Structural Modeling and Molecular Dynamics of the Immune Checkpoint Molecule HLA-G.

HLA-G is considered to be an immune checkpoint molecule, a function that is closely linked to the structure and dynamics of the different HLA-G isoforms. Unfortunately, little is known about the structure and dynamics of these isoforms. For instance, there are only seven crystal structures of HLA-G molecules, being all related to a single isoform, and in some cases lacking important residues associated to the interaction with leukocyte receptors. In addition, they lack information on the dynamics of both membrane-bound HLA-G forms, and soluble forms. We took advantage of in silico strategies to disclose the dynamic behavior of selected HLA-G forms, including the membrane-bound HLA-G1 molecule, soluble HLA-G1 dimer, and HLA-G5 isoform. Both the membrane-bound HLA-G1 molecule and the soluble HLA-G1 dimer were quite stable. Residues involved in the interaction with ILT2 and ILT4 receptors (α3 domain) were very close to the lipid bilayer in the complete HLA-G1 molecule, which might limit accessibility. On the other hand, these residues can be completely exposed in the soluble HLA-G1 dimer, due to the free rotation of the disulfide bridge (Cys42/Cys42). In fact, we speculate that this free rotation of each protomer (i.e., the chains composing the dimer) could enable alternative binding modes for ILT2/ILT4 receptors, which in turn could be associated with greater affinity of the soluble HLA-G1 dimer. Structural analysis of the HLA-G5 isoform demonstrated higher stability for the complex containing the peptide and coupled ß2-microglobulin, while structures lacking such domains were significantly unstable. This study reports for the first time structural conformations for the HLA-G5 isoform and the dynamic behavior of HLA-G1 molecules under simulated biological conditions. All modeled structures were made available through GitHub (https://github.com/KavrakiLab/), enabling their use as templates for modeling other alleles and isoforms, as well as for other computational analyses to investigate key molecular interactions.

HLA-G1 increases the radiosensitivity of human tumoral cells.

Different molecules regulate the response of tumoral tissues to ionizing radiation. The objective of this work was to determine if HLA-G1 expression modulates the radiosensitivity of human tumoral cell lines. To this end, human melanoma M8 and human erythroleukemia K562 cell lines, with their correspondent HLA-G1 negative and positive variants, were gamma irradiated and the survival frequency was determined by clonogenic assay. The survival fraction of HLA-G1 expressing cells was around 60% of HLA-G1 negative cells. The generation of acidic vesicular organelles was higher in HLA-G1 positive cells. Apoptosis levels showed statistically significant differences only in K562 cells, whereas the variation in G2/M cycle progression was only significant in M8 cells. In addition, irradiation diminished cell-surface HLA-G1 and increased soluble HLA-G1 levels. Soluble HLA-G1 has no influence on cell survival in any cell line. In summary, we could demonstrate that HLA-G1 confers higher radiosensitivity to HLA-G1 expressing cells.