Allosteric disulfide control of ligand binding and endocytosis of KIR2DL4, the natural killer cell receptor for HLA-G
- Molecular and Cellular Immunology Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States
- Centenary Institute, The University of Sydney, Australia
- Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Australia
- Structural Immunology Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States
- Department of Biochemistry and Molecular Biology, University of Chicago, United States
- School of Life Sciences, University of Technology Sydney, Australia
Figures
Figure 1 with 1 supplement
Random mutagenesis screen.
(A) Soluble HLA-G (sG) is internalized with HA-tagged KIR2DL4 into endosomes, as shown by incubation for 2 hr 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. Scale bar is 10 μm. (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 location of detectable HLA-G. Confocal images representative of the five categories of mutants are shown. A DIC image is shown in the upper right panel. Scale bar is 10 μm. 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.
Figure 1—figure supplement 1
Random mutagenesis screen.
(A) Strategy for the generation of mutants. (B) Examination of mutants by confocal microscopy. WT = wild-type. (C) In the same assay as in Figure 1C, mutants C10R and P48S were tested in the absence of soluble HLA-G (sG). Confocal images of anti-HA-Alexa-594 revealed their location. A DIC image is shown in the right panel. PM = plasma membrane. Scale bar is 10 μm. (D) Flow chart of mutants distributed among four categories distinct from WT. Several mutants from each category were also tested in the absence of soluble HLA-G (sG). Examples of site-directed mutants and their phenotype are shown on the last line. HLA-G often dictates the location of KIR2DL4. For example, C10R internalization depended on HLA-G, and P48S was not internalized into vesicles in the presence of soluble HLA-G.
Figure 2
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) Solvent accessibility of disulfide bonds visible in crystal structures of the first Ig domain of KIRs was determined by the DSSP (define secondary structure of proteins) algorithm. The first Ig domain of KIR2DL4 and KIR3DL1 is a D0 domain, whereas the first Ig domain of KIR2DL1 is a D1 domain. (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 strand, respectively, as indicated. (D) The disulfide bond configuration in the KIR2DL4 D0 domain corresponds to an –RHstaple.
Figure 3 with 1 supplement
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 the D2 domain. (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 dithiothreitol (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 served as control. (F) Incubation of purified KIR2DL4 with three different protein disulfide isomerases (PDIs). Reduction of the Cys10–Cys28 bond occurred with PDI at a PDI:KIR2DL4 molar ratio of 2 and 10. The redox state of cysteines was quantified by differential cysteine labeling and mass spectrometry. The error bars (SD) were derived from measurements of two to four peptides. ***p ≤ 0.005 as assessed by unpaired, two-tailed Student's t-test.
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Figure 3—source data 1
KIR2DL4 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. *Cys was labeled with 12C-iodoacetanilide or 13C-iodoacetanilide and is in bold. #Peptide score calculated by Byonic analysis software is a primary indicator of peptide-spectrum matches.
- https://cdn.elifesciences.org/articles/111018/elife-111018-fig3-data1-v1.docx
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Figure 3—source data 2
Source data for experiments shown in Figure 3B, D, F .
- https://cdn.elifesciences.org/articles/111018/elife-111018-fig3-data2-v1.xlsx
Figure 3—figure supplement 1
Analysis of disulfide-linked peptides in 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).
Figure 4
Location of KIR2DL4 and HLA-G after inhibition of protein disulfide isomerase (PDI).
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 hr 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. For images in (A) to (D), scale bar is 10 μm. (E) Confocal microscopy images of primary resting human NK cells loaded with labeled HLA-G (green) for 2 hr in the presence of 1 mM DTNB or 30 μM Rutin. The number of HLA-G positive cells was scored in multiple fields (11–18 fields/condition). (F) Enlarged image of (E) (bottom left) with vesicular localization of HLA-G. Scale bar is 10 μm. (G) Quantification of data obtained from analyzing experiments using NK cells from three different donors.
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Figure 4—source data 1
Source data for experiments shown in Figure 4G.
- https://cdn.elifesciences.org/articles/111018/elife-111018-fig4-data1-v1.xlsx
Figure 5 with 3 supplements
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) Quantitative PCR analysis of IFI44L expression in response to HLA-G stimulation of 293T cells with stable expression of WT, C10S, or C74S KIR2DL4. Baseline IFI44L transcription in untreated cells was set at 1. Data are represented as mean ± SD and are from 8 to 15 independent experiments depending on the cell line. ****p ≤ 0.001; ns, p > 0.05 by Wilcoxon test. (E) Reactivity of the antibodies anti-HA tag, mAb #33, and mAb 2238 with wild-type and the three cysteine mutants of KIR2DL4. (F) Flow cytometry analysis of primary NK cells pretreated with DTNB (1 mM) or Rutin (30 μM) for 1 hr at 37°C and stained for surface expression of KIR2DL4 using mAb 2238 at 4°C in the presence of inhibitor for 30 min, followed by staining with goat anti-mouse secondary antibodies coupled to APC at 4°C for 30 min. Data are represented as median fluorescence units and were acquired with NK cells from five donors tested separately.
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Figure 5—source data 1
Source data for experiments shown in Figure 5D.
- https://cdn.elifesciences.org/articles/111018/elife-111018-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Flow cytometry profiles of 293T cells stably expressing WT, C10S, or C74S mutants of HA-tagged KIR2DL4.
Cells were stained at 37°C for 2 hr with anti-HA tag antibody coupled to Alexa 488. Median fluorescence intensity (MFI) for the red profiles is indicated in the upper right corner. The black profiles represent unstained cells.
