Figures and data
Random mutagenesis screen
(A) Soluble HLA-G (sG) is internalized with HA-tagged KIR2DL4 into endosomes, as shown by incubation for 2 h at 37°C with anti-HA coupled to Alexa-594 and soluble HLA-G coupled to Alexa-647. The loading assay was performed with 293T cells transfected with HA-tagged KIR2DL4. (B) Confocal microscopy showing the location of internalized KIR2DL4 (red) and soluble HLA-G (green). An overlay is shown in the right panel (Merge). As control, HLA-G was replaced by HLA-E, also coupled to Alexa-647 (middle panels). 293T cells transfected with HA-tagged receptor 2B4 were also incubated with HLA-G coupled to Alexa-647 (bottom panels). 2B4 (CD244) resides at the plasma membrane. (C) The loading assay was used to screen KIR2DL4 mutants and test for HLA-G binding. The main parameter measured was presence or absence of HLA-G and the location of detectable HLA-G. Confocal images of the five categories of mutants are shown. A DIC image is shown in the upper right panel. The parameters indicated under the images include HLA-G presence and location, location of KIR2DL4, the number of mutants in each of the five categories, and an example of a mutation for each category.
A stressed disulfide bond typical of an allosteric bond in the crystal structure of KIR2DL4
(A) The KIR2DL4 D0 domain has a third Cys residue not found in other D0 domains. Sequence alignment of D0 domains with Cys residues indicated by an arrow and shown in red (B) Disulfide bonds visible in crystal structures of KIRs exhibit a higher solvent accessibility in the Cys10-Cys28 bond unique to KIR2DL4. Solvent accessibility of disulfide bonds was determined by DSSP (define secondary structure of proteins) algorithm. (C) Position of the three Cys residues in the crystal structure of KIR2DL4 (3WYR). Cys10, Cys28 and Cys74 are on the A strand, B strand and F strands, respectively, are as indicated. (D) The disulfide bond configuration in the KIR2DL4 D0 domain is an –RHstaple.
Detection and quantification of disulfide bonds in KIR2DL4 isolated from cells
(A) Disulfide bonds in KIR2DL4. The Cys10-Cys28 disulfide bond and the unpaired Cys74 in the D0 domain and the stable Cys123-Cys172 disulfide bond in the D2 domain are shown in yellow in the ribbon structure of KIR2DL4. PDB identifier is 3WYR. (B) Quantification of disulfide-linked peptides in recombinant KIR2DL4 by mass spectrometry. Peptide abundance was expressed relative to the peptide linked by Cys123 and Cys172 in D2 of the receptor. (C) Protocol to determine the redox state of KIR2DL4 disulfide bonds, as measured by differential cysteine alkylation and mass spectrometry. Reduced disulfide bond cysteines in KIR2DL4 were alkylated with 12C-IPA and the oxidized cysteine thiols with 13C-IPA following reduction with DTT. The ratio of 12C-IPA to 13C-IPA alkylation represents the proportion of the disulfide bonds in the population that are in the reduced state. (D) Quantification of cysteine redox states in recombinant KIR2DL4 by differential cysteine alkylation and mass spectrometry. (E) Human cell-derived KIR2DL4 was found in both Cys10-Cys28 and Cys28-Cys74 redox states. The redox state of disulfide Cys123-Cys172 is shown for comparison. (F) Incubation of purified KIR2DL4 with 3 different protein disulfide isomerases. Reduction of the Cys10-Cys28 bond occurred with PDI at a molar ratio of 2 and 10. The redox state of cysteines were quantified by differential cysteine labelling and mass spectrometry. The error bars (SD) were derived from measurements of 2 to 4 peptides. ***<0.005 as assessed by unpaired, two-tailed Students t-test.
Location of KIR2DL4 and HLA-G after inhibition of PDI
(A to D) Confocal microscopy images of 293T cells transfected with HA-KIR2DL4 (anti-HA coupled to Alexa 594, red) and loaded with labeled HLA-G (green) for 2 h in the presence of DTNB (A), pCMPS (B), Rutin (C) and anti-PDI antibody (D), at the indicated concentrations. The time 0 image in (A) and (B) is the same, and so is the time 0 image shown in (C) and (D). The distribution of KIR2DL4 in vesicles or on the cell surface was determined by scoring at least 200 cells per condition.
Cysteine mutants in KIR2DL4 tested for their impact on KIR2DL4 distribution
(A) Distribution (vesicles or plasma membrane) of WT and of the indicated cysteine mutants of KIR2DL4 in transfected 293T cells. (B) Distribution of HLA-G in 293T cells transfected with KIR2DL4 wild-type and with mutations at Cys10. HLA-G was scored for presence or absence in KIR2DL4+ vesicles. (C) Close-up view of the trio of cysteines in the D0 domain of KIR2DL4, as reported in the crystal structure (3WYR, left) and as predicted by AlphaFold (right). (D) Reactivity of the antibodies anti-HA tag, mAb#33 and MAB2238 with wild-type and the 3 cysteine mutants of KIR2DL4.
