Helix-helix interactions between membrane-spanning transmembrane (TM)\ud domains have been shown to drive the assembly of α-helical membrane proteins\ud within biological membranes. However, the rules that determine these interactions\ud are not yet fully understood, despite such interactions being found in an increasing\ud number of proteins. Recent work has implicated TM domain interactions in the\ud formation of the protein complex Ii-MHC, formed from the association of Major\ud Histocompatibility Complex Class II (MHC) and the MHC-associated-Invariant\ud Chain (Ii) proteins. Following biosynthesis, three MHC α/βheterodimers bind to\ud the Ii homotrimer to form a nonameric Ii-MHC complex within the endoplasmic\ud reticulum. This is a critical step in the export of MHC molecules to the antigen\ud presentation system and hence the activation of an immune response to a\ud pathogen. In this study we have explored the TM domain interactions within the\ud Ii-MHC complex. Results from in vivo and in vitro experiments revealed the TM\ud domains of the α- and β-chains of MHC have a propensity to self-associate into\ud homo-dimers and to associate with one another to form hetero-dimers. Highly\ud conserved GxxxG motifs (known to drive dimerization) were implicated in these\ud interactions. The TM domain of Ii was confirmed to self-associate to form trimers\ud by in vivo and in vitro methods, but surprisingly also displayed additional\ud oligomeric states suggesting the interaction is not as specific as was previously\ud thought. Furthermore, we show that in vivo, the TM domain of Ii can associate\ud with those of the α- and β-chains of MHC, whilst in vitro methods suggested Ii\ud preferentially binds to α-chains. Collectively, these findings strongly suggest that\ud the TM domains of Ii and MHC have a role to play in the assembly of the Ii-MHC\ud complex, and hence the very important process of antigen presentation.\ud Additionally, in this study we have undertaken development of NMR\ud spectroscopy methods that have the potential to increase our understanding of not\ud only the Ii-MHC complex, but protein-protein interactions in general
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