The molecular basis of Abelson kinase regulation by its αI-helix
- Structural Biology and Biophysics, Biozentrum, University of Basel, Switzerland
Figures
Figure 1
Domain organization of Abl and ligand-induced conformations of the Abl core.
(A) Domain organization of Abl isoforms 1a,b and of the chronic myeloid leukemia (CML)-inducing fusion protein Bcr-Abl. (B) Crystal structure of the N-terminally myristoylated Abl 1b isoform …
Figure 2
Production of C-terminally truncated Abl regulatory core constructs.
(A) Detailed view of the interaction site between the SH2 domain (yellow) and the C-terminus of the Abl KD with its αH- (dark blue) and αI- (cyan) helices from the crystal structure of the assembled …
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Figure 2—source data 1
Uncropped original image file of western blot in Figure 2B.
- https://cdn.elifesciences.org/articles/92324/elife-92324-fig2-data1-v1.zip
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Figure 2—source data 2
Uncropped western blot of Figure 2B with annotation of relevant bands.
- https://cdn.elifesciences.org/articles/92324/elife-92324-fig2-data2-v1.zip
Figure 3
NMR evidence for reduced imatinib-induced core disassembly in truncated Abl constructs.
(A) Selected 1H-15N TROSY resonances that report on the imatinib-induced disassembly (left and middle panels) and the SH3SH2/KD interface (right panel). The label indicates the last amino acid of …
Figure 4 with 1 supplement
PCAs of resonances comprising the KD or entire Abl core.
Figure 4—figure supplement 1
PCA loadings showing the contributions of the 1H and 15N chemical shifts of individual residues to PC1 and PC2.
(A) PCA of all KD chemical shifts of all investigated complexes corresponding to Figure 4A. (B) PCA of KD chemical shifts for type I and type II inhibitor complexes (Figure 4B and C). Residues with …
Figure 5
In vitro kinase activity assays and effect of the E528K mutation.
(A) Specific kinase activity of Abl83-534, E528K (orange), Abl83-534 (red), Abl83-519 (light blue), Abl83-517 (green), Abl83-515 (magenta), and Abl83-534•asciminib (asc, blue). Sample sizes and …
Figure 6
Correlation between Abl kinase activity, imatinib-induced Abl core disassembly, and αI-helix mutations.
Figure 7
Mechanical model of Abl allosteric regulation.
(A) Transition between closed (left) and open (right) conformation of Abl’s regulatory core during activation. Arrows indicate mobility of the KD N-lobe and the αI-helix. Clashes between SH3 and …
Tables
Table 1
Michaelis-Menten parameters* for the various Abl helix constructs.
| Abl83-534 | Abl83-519 | Abl83-517 | Abl83-515 | Abl83-534•asciminib | Abl83-534,E528K | |
|---|---|---|---|---|---|---|
| vmax† | 89.4±6.7 | 79.5±15.4 | 56.8±13.3 | 3.2±0.2 | 61.9±11.8 | 201.7±16.0 |
| KM‡ | 51.8±8.8 | 91.4±30.9 | 74.8±35.7 | 39.5±9.0 | 336.7±80.3 | 71.6±12.3 |
| N§ | 8 | 3 | 3 | 3 | 3 | 2 |
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*
the Michaelis-Menten parameters and their errors were obtained by Monte Carlo fitting using the sample mean and standard error of the mean of the measured kinase activities shown in Figure 5A.
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†
in nmol Pi min–1μmol–1, where Pi is the transferred phosphate.
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‡
in μM.
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§
total number of activity assay experiments.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Human) | Abl | Uniprot | P00519-2 | |
| Gene (Escherichia phage lambda) | Lambda phosphatase; LPP | Uniprot | P03772 | |
| Strain, strain background (Escherichia coli) | BL21(DE3) | Sigma-Aldrich | CMC0014 | Chemical competent cells |
| Recombinant DNA reagent | Plasmid containing human Abl83-534 | Skora et al., 2013 DOI:10.1073/pnas.1314712110 | ||
| Recombinant DNA reagent | Plasmid containing Lambda phosphatase | Sonti et al., 2018 DOI:10.1021/jacs.7b12430 | ||
| Antibody | Conjugated poly-histidine antibody | Sigma-Aldrich | Cat.# A7058 | Mouse monoclonal (1:10000); peroxidase conjugate |
| Peptide, recombinant protein | Abltide | ProteoGenix | biotin-GGEAIYAAPFKK | |
| Commercial assay or kit | SAM2 Biotin Capture Membrane | Promega | Cat.# V2861 | |
| Chemical compound, drug | Asciminib; asci | Selleck Chemicals | Cat.# S8555 | |
| Chemical compound, drug | GNF-5 | Selleck Chemicals | Cat.# S7526 | |
| Chemical compound, drug | AMP-PNP | Roche | Cat.# 10102547001 | |
| Chemical compound, drug | Bosutinib; bosu | Selleck Chemicals | Cat.# S1014 | |
| Chemical compound, drug | Axitinib; axit | Selleck Chemicals | Cat.# S1005 | |
| Chemical compound, drug | Dasatinib; dasa | Selleck Chemicals | Cat.# S1021 | |
| Chemical compound, drug | Danusertib; danu | Selleck Chemicals | Cat.# S1107 | |
| Chemical compound, drug | Tozasertib; toza | Selleck Chemicals | Cat.# S1048 | |
| Chemical compound, drug | Staurosporine; staur | Selleck Chemicals | Cat.# S1421 | |
| Chemical compound, drug | PD180970 | Sigma-Aldrich | Cat.# PZ0142 | |
| Chemical compound, drug | PD166326 | Sigma-Aldrich | Cat.# PZ0366 | |
| Chemical compound, drug | Rebastinib; reba | Selleck Chemicals | Cat.# S2634 | |
| Chemical compound, drug | Ponatinib; pona | Selleck Chemicals | Cat.# S1490 | |
| Chemical compound, drug | Nilotinib; nilo | Selleck Chemicals | Cat.# S1033 | |
| Chemical compound, drug | Bafetinib; bafe | Selleck Chemicals | Cat.# S1369 | |
| Chemical compound, drug | Imatinib; ima | Selleck Chemicals | Cat.# S2475 | |
| Software, algorithm | NMRPipe | Delaglio et al., 1995 DOI:10.1007/bf00197809 | ||
| Software, algorithm | SPARKY | Lee et al., 2015 DOI:10.1093/bioinformatics/btu830 | ||
| Software, algorithm | NumPy | Harris et al., 2020 DOI:10.1038/s41586-020-2649-2 |
