Determination of the physiological 2:2 TLR5:flagellin activation stoichiometry revealed by the activity of a fusion receptor

https://doi.org/10.1016/j.bbrc.2013.04.030Get rights and content

Highlights

  • The chimeric protein fusing flagellin to the TLR5 ectodomain is constitutively active.

  • Mutation P736H within the BB-loop of TLR5 TIR domain renders the receptor inactive.

  • The R90D mutation in flagellin inactivated autoactivation of the chimeric protein.

  • The 2:2 stoichiometry of the TLR5:flagellin complex is physiologically relevant.

Abstract

Toll-like receptor 5 (TLR5) recognizes flagellin of most flagellated bacteria, enabling activation of the MyD88-dependent signaling pathway. The recently published crystal structure of a truncated zebrafish TLR5 ectodomain in complex with an inactive flagellin fragment indicated binding of two flagellin molecules to a TLR5 homodimer, however this complex did not dimerize in solution. In the present study, we aimed to determine the physiological stoichiometry of TLR5:flagellin activation by the use of a chimeric protein composed of an active flagellin fragment linked to the N-terminus of human TLR5 (SF-TLR5). This construct was constitutively active. Inactivation by the R90D mutation within flagellin demonstrated that autoactivation of the chimeric protein depended solely on the specific interaction between TLR5 and flagellin. Addition of wild-type hTLR5 substantially lowered autoactivation of SF-TLR5 in a concentration dependent manner, an effect which was reversible by the addition of exogenous Salmonella typhimurium flagellin, indicating the biological activity of a TLR5:flagellin complex with a 2:2 stoichiometry. These results, in addition to the combinations of inactive P736H mutation within the BB-loop of the TIR domain of TLR5 and SF-TLR5, further confirm the mechanism of TLR5 activation.

Introduction

Toll-like receptors (TLRs) are type I transmembrane glycoproteins structurally characterized by the presence of leucine-rich repeat (LRR) motifs in their horseshoe-shaped ectodomain (ECD), a single transmembrane helix and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain [1], [2]. Binding of ligands to the ectodomain of TLR receptors induces formation of a receptor dimer [3], [4], [5], [6] bringing the C-terminal regions of the ectodomains into close proximity, enabling intracellular TIR domains [7] to trigger downstream activation of signaling. Activation of TLRs proceeds through MyD88-dependent or TRIF-dependent signaling and synthesis of proinflammatory cytokines. Despite the conserved similar ‘m’ shaped TLR dimer formation, the active complexes of different TLRs differ in stoichiometry between the receptors and their ligands. TLR3 forms a symmetric 2:1 complex with dsRNA [3], [8]. 2:1 stoichiometry has been determined for the asymmetric TLR1-TLR2-Pam3CSK4 and TLR2-TLR6-Pam2CSK4 complex [5], [6]. TLR4/MD-2-LPS forms a symmetric complex with 2:2 stoichiometry [4], [9].

TLR5 binds the monomeric form of flagellin which is the main structural component of bacterial flagella and functions as a virulence factor upon host infection. Flagellin consists of four linearly connected domains named D0, D1, D2 and D3, proceeding in this order from the core of the filament towards the exposed region [10], [11]. The N- and C-terminal regions that form the D0–D1 domains are conserved among flagellins of different bacterial species, while amino acid sequences of D2–D3 domains are highly variable in sequence and length [12]. The region of flagellin recognized by TLR5 lies within the conserved D1 domain which is also necessary for filament assembly [13].

The determined crystal structure of the fragment zebrafish TLR5 with a D1–D2 domain of FliC from Salmonella enetrica serovar Dublin revealed that the TLR5-FliC heterodimer forms a 2:2 complex [14]. The buried interaction surface of this complex is quite low and the dimer has not been observed in solution. Moreover, since the flagellin fragment used in the crystal structure is biologically inactive there is a concern if the physiological ligand:receptor complex stoichiometry is the same as in the crystal structure.

The aim of the study was to test TLR5:flagellin activation stoichiometry on living cells. We aimed to solve this problem by the use of a flagellin-TLR5 chimeric protein (SF-TLR5) composed of active flagellin lacking the hypervariable region (termed SF) linked to the N-terminus of human TLR5. The selectivity of intramolecular flagellin:TLR5 interactions was demonstrated by the inactivity of a point mutant within flagellin and within the TIR domain of TLR5. Results of activation assays testing combinations of the chimeric protein and TLR5 demonstrate that the stoichiometry of the active TLR5:flagellin complex is 2:2.

Section snippets

Cell cultures

The human embryonic kidney cell lines HEK293 and HEK293T were cultured in complete media (DMEM; 1 g/l glucose, 2 mM l-glutamin, 10% heat-inactivated FBS (Gibco)) in 5% CO2 at 37 °C.

Plasmids and DNA construct preparation

Plasmid pUNO-hTLR5 coding for human TLR5 (InvivoGen) and control vector pcDNA3 (Invitrogen) were used. Salmonella typhimurium flagellin (bacteria provided by prof. Ignacio Moriyόn, University of Navarra, Spain) was cloned into the XhoI and BamHI sites of the pET-19b expression vector (Novagen) containing an N-terminal

Chimeric TLR5 with linked flagellin exhibits constitutive cell activation

The recently determined crystal structure TLR5-N14VLR/FliC-ΔD0, in which the C-terminal region of the TLR5 ectodomain and the flagellin D0 domain are missing, implies that one flagellin molecule brings two TLR5 receptors together through binding to the primary binding interface of one TLR5 and through additional weaker interactions with the opposite TLR5 receptor. To define if only one flagellin molecule might be sufficient for TLR5 dimer activation (2:1 complex) or two are necessary as

Acknowledgments

This research was supported by the Slovenian Research Agency and EN-FIST Centre of Excellence financed in part by the European structural funds. We thank Irena Škraba and Robert Bremšak for isolation of recombinant flagellins and Vida Forstnerič for help in correcting the manuscript.

References (20)

There are more references available in the full text version of this article.

Cited by (18)

View all citing articles on Scopus
View full text