Phylogenetic analysis of amino acid sequences indicates a central role of transmembrane domain structural elements in MHC class II function.

Human leukocyte antigen class II genes are highly polymorphic. This raises the question of how polymorphisms are distributed across the entirety of the class II molecule and how this distribution affects function. We performed a comprehensive analysis of amino acid variability across the entire class II molecule, using class II amino acid sequences ranging from humans to ancient species such as cartilaginous fish. This analysis is unique in its scale and reveals that while the membrane distal antigen recognition domain exhibits a high frequency of amino acid variability, other domains exhibit much lower degrees of variability. Notably, the transmembrane domain (TMD) exhibits the lowest amino acid variability, consistent with its role in driving core class II functions. Further analysis of the TMD revealed that a cysteine residue and 3 GxxxG (GG4) motifs are 100% conserved throughout evolution. Mutagenesis and monoclonal antibody binding studies revealed that both the GG4 motifs and cysteine residue (the latter a site of class II palmitoylation) are involved in conformer formation. Molecular dynamics simulation of the palmitoylated class II TMD provides a framework for understanding the contribution of GG4 motif pairing and cysteine palmitoylation to class II structure. These results reveal a domain-specific organization of polymorphism within class II and a key role for the TMD and its functional residues/motifs in major histocompatibility complex class II immunobiology.