TAPBPR promotes antigen loading on MHC-I molecules using a peptide trap

Andrew C Mcshan,Nikolaos G Sgourakis,Sarah A Overall,Erik Procko,Christine A Devlin,Neha Akella,Giora I Morozov,Danai Moschidi

doi:10.1038/s41467-021-23225-6

Abstract

Chaperones Tapasin and TAP-binding protein related (TAPBPR) perform the important functions of stabilizing nascent MHC-I molecules (chaperoning) and selecting high-affinity peptides in the MHC-I groove (editing). While X-ray and cryo-EM snapshots of MHC-I in complex with TAPBPR and Tapasin, respectively, have provided important insights into the peptide-deficient MHC-I groove structure, the molecular mechanism through which these chaperones influence the selection of specific amino acid sequences remains incompletely characterized. Based on structural and functional data, a loop sequence of variable lengths has been proposed to stabilize empty MHC-I molecules through direct interactions with the floor of the groove. Using deep mutagenesis on two complementary expression systems, we find that important residues for the Tapasin/TAPBPR chaperoning activity are located on a large scaffolding surface, excluding the loop. Conversely, loop mutations influence TAPBPR interactions with properly conformed MHC-I molecules, relevant for peptide editing. Detailed biophysical characterization by solution NMR, ITC and FP-based assays shows that the loop hovers above the MHC-I groove to promote the capture of incoming peptides. Our results suggest that the longer loop of TAPBPR lowers the affinity requirements for peptide selection to facilitate peptide loading under conditions and subcellular compartments of reduced ligand concentration, and to prevent disassembly of high-affinity peptide-MHC-I complexes that are transiently interrogated by TAPBPR during editing.

Highlights

Chaperones Tapasin and TAP-binding protein related (TAPBPR) perform the important functions of stabilizing nascent major histocompatibility complex (MHC-I) molecules and selecting high-affinity peptides in the MHC-I groove
A re-examination of the peptide-loading complex (PLC) cryo-EM structure[18] shows that the tapasin loop modeled as projecting into the F pocket falls outside the electron density and has B factors fixed at 30.00 Å2, indicating it was excluded from refinement
We found that incubation of pMHC-I with TAPBPR containing G24-R36 loop deletions resulted in ~3 to 4-fold increase of free TAX9 peptide relative to samples containing the equivalent concentrations of wild-type TAPBPR (Fig. 6e), suggesting that a longer loop results in reduced peptide unloading activity, consistent with our previous fluorescence polarization (FP) and isothermal titration calorimetry (ITC) results (Fig. 5)

Summary

Introduction

Chaperones Tapasin and TAP-binding protein related (TAPBPR) perform the important functions of stabilizing nascent MHC-I molecules (chaperoning) and selecting high-affinity peptides in the MHC-I groove (editing). In addition to chaperoning MHC-I, tapasin and TAPBPR function as catalytic enhancers of peptide association and dissociation within the MHCI groove (peptide exchange) and participate in peptide editing and quality control of assembled pMHC-I molecules[5,6,7,8]. Additional quality control is achieved by the association of TAPBPR with UDP-glucose:glycoprotein glucosyltransferase (UGGT1), which promotes reglycosylation of empty or suboptimally loaded MHC-I towards their recycling to the PLC9,10 These processes ensure the correct folding, trafficking and prolonged cell surface display of pMHC-I antigens[11]. In one of the TAPBPR crystal structures[17], a protruding loop (residues G24-R36) of TAPBPR (Fig. 1a) was modeled in an α-helical conformation extending from the luminal tip of the a

Methods

Results

Conclusion