Allosteric disulfide control of ligand binding and endocytosis of the natural killer cell receptor for HLA-G
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eLife Assessment
This study presents important findings on the molecular mechanisms governing how the natural killer cell receptor KIR2DL4 interacts with HLA-G and undergoes internalization. The authors provide solid evidence for an allosteric disulfide-bond switch that regulates receptor activity, using a multifaceted approach that includes mutagenesis, mass spectrometry, and imaging. The work would be further strengthened by validating these mechanisms in primary immune cells and providing direct structural evidence for the proposed ligand-binding interface.
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Abstract
Human Leukocyte Antigen (HLA)-G is selectively expressed by fetal trophoblast cells that invade maternal tissue and encounter maternal natural killer (NK) cells early in pregnancy. In NK cells, the endosomal receptor KIR2DL4 responds to soluble HLA-G by inducing a broad transcriptional program to support placental development. Structural features of KIR2DL4 that control ligand binding and endocytosis are unknown. Random mutagenesis revealed that three cysteines in the first immunoglobulin domain of KIR2DL4 regulated endocytosis and uptake of HLA-G. We found that the Cys10-Cys28 bond visible in the KIR2DL4 crystal structure is an allosteric disulfide with potential to switch to a Cys28-Cys74 bond. Mass spectrometry analysis showed that KIR2DL4 in human cells exists in both disulfide-bonded states. The Cys10-Cys28 bond in purified KIR2DL4 was reduced by protein disulfide isomerase (PDI) in vitro. Inhibition of PDI caused retention of KIR2DL4 at the plasma membrane and prevented HLA-G uptake. Mutants in the Cys10-Cys28 configuration endocytosed spontaneously but did not bind HLA-G. Conversely, KIR2DL4 with a Cys28-Cys74 bond was at the plasma membrane and endocytosed HLA-G. A structural change predicted by AlphaFold upon disulfide switching to the Cys28-Cys74 form reorients the D0 domain into a conformation that binds HLA-G. Thus, conversion of KIR2DL4 from an inactive state to an HLA-G binding form can regulate NK cell function to promote fetal development.
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eLife Assessment
This study presents important findings on the molecular mechanisms governing how the natural killer cell receptor KIR2DL4 interacts with HLA-G and undergoes internalization. The authors provide solid evidence for an allosteric disulfide-bond switch that regulates receptor activity, using a multifaceted approach that includes mutagenesis, mass spectrometry, and imaging. The work would be further strengthened by validating these mechanisms in primary immune cells and providing direct structural evidence for the proposed ligand-binding interface.
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Reviewer #1 (Public review):
Summary:
This paper asks how the NK cell receptor KIR2DL4 binds HLA-G and undergoes endocytosis. The authors propose that an allosteric disulfide-bond switch controls whether the receptor is in a ligand-binding or non-binding state, and they support this model using mutagenesis, imaging, mass spectrometry, and structural prediction.
Strengths:
A major strength is the use of diverse, complementary approaches to validate the central claim. The authors combined unbiased random mutagenesis to identify key residues, confocal microscopy to track cellular localization , and mass spectrometry to quantify the redox states of specific disulfide bonds. These methods consistently support a single model: an allosteric disulfide switch. The transition between a Cys10-Cys28 bond and a Cys28-Cys74 bond serves as a …
Reviewer #1 (Public review):
Summary:
This paper asks how the NK cell receptor KIR2DL4 binds HLA-G and undergoes endocytosis. The authors propose that an allosteric disulfide-bond switch controls whether the receptor is in a ligand-binding or non-binding state, and they support this model using mutagenesis, imaging, mass spectrometry, and structural prediction.
Strengths:
A major strength is the use of diverse, complementary approaches to validate the central claim. The authors combined unbiased random mutagenesis to identify key residues, confocal microscopy to track cellular localization , and mass spectrometry to quantify the redox states of specific disulfide bonds. These methods consistently support a single model: an allosteric disulfide switch. The transition between a Cys10-Cys28 bond and a Cys28-Cys74 bond serves as a functional switch that controls whether the receptor resides at the plasma membrane to bind ligand or remains inactive in endosomes.
Weaknesses:
The core model is interesting, but some of the strongest mechanistic claims still rely heavily on structure prediction rather than direct structural evidence, especially the proposed HLA-G contact surface in Figure 6.
The paper supports an effect of the disulfide state on trafficking and uptake, but the case for direct KIR2DL4-HLA-G binding still feels somewhat indirect. The manuscript itself notes that direct binding had not been previously shown, and the current explanation partly depends on inference about which disulfide state is present.
Most of the main experiments are done in transfected 293T cells, so it is still not fully clear how strongly this mechanism carries over to the more relevant NK-cell setting discussed in the paper.
The cellular evidence for the PDI story is not specific, since it depends a lot on inhibitor and blocking experiments that could affect the broader extracellular redox environment.
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Reviewer #2 (Public review):
Summary:
Rajagopalan et al show how extracellular domain features regulate KIR2DL4 internalization. The trafficking phenotypes of cysteine mutants are logically organized, and well-summarized in a Table. The disulfide mapping and differential alkylation strategy are appropriate and provide strong support for alternative disulfide configurations in D0. The higher accessibility or more selective reduction of Cys10-Cys28 as compared to Cys28-Cys74 by PDI is a key mechanistic anchor.
Strengths:
The identification of a conformational switch in KIR2DL4 is conceptually novel. Experimental elegance, detailed and well-written.
Weaknesses:
Most of the mechanistic work was shown in HEK293. The authors should exhibit relevance using primary NK cells (using primary NK)
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