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| Status |
Public on Sep 01, 2019 |
| Title |
Critical residues within the TAPBPR scoop loop for HLA-A2 binding and peptide loading are determined by deep mutational scanning |
| Organism |
Homo sapiens |
| Experiment type |
Other
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| Summary |
The loading of high affinity peptides onto nascent class I MHC (MHC-I) molecules is facilitated by chaperones, including the class I-specific chaperone TAP-binding protein-related (TAPBPR). TAPBPR features a loop (amino acids 24-35) that projects towards the empty MHC-I peptide binding groove and rests above the F pocket. The 24-35 loop is much shorter in the closely related homologue tapasin, and therefore may be partly responsible for the unique antigen editing properties of TAPBPR. Previously we reported a deep mutational scan of human TAPBPR focused on the 24-35 loop, and determined the relative effects of single amino acid mutations on binding and peptide-mediated release of the murine H2-Dd MHC-I allomorph. Here, we extend our studies to determine the mutational landscape of the 24-35 loop when TAPBPR binds a human MHC-I allomorph, HLA-A*02:01. The data highlights how TAPBPR affinity can be increased or decreased for different MHC-I allomorphs by tuning the electrostatic complementarity of the 24-35 loop for surfaces on the rim of the peptide-binding groove. By changing the selection pressure from HLA-A2 binding to HLA-A2 loading and processing, we find that TAPBPR is reasonably tolerant of mutations in the 24-35 loop for efficient peptide-MHC-I processing and surface trafficking.
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| Overall design |
For investigating binding interactions, the extracellular domains of human TAPBPR were fused to Aga2p at the N-terminus for yeast surface display, and fused to a c-myc tag at the C-terminus for measuring expression. The TAPBPR 24-35 loop (a.a. G27-D38 in the deposited files based on an earlier numbering scheme) was diversified by single-site saturation mutagenesis to encode all possible 240 amino acid substitutions. The library was expressed on the yeast cell surface and sorted by fluorescence-activated cell sorting (FACS) for high binding signal to fluorescent HLA-A*02:01 tetramers, in the absence or presence of competing TAX peptide. For investigating HLA-A2 loading and processing in human cells, TAPBPR was modified with an N-terminal FLAG tag to detect expression, and the transmembrane and cytosolic regions were replaced with the equivalent residues of tapasin. We hypothesize that the chimeric construct may bring TAPBPR into the the peptide-loading complex for enhanced activity. Again, a single-site saturation mutagenesis library focused on the 24-35 loop was constructed, and expressed in Expi293F cells in which the endogenous tapasin gene was knocked out using CRISPR-based genome editing. These cells express little surface HLA-A2, yet MHC-I processing and surface trafficking can be partially rescued by transfecting in MHC-I-specific chaperones tapasin or TAPBPR. Cells transfected with the TAPBPR library and displaying increased surface HLA-A2 were collected by FACS. The enrichment or depletion of every TAPBPR mutation was calculated by comparing frequencies in the naive libraries with the sorted populations.
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| Contributor(s) |
Procko E, Devlin CA |
| Citation(s) |
34039964 |
| Submission date |
Feb 07, 2019 |
| Last update date |
Jun 15, 2021 |
| Contact name |
Erik Procko |
| E-mail(s) |
procko@illinois.edu
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| Phone |
217-300-1454
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| Organization name |
University of Illinois
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| Department |
Biochemistry
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| Lab |
RAL 318G
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| Street address |
601 S Goodwin Ave
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| City |
Urbana |
| State/province |
IL |
| ZIP/Postal code |
61801 |
| Country |
USA |
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| Platforms (2) |
| GPL15520 |
Illumina MiSeq (Homo sapiens) |
| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
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| Samples (11)
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| GSM3593593 |
TAPBPR-tapasin chimera naive library |
| GSM3593594 |
Wild type TAPBPR-tapasin chimera: Control for sequencing errors |
| GSM3593595 |
Tapasin-KO human cells expressing TAPBPR library sorted for high surface HLA-A2 (Replicate 1) |
| GSM3593596 |
Tapasin-KO human cells expressing TAPBPR library sorted for high surface HLA-A2 (Replicate 2) |
| GSM3593597 |
Exon 2 of tapasin gene in wild type Expi293F cells |
| GSM3593598 |
Exon 2 of tapasin gene in tapasin-knock out Expi293F cells |
| GSM3593599 |
TAPBPR naive library for yeast display |
| GSM3593600 |
TAPBPR yeast display library sorted for binding 300 ng/ml HLA-A*02:01 |
| GSM3593601 |
TAPBPR yeast display library sorted for binding 100 ng/ml HLA-A*02:01 |
| GSM3593602 |
TAPBPR yeast display library sorted for binding 2000 ng/ml HLA-A*02:01 in presence of 3000 nM Tax peptide |
| GSM3593603 |
TAPBPR yeast display library sorted for binding 2000 ng/ml HLA-A*02:01 in presence of 300 nM Tax peptide |
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| Relations |
| BioProject |
PRJNA521310 |
| SRA |
SRP184465 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE126206_enrichment_ratios_TAPBPR_HLA-A2.xlsx.gz |
59.6 Kb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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