Cross-linking of the human MHC-I peptide-loading complex

The peptide-loading complex (PLC) is a transient, multisubunit membrane complex in the endoplasmic reticulum (ER), essential for establishing a hierarchical immune response. The PLC coordinates peptide translocation into the ER with loading and editing of major histocompatibility complex class I molecules (MHC-I). After final proofreading in the PLC, stable peptide/MHC-I complexes are released to the cell surface to evoke a T-cell response against infected or malignant cells. Sampling of different MHC-I allomorphs requires the precise coordination of seven different subunits into a single macromolecular assembly, including the transporter associated with antigen processing TAP1/2, the oxidoreductase ERp57, the MHC-I heterodimer, and the chaperones tapasin and calreticulin. The molecular organization and mechanistic events in the PLC are unknown due to the compositional heterogeneity and intrinsic dynamic nature of the complex. Here, PLC from Burkitt’s lymphoma cells was isolated using an engineered viral inhibitor as bait, followed by cross-linking mass spectrometry to aid EM structure elucidation.