Duplicated TLR5 of zebrafish functions as a heterodimeric receptor

Abstract

Toll-like receptor 5 (TLR5) of mammals, birds, and reptiles detects bacterial flagellin and signals as a homodimeric complex. Structural studies using truncated TLR5b of zebrafish confirm the homodimeric TLR5-flagellin interaction. Here we provide evidence that zebrafish () TLR5 unexpectedly signals as a heterodimer composed of the duplicated gene products drTLR5b and drTLR5a. Flagellin-induced signaling by the zebrafish TLR5 heterodimer increased in the presence of the TLR trafficking chaperone UNC93B1. Targeted exchange of drTLR5b and drTLR5a regions revealed that TLR5 activation needs a heterodimeric configuration of the receptor ectodomain and cytoplasmic domain, consistent with ligand-induced changes in receptor conformation. Structure-guided substitution of the presumed principal flagellin-binding site in human TLR5 with corresponding zebrafish TLR5 residues abrogated human TLR5 activation, indicating a species-specific TLR5-flagellin interaction. Our findings indicate that the duplicated TLR5 of zebrafish underwent subfunctionalization through concerted coevolution to form a unique heterodimeric flagellin receptor that operates fundamentally differently from TLR5 of other species.

Structure of fish Toll-like receptors (TLR) and NOD-like receptors (NLR)

“…A heterodimeric model of TLR1-TLR2 complex is built using comparative modeling in common carp that shares a good fitting with the template human TLR1-TLR2 complex crystal structure [ 116 ]. Horseshoe structure is also observed for other fish TLRs that include Tibet fish TLR4 [ 117 ], zebrafish TLR5a and TLR5b [ 82 ] and miiuy croaker TLR28 [ 118 ]. The modeled zebrafish TLR5a-TLR5b heterodimer structure was reported to have a very similar structure when compared with the homodimer TLR5-flagellin complex structure resolved using X-ray crystallography [ 82 , 93 ].…”

“…TLR2 expression in fish has shown to be up-regulated in response to PGN and LTA [ 80 ]. TLR4 and TLR5 respectively have been identified to recognize LPS and flagellin in higher eukaryotes, but some fishes lacks TLR4 and do not recognize LPS and have multi-copies of TLR5 indicating their PAMP selectivity [ [81] , [82] , [83] ]. Viral and bacterial RNA is shown to be a molecular target for TLR3, TLR9, TLR13 and TLR22 in fish [ 31 , [84] , [85] , [86] , [87] , [88] ].…”

“…They compared the structural differences in the TLR5-flagellin hetero-complex composed of two variable copies of TLR5 (TLR5a and TLR5b) discovered in zebrafish with the experimental homodimer TLR5 structure. This study identified that flagellin binding induces conformational change in the loop connecting LRR9–10, while the rest of the TLR5-ECD structure is highly similar to that of unbound flagellin structure [ 82 ]. Studies on rohu TLR2 interaction with different variants of PGN, LTA and zymosan also reported a conserved predicted binding site by AutoDock, FlexX and GOLD [ 46 ].…”

Characterization of the TLR Family in Branchiostoma lanceolatum and Discovery of a Novel TLR22-Like Involved in dsRNA Recognition in Amphioxus

“…Other authors also have obtained similar fold-changes in the NF-κB reporter assay ( 65 , 66 ). On the other hand, Voogdt et al showed an extremely high activation of the NF-κB signaling pathway after flagellin stimulation but the main difference with our approach is that they used cells stably expressing NF-κB reporter ( 67 ). Poly I:C is a specific ligand of vertebrate TLR3 including many fish species ( 20 , 23 , 65 , 68 ), of M. croaker TLR13 ( 19 ) and of different fish TLR22 ( 20 – 22 , 24 , 69 ).…”

Evolutionary Regression and Species-Specific Codon Usage of TLR15

“…The purified ancaTLR15 gene was next amplified to add a KpnI restriction and kozak site at the 5′ end of the gene and an overlap on a 3 × Hemagglutinin-epitope (HA) sequence on the 3′ end of the gene. A 3 × HA sequence was amplified from a pTracer-CMV2ΔGFP/3 × HA vector ( 42 ) to add an overlap to ancaTLR15 at the start of the HA sequence and a PacI restriction site at the end of the sequence. The ancaTLR15 gene and 3 × HA sequence were subsequently fused by standard overlap PCR, digested with KpnI and PacI and ligated in pTracer-CMV2ΔGFP to yield ancaTLR15 carrying a C-terminal 3 × HA-tag.…”