In silico Characterization, Structural Analysis, Evolutionary Conservation and Functional Association of Chicken Toll-like Receptor 4 (chTLR4)

Abstract

Background: Toll-like receptor 4 (TLR4) is a pattern-recognition receptor involved in chicken innate immunity. This study examined the sequence characteristics, predicted structural features, evolutionary conservation and functional associations of chicken TLR4 using bioinformatics tools. Methods: The reference mRNA sequence of Gallus gallus TLR4 was examined through sequence annotation, physicochemical and secondary-structure prediction, conserved-domain and topology analyses, multiple sequence alignment, phylogenetic reconstruction, AlphaFold structural information and STRING functional association analysis. Results: The reference mRNA contained a 2,532-bp coding sequence encoding an 843-amino-acid protein. The predicted protein had a molecular weight of 95.64 kDa and a theoretical isoelectric point of 7.13. Secondary-structure prediction identified 47.09% α-helices, 41.99% random coils and 10.91% extended strands. Leucine-rich repeat and Toll/interleukin-1 receptor domains, an N-terminal signal peptide and a membrane-spanning region were identified. The AlphaFold model had a mean pLDDT score of 86.31. Conserved-motif analysis identified three motifs shared among the seven analysed protein sequences. Pairwise TLR4 sequence identity among seven selected poultry species ranged from 81.73% to 95.26%, with phylogenetic analysis identifying distinct sequence clusters. STRING identified associations with immune signalling proteins and enrichment of Toll-like receptor and lipopolysaccharide-related processes. Conclusion: The results provide a computational characterisation of chicken TLR4 and a basis for experimental examination of its predicted molecular interactions and biological functions.