts as a dominant negative, blocking the interaction of MAL with its downstream target MyD88. The H92P mutation mimics the dominant-negative effects of the C91A mutation, presumably by preventing C91 glutathionylation. The MAL C91A and H92P mutants also display diminished degradation and interaction with interleukin-1 receptor-associated kinase 4 (IRAK4). We conclude that in the cell, MAL is not disulfide-bonded and requires glutathionylation of C91 for signaling.
Keyword Glutathione
Inflammation
MAL/TIRAP
NMR spectrometry
Toll-like receptor
Q-Index Code C1
Q-Index Status Provisional Code
Grant ID 12/IA/1531
1003326
FT10100925
Institutional Status UQ

 
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