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Identification of protein-protein interaction sites in the TLR4/Mal/MyD88 complex

Celia Bovijn (UGent)
(2012)
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(UGent) and (UGent)
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Abstract
Innate immune functions are triggered by recognition of pathogen associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Different PRRs families play crucial roles in this pathogen detection, including the Toll-like receptors (TLRs). TLRs are transmembrane receptors that recognize PAMPs exposed by bacteria, viruses and parasites. After recognition of the invading pathogen, TLRs trigger an immune response by inducing transcription of pro-inflammatory genes and type I interferons. TLRs guide the presentation of ingested and processed pathogenic molecules on major histocompatibility (MHC) receptors, which subsequently activates the adaptive immune system. TLRs also recognize danger associated molecular patterns (DAMPs) released by cells upon tissue damage, microbial invasion or in response to stress. This can lead to inflammatory and healing responses including autophagy to preserve cellular homeostasis. During the first stage of infection, negative regulation of TLR signaling by decoy receptors are important for dampening acute responses to infection. Once the TLR is activated, signaling can be further negatively controlled by intracellular modulators. Sometimes, TLRs become hyper-activated or particular feedback mechanisms fail leading to chronic inflammation. A profound insight in the function, the mechanism and the structure of TLRs can guide the development of novel medicines to fight these disorders. The TLRs are composed of an extracellular leucine rich repeat (LRR) domain, a transmembrane helix and an intracellular toll/IL-1 receptor (TIR) domain. So far, the crystal structures of the human TLR1, TLR2 and the TLR10 TIR domain have been determined (Nyman et al., 2008; Tao et al., 2002; Xu et al., 2000). Initiation of the TLR signaling cascade upon ligand induced dimerization requires the interaction of TLR TIR domains with adapter TIR domains. Four adapter TIR proteins mediate TLR signal propagation: myeloid differentiation primary response gene 88 (MyD88), MyD88 adapter-like protein (Mal), TIR domain-containing adapter protein inducing IFN-β (TRIF) and TRIF related adapter molecule (TRAM). The structures of MyD88 and Mal have been determined as well (Lin et al., 2012; Ohnishi et al., 2009; Valkov et al., 2011; Woo et al., 2012). Alanine scanning mutagenesis showed that mutations in two conserved putative interaction sites in the TLR4 TIR domain affect TLR4 reporter assays (Ronni et al., 2003). In this work, we studied the interactions of TLR4 with the adapter proteins Mal, TRAM and MyD88. For this purpose, we combined mutagenesis and the mammalian protein-protein interaction assay (MAPPIT) to identify mutations in the TIR domain of TLR4 and Mal that affect their TIR-TIR interactions. We mapped these results on homology models of TLR4 and the structure of Mal to deduce possible interaction sites. To identify possible interaction sites on TLR4, we first generated and selected mutants based on sequence conservation and the alanine scan performed by Ronni et al. Next to the two previously reported interaction sites, our results suggested the existence of a third interaction site. Moreover, our MAPPIT data allowed us to assign these binding sites to specific interactions. More specifically, we identified the dimerization site and the adaptor binding site on the TLR4 TIR domain. However, the function of the third interaction site remains unclear. We further investigated possible interaction sites on the TLR4-binding partner Mal. Here, mutants were obtained via random mutagenesis. The data were mapped on the recently published crystal structures of Mal. In these experiments, we functionally confirmed the symmetrical dimer interface as observed in the crystal structure. In addition, we found three new interaction sites. Two of the interaction sites are juxtaposed on the Mal dimer and form one big surface for binding of TLR4 and MyD88. The other interaction site seems to be mainly involved in TLR4 interactions. Our data suggest that Mal is dimerized in the TLR4 TIR complex. Our MAPPIT data provide strong evidence for cooperative binding of the TIR domains. In conclusion, we identified three interaction sites on the TLR4 TIR domain and four interaction sites on the Mal TIR domain and could assign these interaction sites to specific interactions. Additionally, our results suggest that Mal dimerization is probably needed for the formation of the TIR complex and that it is only through simultaneous binding of different TIR domains that the TIR complex can be assembled.

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MLA
Bovijn, Celia. Identification of Protein-Protein Interaction Sites in the TLR4/Mal/MyD88 Complex. Ghent University. Faculty of Medicine and Health Sciences, 2012.
APA
Bovijn, C. (2012). Identification of protein-protein interaction sites in the TLR4/Mal/MyD88 complex. Ghent University. Faculty of Medicine and Health Sciences, Ghent, Belgium.
Chicago author-date
Bovijn, Celia. 2012. “Identification of Protein-Protein Interaction Sites in the TLR4/Mal/MyD88 Complex.” Ghent, Belgium: Ghent University. Faculty of Medicine and Health Sciences.
Chicago author-date (all authors)
Bovijn, Celia. 2012. “Identification of Protein-Protein Interaction Sites in the TLR4/Mal/MyD88 Complex.” Ghent, Belgium: Ghent University. Faculty of Medicine and Health Sciences.
