Systematic genetic characterization of the human PKR kinase domain highlights its functional malleability to escape a poxvirus substrate mimic
- Center for Genomics and Data Science Research, National Human Genome Research Institute, National Institutes of Health, United States
- Department of Microbiology & Immunology, Georgetown University, United States
Figures
Figure 1 with 7 supplements
Exploring genetic variants of human PKR against the pseudosubstrate antagonist vaccinia K3.
(A) (Left) AlphaFold2 model of the PKR kinase domain (green) bound to its target, eIF2α (gray). (Right) Rotated 90°, the eIF2α binding surface of PKR is shaded gray and outlined in white dashed …
Figure 1—figure supplement 1
Composition of the PKR variant library.
(A) Barchart depicts the number of variants made at each of the four PKR windows categorized by amino acid properties: Positive Charge = H, K, R; Negative Charge = D, E; Polar-Neutral=C, N, Q, S, T; …
Figure 1—figure supplement 2
Calculation of PKR functional scores from yeast growth assay.
(A) Line plot of mock barcode read count data over time for cells expressing nonfunctional (red) or functional (blue) PKR across four sampled timepoints. As PKR activity is toxic to yeast, the …
Figure 1—figure supplement 3
Replication of PKR variants paired with K3 alleles.
(A) Scatter plot of PKR functional scores for variants paired with K3-WT for two biological replicate experiments. Functional scores were calculated across four timepoints as the area under the …
Figure 1—figure supplement 4
Systematic generation of PKR variants using mixed-base primer tile sets.
(A) Each variant primer is composed of a homology arm, variant region, and priming region. (B) Nonsynonymous SNP-accessible variants are generated by altering the codon in the variant region of the …
Figure 1—figure supplement 5
Multiple unique barcode sequences were attached to each PKR variant.
(A) The barcode primer is composed of a homology arm, barcode region, and priming region. The barcode primer is used as the reverse primer in the variant-generating PCR reactions to attach a unique …
Figure 1—figure supplement 6
Assembly of PKR variant library using variant tile sets and barcode primers.
(A) 15 variant primer tile sets were designed to generate variants across four windows of interest in PKR. The full-length PKR sequence is denoted in green, with Windows 1–4 overlaid in yellow, …
Figure 1—figure supplement 7
Alphafold2 multimer predictions used to identify PKR sites proximal to eIF2α and K3.
(A) AlphaFold2 multimer prediction of PKR in complex with eIF2α (Left) and K3 (Right). Residues are colored by AlphaFold2 pLDDT confidence scores per residue. In both predicted models, PKR is on the …
Figure 2 with 2 supplements
PKR variants that evade K3 and maintain kinase function are enriched at positive selection sites and helices αD and αG.
(A, C) PKR functional scores versus K3 are colored ranging from susceptible (red) to WT-like (white) to resistant (blue). (A) Heatmap of PKR variants with cells colored by the PKR functional score …
Figure 2—figure supplement 1
Experimental validation of the K3 resistance phenotypes of PKR variants.
Select PKR variants were generated and screened against K3Δ58, WT, and H47R alleles using a yeast growth assay visualized through serial dilution (Kawagishi-Kobayashi et al., 1997). PKR is under the …
Figure 2—figure supplement 2
Bimodal distribution of PKR functional scores at K3 contact sites.
Strip plot of PKR functional scores versus K3 partitioned by predicted contact with K3. Black dashed line denotes the threshold at which variants were separated as nonfunctional-like or functional. …
Figure 3
Few nonfunctional PKR variants identified in the absence of K3 inhibition.
(A) Scatter plot showing each variant’s PKR functional score versus wild-type K3 plotted against its PKR functional score versus K3∆58. The data point for PKR-WT is colored green and a data point …
Figure 4 with 1 supplement
Identification of PKR sites highly susceptible to K3 inhibition.
(A) A line was drawn connecting the data point for PKR-WT to the position of the average data point for the four nonsense variants (PKR∆) from the data in the scatter plot from Figure 3A, with all …
Figure 4—figure supplement 1
Sequence similarity between human eIF2α and vaccinia K3.
Alignment of human eIF2α (RefSeq Accession NP_004085.1, residues 0–117) to vaccinia K3 (RefSeq Accession YP_232916.1). The Ser51 site of phosphorylation in eIF2α is indicated by #. Sequences were …
Figure 5 with 2 supplements
PKR variants that are K3-resistant are also largely K3-H47R-resistant.
(A) Scatter plot of PKR functional scores versus wild-type K3 plotted against PKR functional scores versus K3-H47R. The data point for PKR-WT is colored green and a datapoint representing the …
Figure 5—figure supplement 1
Differing patterns of resistance between wild-type K3 and K3-H47R.
(A) A nonlinear exponential curve (black line) was fitted to the data in the scatter plot from Figure 5A. Points are colored by their residuals from that curve (K3 /K3 H47R Residuals), ranging from …
Figure 5—figure supplement 2
Additional variants at the sites of previously identified K3-H47R-resistant variants often also conferred resistance to K3.
Strip plots of PKR functional scores of variants paired with K3-H47R (top) and K3-WT (bottom). Each plot highlights PKR variants made at sites where an improved PKR variant was previously identified …
Figure 6
Unified spatial view of highlighted PKR sites across the K3-binding interface.
(A) Positions of sites classified as essential residues (black), K3-resistant (blue), and K3-susceptible (red) based upon the above analyses. All other tested sites are marked white. (Left) The …
Additional files
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Supplementary file 1
Oligonucleotides used in the study.
- https://cdn.elifesciences.org/articles/99575/elife-99575-supp1-v1.xlsx
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Supplementary file 2
Expanded list of oligonucleotides used to generate PKR variants.
- https://cdn.elifesciences.org/articles/99575/elife-99575-supp2-v1.xlsx
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Supplementary file 3
Plasmids used in this study.
- https://cdn.elifesciences.org/articles/99575/elife-99575-supp3-v1.xlsx
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Supplementary file 4
- https://cdn.elifesciences.org/articles/99575/elife-99575-supp4-v1.csv
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Supplementary file 5
Root mean square deviation between AlphaFold2-predicted complexes and experimentally determined structures.
- https://cdn.elifesciences.org/articles/99575/elife-99575-supp5-v1.xlsx
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Supplementary file 6
PDB-formatted atomic coordinate file for the AlphaFold2-predicted structure of the kinase domain of human PKR bound to human eIF2α.
- https://cdn.elifesciences.org/articles/99575/elife-99575-supp6-v1.zip
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Supplementary file 7
PDB-formatted atomic coordinate file for the AlphaFold2-predicted structure of the kinase domain of human PKR bound to vaccinia K3.
- https://cdn.elifesciences.org/articles/99575/elife-99575-supp7-v1.zip
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MDAR checklist
- https://cdn.elifesciences.org/articles/99575/elife-99575-mdarchecklist1-v1.pdf