Cysteine mutants in the D0 domain of KIR2DL4 and their impact on the distribution of KIR2DL4 and its interaction with HLA-G.
V=vesicular; PM=Plasma membrane.
Predicted outcome of an allosteric disulfide switch on the conformation of KIR2DL4 and its interaction with HLA-G
(A) Overlay of the crystal structure (3WYR; purple) and the structure predicted by AlphaFold DB (blue). The allosteric disulfide Cys10-Cys28 is in yellow and the Cys28-Cys74 structural disulfide is in green. The circle indicated with a blue arrow shows the proline loop shown in B. (B) D0 domain shown with the Proline loop facing up and pointing toward the predicted HLA-G binding site. The position of Val45 and Pro46 in the crystal structure (C10-C28; yellow) is overlayed with that in the AlphaFold predicted structure (C28-C74; green). The distance between the Val45 and Pro46 in the crystal structure and their counterpart in the predicted structure of a Cys28-Cys74 structure is 5.3 and 5.4 Angstrom, respectively. (C) KIR2DL4 (green) D0 domain interaction with HLA-G (blue) as predicted by AlphaFold3. Val45 and Pro48 of KIR2DL4 contact Met76 and Glu19 in HLA-G, respectively. The numbers indicate the distance in Angstrom between side chains on KIR2DL4 (yellow) and side chains on HLA-G (green). (D) Docking of the KIR2DL4 crystal structure (3WYR, pink) and HLA-G (blue), as predicted by AlphaFold3. The shift of the Proline loop moves the side chains Val45 and Pro48 away to a distance greater than 3.5 Angstrom. (E) Predicted interaction of mutant KIR2DL4 C74R (beige) with HLA-G (blue). The structure of this KIR2DL4 in a Cys10-Cys28 configuration resembles that of the crystal structure, with distances greater than 3.5 Angstrom between side chains on C74R (yellow) and HLA-G (green). (F) Predicted interaction between the KIR2DL4 C10R AlphaFold DB structure (orange) and HLA-G (blue). This KIR2DL4 in a Cys28-Cys74 configuration resembles the WT KIR2DL4 (in C) and maintains close contacts with HLA-G via side chains Val45 and Pro48. (G) Predicted interaction of the four D2 Ig domains (shown as an overlay) with HLA-G (blue). Predicted close contacts of KIR2DL4 with HLA-G include the pairs Ser128–Arg145 (3.1 A, left), Asp130–Arg145 (2.7 A, center), and Ser179–Lys146 (2.7 A, right).
Random mutagenesis screen.
(A) Generation of mutants. (B) Analysis of mutants by confocal microscopy. (C) In the same assay shown in Figure 1C, mutants C10R and P48S were tested in the absence of HLA-G. Confocal images of anti-HA-Alexa-594 revealed their location (first panel). A DIC image is shown in the right. (D) Flow chart of mutants distributed among 4 categories distinct from WT. Several mutants from each category were also tested in the absence of soluble HLA-G. The location of KIR2DL4 often depends on HLA-G. For example, C10R was not internalized and P48S was able to internalize into vesicles in the absence of soluble HLA-G.
Disulfide-linked peptide analysis of recombinant KIR2DL4 protein by mass spectrometry.
Tandem mass spectra of the peptides linked by disulfide bond between Cys10 and Cys28 and between Cys28 and Cys74 are shown. The accurate mass of Cys10-Cys28 and Cys28-C74 peptides are shown in the insets (Cys10-Cys28: [M + 3H]3+ = m/z 762.03039 and expected [M + 3H]3+ = m/z 762.0299; Cys28-Cys74 : [M + 3H]3+ = m/z 405.18726 and expected [M + 3H]3+ = m/z 405.1870). Ions with a neutral loss of small molecules are labelled with an asterisk (loss of ammonia) and a prime (loss of water).
Flow cytometry profiles of 293T cells expressing HA-tagged KIR2DL4 WT and, in parallel, the 3 Cys to Ser mutants loaded for 2 h at 37°C with antibodies to the HA-tag and KIR2DL4.
Staining with the control anti-HA antibody coupled with Alexa 488 is shown on the x axis versus no antibody (top), MAB2238-Alexa 647 (center), and mAb #33-APC (bottom) on the y axis. The lower left quadrant was set to include and gate out 98% of unstained control cells.
Full images of the D0 domains shown in Figure 6 (C-F).
The Cys28-Cys74 disulfide bond is shown in blue (A, D).The Cys10-Cys28 disulfide bond is shown in red (B, C).
2DL4 peptides analyzed to determine disulfide bond redox state.
Peptides were detected by Byonic analysis software, confirmed by MS/MS and have errors <6 ppm. Only peptides with peak areas >10 million for a given Cys were included in the analysis.
