A miniaturized MR1 metabolite display system with native-like protein features

Reviewer #1 (Public review):

Summary:

This study presents an Important tool for the study of MR1 antigen binding, opening new possibilities, and cutting-edge techniques. The evidence supporting the claims of the authors is solid, although including some functional experiments using primary T-cells would also provide a more complete physiologic evaluation. The work will be of interest to T cell immunologists, in general, especially those studying unconventional T cells.

Strengths:

In this study, the authors developed a single-chain MR1-derived protein by exchanging the α3 domain and β2-microglobulin for a helical stabilizing domain that they had previously developed. The aim was to generate a more compact structure that would still fold properly, without the risk of losing β2-microglobulin. This overall more robust structure would facilitate ligand exploration using various cutting-edge biophysical techniques.

The authors successfully demonstrated that their construct folds similarly to native MR1 and retains the ability to bind MAIT TCR in solution, as shown by cryo-EM experiments. Its melting temperature was equivalent to that of the native protein. Importantly, the construct enables the use of differential scanning fluorometry and transverse relaxation-optimized spectroscopy, which represent the main strengths of this work. These approaches should greatly facilitate the screening of additional unknown ligands and enable interaction mapping.

Weaknesses:

One possible area for improvement would be to extend the validation to additional known ligands, particularly weaker binders. Furthermore, although the cryo-EM data are highly convincing, including either MAIT cell staining or MAIT activation assays with the generated construct would provide stronger functional validation of its equivalence to the wild-type protein with respect to ligand-binding properties.

Overall, this work is of great interest to the field, as several groups worldwide are seeking to identify endogenous/tumour-derived MR1 ligands. In addition, some pathogens lacking the capacity to produce 5-OP-RU have been shown to activate MAIT cells, raising the possibility that unknown pathogen-derived ligands may also exist.

Reviewer #2 (Public review):

Summary:

The authors develop a miniaturized MR1 construct (SMART-MR1) in which the α1/α2 platform is stabilized by a synthetic domain, and show that it can bind ligands, engage a cognate TCR, and recapitulate native-like recognition by cryo-EM.

Strengths:

The work is well-written, technically strong and carefully executed. The authors combine biochemical, biophysical and structural approaches, including ITC, NMR and cryo-EM, to show that SMART-MR1 behaves in a manner closely resembling native MR1. The reduction in size and the demonstration of solution NMR are clear practical advantages for certain types of mechanistic studies.

Weaknesses:

The main limitation is that the manuscript does not clearly establish a practical advantage over existing MR1 formats, such as single-chain MR1-β2M or previously described stabilized constructs. The comparison is largely framed against native MR1, which risks overstating the problem, and on the basis of the data presented, it is unlikely that other researchers will adopt this system. In addition, the choice of the A-F7 TCR as a validation reagent may overestimate the generality of the approach, as this receptor is known to exhibit relatively broad ligand tolerance, including recognition of MR1 presenting vitamin B6 metabolites (PDB 9CGR) and structurally diverse synthetic ligands. The extent to which SMART-MR1 supports recognition by a broader range of MR1-restricted TCRs is not addressed.

Reviewer #3 (Public review):

Summary:

This manuscript describes the engineering, production and validation of an MR1 variant with enhanced suitability for screening of ligands and biophysical and structural analysis. The authors utilize a previous advance from their laboratory on a classical MHC (HLA-A2) whereby the alpha 3 and b2m domains are replaced by a helical stabilizing domain.

Strengths:

This variant has a smaller molecular weight than the native MR1, can be produced easily through refolding and is thus much more suitable for NMR analysis. The authors provide data demonstrating that many of the parameters typically evaluated in protein biochemistry/biophysics are similar to reported values between this engineered variant and the wild-type protein. Overall, this is a significant advance to the MR1 field and more broadly to MR1 relevance in immunology and cancer biology, as this will accelerate high-throughput screening and discovery of disease-relevant ligands for MR1, which have been overshadowed by the misguided fixation on 5-OP-RU.

Weaknesses:

Minor concerns about the lack of comparison with the native MR1 extracellular domain construct in the validation of this engineered construct.