Figure 5—figure supplement 2
Reactivity of 293T cells expressing HA-tagged WT and mutant KIR2DL4 to mAbs specific for KIR2DL4.
Flow cytometry profiles of 293T cells expressing HA-tagged KIR2DL4 WT and the 3 Cys to Ser mutants, as indicated, after incubation for 2 hr at 37°C with the following antibodies: an antibody to the HA-tag (x axis) and the two antibodies to KIR2DL4 mAb 2238-Alexa 647 (center) and mAb #33-APC (bottom) shown on the y axis. No antibody is shown at the top. The lower left quadrant was set to include 98% of unstained control cells.
Figure 5—figure supplement 3
Staining of primary NK cells with mAbs specific for KIR2DL4.
Flow cytometry profiles of primary NK cells from two different donors stained at the cell surface with mAb #33 (blue profiles) or mAb 2238 (red profiles) followed by APC-coupled secondary antibodies. Control staining with secondary antibodies alone is also shown (black profiles).
Figure 6 with 1 supplement
Surface plasmon resonance (SPR) affinity measurements.
(A) Representative sensorgrams from single dilution series of KIR2DL4, KIR2DL4 (C10L), and KIR3DL1 binding to HLA-GVPLH (top panel) and interaction analysis of this binding (bottom panel). (B) Analysis of KIR2DL4 and KIR2DL4(C10L) interaction with non-binding epitopes of HLA-G refolded with two other peptides. Error bars denote standard deviation. Equilibrium dissociation constants KD were determined using a 1:1 binding model from two independent experiments performed in duplicate.
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Figure 6—source data 1
Summary of surface plasmon resonance (SPR) measurements.
KIR binding to the immobilized HLA-G epitopes was measured in two independent experiments. KD was calculated from all data using one site-specific binding model. N.B., no binding.
- https://cdn.elifesciences.org/articles/111018/elife-111018-fig6-data1-v1.docx
Figure 6—figure supplement 1
Size exclusion chromatography (SEC) profiles.
Overlay of size exclusion profiles with elution volume of purified KIR2DL4 wt (blue), KIR2DL4 C10L (orange), and KIR2DL4 C74S (green).
Figure 7 with 1 supplement
Predicted outcome of an allosteric disulfide switch on the conformation of KIR2DL4 and its predicted 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 region indicated with a blue arrow corresponds to the loop highlighted in B. (B) D0 domain shown with the Pro46–Pro48 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 overlaid with that in the AlphaFold-predicted structure (C28–C74; green). The distance between Val45 and Pro46 in the crystal structure and their counterpart in the predicted structure of a Cys28–Cys74 KIR2DL4 is 5.3 and 5.4 Å, respectively. (C) Docking of the KIR2DL4 crystal structure (3WYR, pink) with HLA-G (blue), as predicted by AlphaFold 3. (D) Docking of the KIR2DL4 structure predicted by AlphaFold 2 (green) with HLA-G (blue), as predicted by AlphaFold 3. In the Cys28–Cys74 KIR2DL4 bonded form, the Val45–Pro48 loop is in a different orientation, suggesting potential contacts with Met76 and Glu19 of HLA-G.
Figure 7—figure supplement 1
Potential interaction of KIR2DL4 with HLA-G, as predicted by AlphaFold.
(A) Docking of the C74R KIR2DL4 (beige) with HLA-G (blue), as predicted by AlphaFold 3. The structure of KIR2DL4 in this Cys10–Cys28 configuration is similar to that of the crystal structure (Figure 7C). (B) C10R KIR2DL4 (orange) with HLA-G (blue). The predicted structure of this Cys28–Cys74 KIR2DL4, with the Val45–Pro48 loop closer to HLA-G, resembles that of WT KIR2DL4 predicted by AlphaFold (Figure 7D). (C) Folding of the D2 domain in the four KIR2DL4 isoforms 3WYR crystal structure of WT (pink), and AlphaFold predictions of WT (green), C74R (beige), and C10R (orange). examined here, indicate a conserved D2 domain folding (shown as an overlay). Potential contacts of KIR2DL4 with HLA-G (blue), such as Ser128–Arg145 (3.1 Å, left), Asp130–Arg145 (2.7 Å, center), and Ser179–Lys146 (2.7 Å, right) are similar to those of the KIR3DL1 D2 domain with HLA-B.
Tables
Table 1
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.
| KIR2DL4 Cys mutants | KIR2DL4 location | KIR2DL4 location upon sHLA-G load | sHLA-G location | Disulfide bonds in D0 domain |
|---|---|---|---|---|
| WT | V | V | V | 3 possible |
| C10S | PM | PM and V | V | C28–C74 |
| C10A | PM | PM and V | V | |
| C10R | PM | PM and V | V | |
| C28S | PM | PM | None | C10–C74 |
| C28A | PM | PM | None | |
| C28R | PM | PM | None | |
| C74S | V | V | Few V | C10–C28 |
| C74A | V | V | None | |
| C74R | V | V | Few V | |
| C74W | V | V | None |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/111018/elife-111018-mdarchecklist1-v1.pdf
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Source data 1
Mutants generated in the random mutagenesis screen.
- https://cdn.elifesciences.org/articles/111018/elife-111018-data1-v1.xlsx
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Allosteric disulfide control of ligand binding and endocytosis of KIR2DL4, the natural killer cell receptor for HLA-G
eLife 15:RP111018.
https://doi.org/10.7554/eLife.111018.3
