Evidence for role of transmembrane helix–helix interactions in the assembly of the Class II major histocompatibility complex

Abstract

The Major Histocompatibility Complex Class II (Class II MHC) and invariant chain (Ii) proteins are key initiators of an immune response to invading pathogens. Following biosynthesis, three MHCalpha/beta hetero-dimers associate with an Ii homotrimer to form a nine-chain protein complex. Only as part of this complex are the MHC molecules exported to the cell surface to trigger an immune response. Previous reports implicate the transmembrane (TM) domains of all three proteins in correct assembly, ligand binding and function of Class II MHC. Building on our previous work that revealed the Ii TM domain may contribute significantly to correct assembly of the full-length protein, we have used a variety of genetic, biophysical and computational methods to investigate the role of the TM domains in stabilizing MHCalpha/beta heterodimers. Using the in vivo GALLEX assay, we find that the TM domains of both proteins form strong homo- and hetero-oligomers in natural membranes that are stabilized by GXXXG motifs within the sequence. Förster resonance energy transfer (FRET) measurements, using fluorescently-tagged peptides derived from the TM domains of each protein, were then employed to confirm the presence of TM helix-helix hetero-interactions in detergent micelles, as well as the stoichiometry of these interactions. Our results are summarized in a revised model of Class II MHC-Ii complex formation that illustrates key protein-protein contacts. This work provides the first evidence that the TM domains of the Class II MHC molecules are capable of significant protein-protein interactions that may help to stabilize or even initiate formation of the MHC-Ii complex.

Differential Transmembrane Domain GXXXG Motif Pairing Impacts Major Histocompatibility Complex (MHC) Class II Structure

“…Lipid raft-resident Ia.2 ϩ class II molecules drive Src family kinase-dependent calcium signaling in mature B cells and, consistent with the work of Roche and co-workers (17)(18)(19), are critically important for B cell antigen presentation and formation of B cell-T cell conjugates (5). Previous studies also suggested a role for TM domain GXXXG dimerization motifs in MHC class II ␣-␤ chain pairing (15). In this study, we provide a molecular explanation for the role of these motifs in determining MHC class II conformer formation by showing that differential pairing of the class II ␤ chain GXXXG motif with one of two ␣ chain motifs drives or enables formation of Ia.…”

“…However, there is at least one potential explanation for this finding. Previous studies established that ␤ chain pairing to the ␣ chain M1 motif is significantly less robust than pairing to the M2 motif (15). Therefore, it is possible that the presence of two large side chain residues (e.g.…”

“…However, our previous work demonstrated that the ␣ chain N-terminal M1 motif pairs weakly with the ␤ chain motif (15), so if non-GXXXG motif paired class II molecules can exit the ER, the A␣k M2 mutant may not exhibit enhanced Ia.2 ϩ conformer formation.…”

“…However, we now know that the Ia.2 epitope marks a subset of signaling-competent lipid raft-tropic I-A k class II conformers (5). Moreover, our previous work established a general role for TM domain GXXXG dimerization motifs (originally identified and characterized in glycophorin (14)) in MHC class II ␣-␤ chain pairing (15).…”

Assembly of the TLR2/6 Transmembrane Domains Is Essential for Activation and Is a Target for Prevention of Sepsis

“…The hetero-interactions of the TMDs within a natural membrane environment were studied using the GALLEX assay (24,25 Fig. 1 (without the two Lys at both termini), and those of glycophorin A (GpA) and its G83I mutant, used as positive and negative controls, respectively, are ITLIIFGVMAGVIGTILLI and ITLIIFGVMAIVIGTILLI.…”