Crystal structure of suboptimal viral fragments of Epstein Barr Virus Rta peptide-HLA complex that stimulate CD8 T cell response

Huan, Xuelu; Zhuo, Ziyi; Xiao, Ziwei; Ren, Ee Chee

doi:10.1038/s41598-019-53201-6

Cited by 3 publications

(2 citation statements)

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“…22). Interestingly, the two peptide fragments, resulting from the photocleavage, remain bound to the MHC I hc, as seen for a crystal structure of HLA-A*11:01 carrying a 4mer and 5mer peptide 120 . The study demonstrates that the peptide fragments complement each other to form a stable pMHC I complex 120 .…”

Section: Refolding Of Mhc I Allomorphsmentioning

confidence: 87%

“…Interestingly, the two peptide fragments, resulting from the photocleavage, remain bound to the MHC I hc, as seen for a crystal structure of HLA-A*11:01 carrying a 4mer and 5mer peptide 120 . The study demonstrates that the peptide fragments complement each other to form a stable pMHC I complex 120 . In this case, the structure provides direct information on the photocleavage that occurs in the MHC I binding pocket, highlighting the two peptide fragments which remain tightly bound.…”

Section: Refolding Of Mhc I Allomorphsmentioning

confidence: 87%

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Structural and functional analysis of the MHC I–tapasin–ERp57 multi-chaperone complex in antigen processing and presentation

Müller¹

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The health status of every nucleated cell in the human body is monitored through peptides presented by major histocompatibility complex class I (MHC I) to T-cell receptors of CD8+ T-cells. Thereby, the adaptive immune system ensures the recognition and elimination of infected or cancerous cells. MHC I molecules comprise the polymorphic heavy chain (hc) and the light chain β2-microglobulin (β2m). More than 13,000 allomorphs of the MHC I hc have been identified. All MHC I hcs associate with β2m but differ in their binding preferences for peptides, ensuring the presentation of a large peptide pool. After maturation of MHC I hc/β2m heterodimers in the endoplasmic reticulum (ER), most of the peptide-deficient MHC I molecules are recruited to the peptide-loading complex (PLC). There, they go through peptide loading and editing before they are released as stable peptide-MHC I (pMHC I) complexes and traffic to the cell surface for antigen presentation. During the stringent quality control of MHC I peptide loading and editing within the PLC, the chaperone tapasin in conjunction with the oxidoreductase ERp57 stabilizes peptide-receptive MHC I molecules and alters the peptide cargo for high immunogenicity by catalyzing peptide-exchange. The tapasin-homologue TAP-binding protein related (TAPBPR) is involved in downstream quality control, editing the peptide repertoire of MHC I molecules that slipped through peptide proofreading by tapasin. Both chaperones were shown to adopt similar binding-modes for MHC I, suggesting related mechanisms of peptide editing. Nevertheless, the MHC I specific chaperones operate in different subcellular locations with differing assistance. While TAPBPR mediates peptide-exchange solely in the peptide-poor environment of the cis-Golgi and ER-Golgi intermediate compartment (ERGIC), tapasin functions mainly within the PLC together with ERp57 and the lectin-like chaperone calreticulin. Calreticulin with its lectin-, arm- and C-terminal domain contacts the MHC I heterodimer, ERp57 and the C-terminal domain of tapasin, respectively. Notably, the interaction site between calreticulin and tapasin has not yet been elucidated experimentally at molecular detail. The depletion of tapasin leads to a compromised immune response and a change in the pool of peptide cargo. The numerous MHC I allomorphs vary in their plasticity and their dependence on tapasin for the loading of optimal peptides. Moreover, the conformational plasticity of MHC I correlates with their dependence on tapasin. However, the molecular basis on how tapasin edits the various MHC I allomorphs and the structural features that are essential for peptide exchange catalysis at atomic resolution remained elusive. In the first part of this thesis, the trimeric complex of tapasin–ERp57/calreticulin was analyzed. To this end, laser induced liquid bead ionization mass spectrometry (LILBID-MS) was performed as part of a collaboration and revealed the trimeric assembly for tapasin–ERp57 and calreticulin. Furthermore, additional to a wildtype construct of calreticulin, a second construct, lacking the acidic helix of calreticulin that was found to come to close contact with tapasin, was utilized for isothermal titration calorimetry (ITC). A micromolar affinity of wildtype calreticulin to tapasin–ERp57 was determined. Previous biochemical and NMR studies utilizing the P-domain of calreticulin and solely ERp57 provided a micromolar affinity for the complex of calreticulin and ERp57. In this study, no interaction of calreticulin lacking the acidic helix with tapasin–ERp57 could be measured by ITC. However, these results undergo with findings that calreticulin lacking the acidic helix impairs the function of the PLC. Most likely, the negatively charged acidic helix is located in a groove of tapasin, carrying a more positive charge. Taken together, the functional data demonstrates the importance of the acidic helix of calreticulin for assembly of the trimeric subunit of calreticulin/tapasin–ERp57. In the main part of this study an MHC I–tapasin–ERp57 complex was structurally analyzed. Therefore, a photo-triggered approach was chosen to assemble the transient complex of MHC I–tapasin–ERp57. Various allomorphs were screened for complex formation with the tapasin–ERp57 heterodimer after photocleavage by size exclusion chromatography (SEC), resulting in mouse MHC I H2-Db as the suited allomorph. Microseed matrix screening was performed. Crystals diffracting X-rays to a resolution of 2.7 Å were obtained showing one tetrameric tapasin–ERp57–MHC I complex per asymmetric unit. The MHC I-chaperone structure shows molecular rearrangements upon MHC I engagement and unveils structural features of tapasin, involved in peptide-exchange catalysis...

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Section: Refolding Of Mhc I Allomorphsmentioning

confidence: 87%

Section: Refolding Of Mhc I Allomorphsmentioning

confidence: 87%