Differential pairing of transmembrane domain GxxxG dimerization motifs defines two HLA-DR MHC class II conformers

Abstract

MHC class II molecules function to present exogenous antigen-derived peptides to CD4 T cells to both drive T cell activation and to provide signals back into the class II antigen-presenting cell. Previous work established the presence of multiple GxxxG dimerization motifs within the transmembrane domains of MHC class II α and β chains across a wide range of species and revealed a role for differential GxxxG motif pairing in the formation of two discrete mouse class II conformers with distinct functional properties (i.e., M1-and M2-paired I-A^k class II). Biochemical and mutagenesis studies detailed herein extend this model to human class II by identifying an anti-HLA-DR mAb (Tü36) that selectively binds M1-paired HLA-DR molecules. Analysis of the HLA-DR allele reactivity of the Tü36 mAb helped define other HLA-DR residues involved in mAb binding. In silico modeling of both TM domain interactions and whole protein structure is consistent with the outcome of biochemical/mutagenesis studies and provides insight into the possible structural differences between the two HLA-DR conformers. Cholesterol depletion studies indicate a role for cholesterol-rich membrane domains in the formation/maintenance of Tü36 mAb reactive DR molecules. Finally, phylogenetic analysis of the amino acid sequences of Tü36-reactive HLA-DR β chains reveals a unique pattern of both Tü36 mAb reactivity and key amino acid polymorphisms. In total, these studies bring the paradigm M1/M2-paired MHC class II molecules to the human HLA-DR molecule and suggest that the functional differences between these conformers defined in mouse class II extend to the human immune system.

Keywords: HLA; MHC class II; antigen presentation; major histocompatibility complex (MHC); monoclonal antibody; protein conformation; structural model.

Figure 1

L243 mAb recognizes all HLA-DR class II molecules. The indicated cells were lysed in RIPA buffer. Cleared WCL was subjected to three rounds of IP with L243 and Protein A–agarose beads (“L243”). As a control, WCL was IP-ed with just Protein A–agarose (“none”). IP and supernatant (SN) from each round were probed for DRA by Western blot. The gray bar to the right of each blot indicates the position of the 34 kDa molecular weight standard. Shown are representative results from one of three or more independent experiments for each cell type. IP, immunoprecipitations; WCL, whole cell lysate.

Figure 2

Mutation of DRA transmembrane domain GxxxG dimerization motif selectively inhibits Tü36 mAb binding.A, amino acid sequence of tested DRA TM domains. GxxxG dimerization motifs are highlighted. Mutated amino acid residues are in red. B–D, 293T cells were transfected with the indicated DRA chains along with DRB1∗04.03. Cells were stained with the indicated anti-DR mAb and analyzed by flow cytometry. The gray histogram in each panel illustrates the staining of untransfected 293T cells. E, the level of mAb binding across multiple experiments was compared. In each experiment, binding of the indicated mAb to cells expressing WT DR was set to 1.0 and mAb binding to other cells normalized to this value. Bars indicate ±1 SD from across three independent experiments. TM, transmembrane.

Figure 3

Tü36 recognizes a subset of HLA-DR4 molecules. WCL from the indicated cells was prepared as in Figure 1 and subject to two rounds of sequential IP with the indicated mAbs. SN from the first round of IP was used as input for round two. IPs were analyzed for DRA by Western blot as in Figure 1. Shown are representative results from one of two (transfected 293T) to three (1122 B cells) independent experiments. Gray bar to the right of each blot indicates the position of the 34 kDa molecular weight standard. See the text for an explanation of red and blue arrows. Densitometry analysis of blots from 1122 B cell samples reveals that Tü36 recognizes 6.7% of class II recognized by the pan-reactive L243 mAb (4%, 5%, and 11% across three independent experiments). IP, immunoprecipitations; WCL, whole cell lysate.

