Allosteric disulfide control of ligand binding and endocytosis of the natural killer cell receptor for HLA-G

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.

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)