Chaperones TAPBPR and tapasin associate with class I major histocompatibility complexes (MHC-I) to promote optimization (editing) of peptide cargo. Here, we use solution NMR to investigate the mechanism of peptide exchange. We identify TAPBPR-induced conformational changes on conserved MHC-I molecular surfaces, consistent with our independently determined X-ray structure of the complex. Dynamics present in the empty MHC-I are stabilized by TAPBPR and become progressively dampened with increasing peptide occupancy. Incoming peptides are recognized according to the global stability of the final pMHC-I product and anneal in a native-like conformation to be edited by TAPBPR. Our results demonstrate an inverse relationship between MHC-I peptide occupancy and TAPBPR binding affinity, wherein the lifetime and structural features of transiently bound peptides control the regulation of a conformational switch located near the TAPBPR binding site, which triggers TAPBPR release. These results suggest a similar mechanism for the function of tapasin in the peptide-loading complex.
Genomic stability in proliferating cells critically depends on telomere maintenance by telomerase reverse transcriptase. Here we developed a real-time single-molecule RNA sequencing approach that visualizes telomerase catalysis and structural dynamics at single-nucleotide resolution using FRET and zero-mode waveguides. The method permits direct detection of dynamic steps and structural states throughout the telomerase catalytic cycle and can be generalized to other nucleic acid polymerase systems.
Molecular chaperones TAPBPR (TAP-binding protein related) and tapasin associate with class-I major histocompatibility complex (MHC-I) molecules to promote optimization (editing) of peptide cargo. Using solution NMR, we investigate the molecular mechanism of peptide exchange performed by the 90 kDa chaperone protein complex. We identify TAPBPR-induced conformational changes on conserved MHC-I surfaces, consistent with our independently determined X-ray structure of the empty complex. Conformational dynamics present in the empty MHC-I are stabilized by TAPBPR in a peptide-deficient complex, and become progressively dampened with increasing peptide occupancy. Incoming peptides are recognized by the chaperoned groove according to the global stability of the final pMHC-I product, and anneal in a native-like conformation. Our results demonstrate an inverse relationship between MHC-I occupancy by peptide and the affinity of TAPBPR for such pMHC-I molecules, where the lifetime of transiently bound peptides controls the dynamic regulation of a conformational switch, located near the TAPBPR binding site, which triggers TAPBPR release. We further discuss the role of protein dynamics in shaping chaperone specificity towards different human and murine class-I MHC allotypes, and present the high-resolution cryoEM structure of a human A*02/TAPBPR complex with novel insights into the antigen editing mechanism. These results suggest a similar mechanism for the editing function of tapasin in the peptide-loading complex, and provide a molecular blueprint for the design of novel chaperones with tailored antigen editing functions.
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