Invariant Chain Transmembrane Domain Trimerization:  A Step in MHC Class II Assembly

Abstract

The transmembrane (TM) domain of the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) has long been implicated in both correct folding and function of the MHC class II complex. To function correctly, Ii must form a trimer, and the TM domain is one of the domains thought to stabilize the trimeric state. Specific mutations in the TM domain have been shown previously to disrupt MHC class II functions such as mature complex formation and antigen presentation, possibly due to disruption of Ii TM helix-helix interactions. Although this hypothesis has been reported several times in the literature, thus far no experimental measurements have been made to explore the relationship between TM domain structure and TM mutations that affect Ii function. We have applied biophysical and computational methods to study the folding and assembly of the Ii TM domain in isolation and find that the TM domain strongly self-associates. According to analytical ultracentrifugation analyses, the primary oligomeric state for this TM domain is a strongly associated trimer with a dissociation constant of approximately 120 nM in DPC micelles. We have also examined the effect of functionally important mutations of glutamine and threonine residues in the TM domain on its structure, providing results that now link the disruption of TM helix interactions to previously reported losses of Ii function.

A Transmembrane Domain and GxxxG Motifs within L2 Are Essential for Papillomavirus Infection

“…Notably, the G63V mutant was only mildly impaired, supporting our observations with the A64 mutant (Fig. 3C) and suggesting that the two N-terminal 52 GxxxG 56 and 57 GxxxG 61 motifs are important and that only Gly59 of the 59 GxxxG 63 motif is critical. Three inversion mutants, Inv55, Inv57, and Inv59, were also generated whereby select glycine res- idues were switched in position with neighboring amino acids ( Table 2).…”

“…The inversion mutants disrupt GxxxG motifs by altering the primary sequence of the TM domain without affecting the chemical composition or hydrophobicity of the region. Infectivity experiments again reveal an important role for the two N-terminal 52 GxxxG 56 and 57 GxxxG 61 motifs as well as Gly59 in HPV16 infection (Fig. 3F).…”

“…3A). Upon further inspection of the primary sequence, we discovered that it contains three highly conserved GxxxG motifs, a single 52 (Fig. 3B).…”

“…A55 is predicted to be the most deleterious mutation, disturbing all the TM domain GxxxG motifs. A60 preserves the 52 GxxxG 56 motif but disrupts all downstream motifs, and A64 disrupts only the C-terminal 61 GxxxG 65 and 63 GxxxG 67 motifs. The A55 mutant was completely noninfectious, the A60 mutant had drastically reduced infectivity, and the A64 mutant had no reduction in infectivity (Fig.…”

“…3D). The severely reduced infectivity of A55 and A60 combined with the unaltered infectivity of A64 strongly suggests that only the N-terminal 52 GxxxG 56 , 57 GxxxG 61 , and 59 GxxxG 63 motifs are essential for infection. To further verify a role for these N-terminal GxxxG motifs in HPV16 infection, individual glycine-to-valine mutant viruses were generated and tested.…”