Vancouver
1.
Bovijn C. Identification of protein-protein interaction sites in the TLR4/Mal/MyD88 complex. [Ghent, Belgium]: Ghent University. Faculty of Medicine and Health Sciences; 2012.
IEEE
[1]
C. Bovijn, “Identification of protein-protein interaction sites in the TLR4/Mal/MyD88 complex,” Ghent University. Faculty of Medicine and Health Sciences, Ghent, Belgium, 2012.
@phdthesis{3077608,
  abstract     = {{Innate immune functions are triggered by recognition of pathogen associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Different PRRs families play crucial roles in this pathogen detection, including the Toll-like receptors (TLRs). TLRs are transmembrane receptors that recognize PAMPs exposed by bacteria, viruses and parasites. After recognition of the invading pathogen, TLRs trigger an immune response by inducing transcription of pro-inflammatory genes and type I interferons. TLRs guide the presentation of ingested and processed pathogenic molecules on major histocompatibility (MHC) receptors, which subsequently activates the adaptive immune system. TLRs also recognize danger associated molecular patterns (DAMPs) released by cells upon tissue damage, microbial invasion or in response to stress. This can lead to inflammatory and healing responses including autophagy to preserve cellular homeostasis. During the first stage of infection, negative regulation of TLR signaling by decoy receptors are important for dampening acute responses to infection. Once the TLR is activated, signaling can be further negatively controlled by intracellular modulators. Sometimes, TLRs become hyper-activated or particular feedback mechanisms fail leading to chronic inflammation. A profound insight in the function, the mechanism and the structure of TLRs can guide the development of novel medicines to fight these disorders. The TLRs are composed of an extracellular leucine rich repeat (LRR) domain, a transmembrane helix and an intracellular toll/IL-1 receptor (TIR) domain. So far, the crystal structures of the human TLR1, TLR2 and the TLR10 TIR domain have been determined (Nyman et al., 2008; Tao et al., 2002; Xu et al., 2000). Initiation of the TLR signaling cascade upon ligand induced dimerization requires the interaction of TLR TIR domains with adapter TIR domains. Four adapter TIR proteins mediate TLR signal propagation: myeloid differentiation primary response gene 88 (MyD88), MyD88 adapter-like protein (Mal), TIR domain-containing adapter protein inducing IFN-β (TRIF) and TRIF related adapter molecule (TRAM). The structures of MyD88 and Mal have been determined as well (Lin et al., 2012; Ohnishi et al., 2009; Valkov et al., 2011; Woo et al., 2012). Alanine scanning mutagenesis showed that mutations in two conserved putative interaction sites in the TLR4 TIR domain affect TLR4 reporter assays (Ronni et al., 2003). In this work, we studied the interactions of TLR4 with the adapter proteins Mal, TRAM and MyD88. For this purpose, we combined mutagenesis and the mammalian protein-protein interaction assay (MAPPIT) to identify mutations in the TIR domain of TLR4 and Mal that affect their TIR-TIR interactions. We mapped these results on homology models of TLR4 and the structure of Mal to deduce possible interaction sites. To identify possible interaction sites on TLR4, we first generated and selected mutants based on sequence conservation and the alanine scan performed by Ronni et al. Next to the two previously reported interaction sites, our results suggested the existence of a third interaction site. Moreover, our MAPPIT data allowed us to assign these binding sites to specific interactions. More specifically, we identified the dimerization site and the adaptor binding site on the TLR4 TIR domain. However, the function of the third interaction site remains unclear. We further investigated possible interaction sites on the TLR4-binding partner Mal. Here, mutants were obtained via random mutagenesis. The data were mapped on the recently published crystal structures of Mal. In these experiments, we functionally confirmed the symmetrical dimer interface as observed in the crystal structure. In addition, we found three new interaction sites. Two of the interaction sites are juxtaposed on the Mal dimer and form one big surface for binding of TLR4 and MyD88. The other interaction site seems to be mainly involved in TLR4 interactions. Our data suggest that Mal is dimerized in the TLR4 TIR complex. Our MAPPIT data provide strong evidence for cooperative binding of the TIR domains. In conclusion, we identified three interaction sites on the TLR4 TIR domain and four interaction sites on the Mal TIR domain and could assign these interaction sites to specific interactions. Additionally, our results suggest that Mal dimerization is probably needed for the formation of the TIR complex and that it is only through simultaneous binding of different TIR domains that the TIR complex can be assembled.}},
  author       = {{Bovijn, Celia}},
  language     = {{eng}},
  pages        = {{183}},
  publisher    = {{Ghent University. Faculty of Medicine and Health Sciences}},
  school       = {{Ghent University}},
  title        = {{Identification of protein-protein interaction sites in the TLR4/Mal/MyD88 complex}},
  year         = {{2012}},
}