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

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 β-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.MAL/TIRAP | glutathione | Toll-like receptor | NMR spectrometry | inflammation

A survey of TIR domain sequence and structure divergence

“…d H73 is on the opposite side of αC″ from W71 and E75, which bind ligand, and interacts with the other group 24 residues shown in blue; this cluster likely stabilizes this SARM1-specific structural motif residues relative to other TIR domains, resulting in the absence of αB and in an abnormally long, unstructured AB loop. NMR spectroscopy reveals, however, that in solution, the size and backbone conformation of the TIRAP TIR βB region is similar to that of groups 3 and 4 (Hughes et al 2017). Highresolution cryo-EM of the tertiary structure of self-assembled, oligomeric complexes of TIRAP TIR domains in solution ) revealed an open-ended, multifilamentous organization of TIR oligomers, with monomers of the assembly having a rather typical TIR structure, which resembles the NMR structure.…”

“…5c, 6d, and not shown). Many lines of evidence indicate that the region near βB is conformationally flexible (see, for example, Hughes et al 2017); therefore, it is not surprising that these interactions are absent from some conformation variants (e.g., see Fig. 9f).…”

“…In available structures of human TIRAP, C5 forms a disulfide bond with C48 (Lin et al 2012;Snyder et al 2013;Valkov et al 2011;Woo et al 2012) that, however, appears to be a crystallographic artifact, which accounts for TIRAP's significant structural deviation from other TIRs. The NMR structure of human TIRAP and the cryo-EM structures of oligomeric TIRAP complexes (Hughes et al 2017;Ve et al 2017) lack the C5-C48 disulfide bond, and, unlike the BB loop Box 2 motif (-R 9 D 9 -ϕ-ϕ-P 8 G 9 -), C48 is only~50% conserved in TIRAP-related proteins (Fig. 8a).…”

“…8a). C7, which was proposed to be involved in the redox regulation of TIRAP through glutathionylation (Hughes et al 2017), occurs in > 90% of group 10 TIRs and in < 10% of other TIRs ( Figs. 2a and 8a).…”

Metabolic regulation of NLRP3

“…16 In addition to acting as an antioxidant, GSH can posttranslationally modify proteins, and we have recently identified a positive role for glutathionylation in TLR signaling adapter MyD88adapter-like (MAL) activation. 17 Here, we will discuss novel regulators of NLRP3 (Table 1) and potential mechanisms of NLRP3 regulation.…”