Figure 4

Modeling of DR transmembrane domain interactions. For each sequence (apart from panel C), the highest ranked (light gray) and second-highest ranked (dark gray) models obtained from PREDDIMER are overlaid. The two GxxxG motifs in the DRA TM domain are shown as red spheres (M1) and blue spheres (M2), and the GxxxG motif in DRB1 is shown in orange. Additional residues packed at the helix-helix interface are shown as spheres, colored by the element, and labeled. Residues that form part of a G>V mutation are shown in ball and stick representation. A and B, wild-type sequences yield heterodimers stabilized by packing of either the M1 motif (panel A) or the M2 motif (panel B) against the single DRB1 motif. C, mutation of the M1 motif to valine (G198V, G202V) prevents M1 pairing of the TM domains and more broadly prevents packing of the Gly-rich faces of the two TM domains in all structures obtained. The top-ranked model is shown here (additional models detailed in Fig. S1) in which the DRB1 GxxxG motif packs against bulky and/or polar residues in DRA while the M1/M2 motifs are exposed to lipids. D, M2-pairing of the helices is much more tolerant to mutation, yielding models similar to wild-type and further stabilized by close packing of downstream G216 in DRA with G216 in DRB1. E and F, mutation of the M2 motif to valine (G205V, G209V) yields heterodimers very similar to wild-type, with the top-ranked models illustrating both M1-paired (panel E) and M2-paired (panel F) heterodimers.

Figure 5

Simultaneous binding of L243 and Tü36 anti-DR monoclonal antibodies.A and B, human B cells expressing either DR4 or DR1 were stained concurrently with L243-PE and/or Tü36-Alexa₄₈₈ and analyzed by flow cytometry. C and D, the level of mAb binding across multiple experiments is compared. The binding of each mAb in isolation was normalized to a value of 1.0 and the relative binding of that mAb in the presence of the partner was determined. Bars indicate ±1 SD from across three independent experiments.

Figure 6

DR allele reactivity of the Tü36 Anti-DR monoclonal antibody.A, the allele reactivity of the L243 and Tü36 mAbs was determined by Single Antigen Bead analysis (see Experimental procedures). For each mAb the MFI of staining of each bead was normalized to the brightest signal for that reagent. The level of staining was ordered from highest to lowest Tü36 binding (left to right). The vertical red line indicates a “break” in Tü36 mAb reactivity. B, the amino acid sequence (residues 134–151) of each SAB-interrogated DR allele is ordered based on Tü36 reactivity (highest [top] to lowest [bottom]—see Fig. S2 for ordered alignment of complete amino acid sequences). The horizontal red line indicated the break in Tü36 reactivity (vertical red line in panel A). The red and blue arrows indicate amino acid residues that track with Tü36 mAb reactivity (see text).

Figure 7

Double-label analysis of the impact of HLA-DR mutations on Tü36 mAb binding. 293T cells were transfected with the indicated DRA and DRB chains and then stained with both L243-Alexa₄₈₈ and Tü36-PE. Stained cells were analyzed by flow cytometry. Black dots illustrate the staining of non-DR-transfected 293T cells. Shown are representative results from one of three independent experiments. A, TM domain GxxxG motif mutants. B, DRB 140 and DRB 149 mutants. C, combine mutants.

Figure 8

Overlay of alphaFold2 model of wld-type DR4 and DR4 crystal structure. The structure of the AlphaFold2 model of full-length DR4 (see text, gray trace) and crystal structure of the DR4 extracellular domain (pdb file 7NZE, red trace) were aligned using the Matchmaker tool in Chimera X1.4. The alignment yields an RMSD between 179 pruned atom pairs of 0.843 Å. Key residues/regions of interest in the extracellular domain are highlighted as spheres. The limits of a long helical domain, containing the TM domain, are also labeled and the TM GxxxG motifs are colored as in Figure 4.

Figure 9

Impact of MβCD treatment on anti-DR mAb binding. DR4-expressing 1122 human B cells were treated with the indicated concentration of MβCD and then co-stained with L243-FITC and Tü36-PE. Stained cells were analyzed by flow cytometry. A, histograms of mAb staining at each MβCD concentration as indicated. Filled-in gray histogram is from unstained cells. B, the level of mAb binding to 1122 B cells across three experiments is compared. The binding of each mAb in the absence of MβCD treatment was set to a value of 1.0 and other values normalized to that level of mAb binding. Bars indicate ±1 SD from across three independent experiments. C, impact of MβCD treatment on Tü36 and L243 binding to Raji B cells. Results compiled from across three independent experiments as for panel B.