Adaptation to solvent environment in toll-like receptor 5: kernel-based evolutionary analysis of membrane-bound and...

Toll-like receptor 5 (TLR5) is a conserved member of the innate immune system and known for its ability to detect bacterial flagellin. Interestingly, the orange-spotted grouper (Epinephelus coioides), possess both membrane-bound (mTLR5) and soluble (sTLR5) forms of TLR5, which is not observed in mammals. We analyzed sequence characteristics, amino acid composition, and physicochemical properties of both proteins to understand the basis for their differing solubility and polarity. Superposition of Alphafold3 predicted structures quantified the structural similarities with a RMSD value of 1.361 Å. Contrary to the conserved tertiary structures, our results reveal distinct amino acid preferences between sTLR5 and mTLR5. Asymmetric evolutionary dynamics and kernel causality were thereby investigated using generalized correlation coefficients. The dependence analysis revealed that TLR5 evolution shows multidirectional dynamics between soluble and membrane-bound forms, as 2 amino acids (M and V) shift membrane-to-solvent, 2 shift solvent-to-membrane (H and Y). The T < =>V and D > K substitutions required two base changes and therefore introduced a mutational bias. Regression analysis of the homologous sequences indicated T < =>V changes may have supported by alanine intermediates. These findings provide insights into the functional diversification of TLR5 and broader implications on the diversification of immune system proteins. Insight Box This study investigates the evolutionary patterns towards adapting a solvent environment in TLR5 by comparing the membrane-bound (mTLR5) and soluble (sTLR5) forms from the Epinephelus coioides. Contrary to the conserved tertiary structures, our results reveal distinct amino acid preferences between sTLR5 and mTLR5. We show that adaptation is bidirectional and strategically localized, preserving ligand recognition while enabling immune modulation in extracellular space. Understanding these adaptations offer new perspectives on immune responses, particularly in conditions involving dysregulated TLR5 signalling, such as chronic inflammation and autoimmune diseases.