Adaptation to Solvent Environment in Toll-like Receptor 5: A Comparative Evolutionary Analysis of Membrane-bound and Soluble Forms in Epinephelus coioides

Toll-like receptor 5 (TLR5) is a conserved member of the innate immune system and known for its ability to detect bacterial flagellin. Interestingly, some teleost fish, including the orange-spotted grouper ( Epinephelus coioides ), possess both membrane-bound (mTLR5) and soluble (sTLR5) forms of TLR5, which is not observed in mammals. 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 . We analyzed sequence characteristics, amino acid composition, and physicochemical properties of both proteins to understand the basis for their differing solubility and polarity. Two proteins share a substantial part of structural and evolutionary characteristics. Superposition of Alphafold3 predicted structures quantified the structural similarities with a RMSD value of 1.361Å. Contrary to the conserved tertiary structures, our results reveal distinct amino acid preferences between sTLR5 and mTLR5, suggesting that specific mutations may have driven the evolution of the soluble form. Asymmetric evolutionary dynamics and kernel causality were thereby investigated using generalized correlation coefficients (gmc). 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.