Figures and data in The molecular basis of Abelson kinase regulation by its αI-helix

Figures
Tables
Additional files

7 figures, 2 tables and 1 additional file

Figures

Figure 1

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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 (residues 2–531, PDB 2FO0). SH3 domain (green), SH2 domain (yellow), KD-SH2 linker (grey), kinase domain (KD) N- and C-lobes (KD-N and KD-C, respectively, blue), activation loop (A-loop, green), and αI-helix (cyan) are shown in cartoon representation. The myristoyl is shown as yellow spheres. The N-cap (aa 2–82), which is largely not observed in the crystal, is indicated as a dashed brown line. A schematic representation on the right indicates the position of the kink in the αI-αI’ helix. (C) Alignment of the crystal structure of a KD-only construct with unliganded myristoyl pocket (blue, PDB 1M52) with the assembled Abl core structure (PDB 2FO0). The SH3 and SH2 domains of the latter are displayed as in panel B. (D) Model of a single conformation of the dynamical imatinib-bound, disassembled Abl core as derived by NMR and SAXS (Skora et al., 2013). The coloring follows panel B with the exception of the activation loop (magenta). Imatinib is shown as magenta sticks. Blue arrows indicate relative motions of the SH3, SH2, and KD domains.

Figure 2

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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 Abl core (PDB 2FO0). The C^α-atoms of M515, Q517, and S519 are shown as cyan spheres (B) Top: western blot of *E. coli*-expressed, soluble truncated Abl constructs in the supernatant after cell lysis. Bottom: expression yields of the corresponding Abl constructs as quantified from the gel bands. The numbers on the horizontal axis denote the last amino acid of the respective construct Abl^83-X. (C) Relative yields of the purified truncated Abl constructs and Abl^83-534,E528K as quantified by OD₂₈₀.

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
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Figure 3

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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 the respective Abl^83-X construct. Abbreviations: ima:imatinib, PD16:PD166326.(B) Scores plot of the PCA of the SH3 and SH2 domain chemical shifts of various Abl core complexes and apo forms. Abbreviations: asci:asciminib, bosu:bosutinib, axi:axitinib, dasa:dasatinib, danu:danusertib, toza:tozasertib, staur:staurosporine, reba:rebastinib, pona:ponatinib, nilo:nilotinib, bafe:bafetinib. (C) Loadings plot of the PCA shown in panel B indicating contributions of individual ¹H^N and ¹⁵N chemical shifts to the first two PCs. Resonances with absolute PC1 or PC2 loadings larger than 0.15 are highlighted in red and blue, respectively. V92 has large loadings for both PCs and is shown in pink. (D) Residues with large PC1 or PC2 loadings according to panel C indicated as spheres within the assembled Abl core structure (PDB 2FO0). The color coding follows panel C.

Figure 4 with 1 supplement

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PCAs of resonances comprising the KD or entire Abl core.

(A) PCA scores of the KD resonances of all complexes shown in Figure 3B. (B) PCA scores of the KD resonances of only type I and type II inhibitor complexes. The respective loadings are shown in Figure 4—figure supplement 1. (C) Residues with absolute PC1 loadings larger than 0.15 for the analysis in panel B are shown as red spheres in the assembled core structure (PDB 2FO0). The P-loop is shown in orange and the A-loop in its active (green, PDB 2FO0) as well as inactive (magenta, PDB 2HYY Cowan-Jacob et al., 2007) conformation. (D) PCA scores of the resonances of the entire Abl core for the apo forms of all αI-helix variants. The respective loadings are shown in Figure 4—figure supplement 1. (E) Residues with absolute PC1 loadings larger than 0.1 of the analysis in panel D are shown as red spheres in the assembled core structure (PDB 2FO0).

Figure 4—figure supplement 1

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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 absolute PC1 values larger than 0.15 are indicated in red. (C) PCA of all SH3SH2KD shifts of all apo forms (Figure 4D and E). Residues with sizeable absolute PC1 values larger than 0.1 or sizeable PC2 (< -0.1 or > 0.125) are indicated in red and blue, respectively. V151, N316, and K438 have both sizeable PC1 and PC2 and is shown in magenta.

Figure 5

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In vitro kinase activity assays and effect of the E528K mutation.

(A) Specific kinase activity of Abl^{83-534, E528K} (orange), Abl^83-534 (red), Abl^83-519 (light blue), Abl^83-517 (green), Abl^83-515 (magenta), and Abl^83-534•asciminib (asc, blue). Sample sizes and statistical information are given in Table 1. (B) Bar plot of fitted Michaelis-Menten v_max values derived from the kinase assays in panel A. Numerical values are given in Table 1. (C) Detailed view of the E528-R479 salt bridge in the structure of the assembled Abl core (PDB 2FO0). Distances between the E528 carboxylate oxygens and the guanidinium sidechain hydrogen of R479 as well as the backbone amide hydrogen of E485 are displayed as dashed lines and indicated in Angstrom. (D) Superpositions of the ¹H-¹⁵N TROSY resonances of V130, T136, and G149 for apo Abl^83-534,wt (red), apo Abl^83-534,E528K (orange), Abl^83-534,wt•imatinib (blue), and Abl^83-534,E528K•imatinib (magenta) showing the increased imatinib-induced core disassembly of the Abl^83-534,E528K mutant.

Figure 6

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Correlation between Abl kinase activity, imatinib-induced Abl core disassembly, and αI-helix mutations.

(A) Correlation between the PC1 scores of the SH3SH2 resonances (PCA in Figure 3B) of all imatinib complexes and the respective kinase activity (Figure 5B). (B) Correlation between the PC2 scores of the SH3SH2KD resonances of all apo forms (PCA in Figure 4D) and the respective kinase activity (Figure 5B). The color code for the Abl constructs follows panel A. (C) Correlation between the PC1 scores of the SH3SH2 resonances of all imatinib complexes (Figure 3B) and the PC2 scores of the SH3SH2KD resonances of all apo forms (Figure 4D). The color code for the Abl constructs follows panel A. (D) Residues with absolute PC1 loadings larger than 0.15 from the PCA of the SH3SH2 resonances of all imatinib complexes (Figure 3C) indicated as red spheres within the structure of the assembled Abl core (PDB 2FO0). (E) Residues with absolute PC2 loadings larger than 0.1 from the PCA of the SH3SH2KD resonances of all apo forms (Figure 4D, Figure 4—figure supplement 1) indicated as red spheres within the structure of the assembled Abl core. (F) Interface between the αI-helix and the SH2 domain. The bent αI-helix of the assembled core structure is shown in cyan. The straight αI-helix of the isolated Abl kinase domain (PDB 1M52) is shown in magenta stick representation, while residues that clash with the SH2 domain of the assembled Abl core being shown in green stick representation. SH2 residues are displayed as colored spheres. Red: residues with absolute PC2 loadings >0.1 as in panel E, blue: residues with absolute PC2 loadings smaller than 0.1, yellow: residues with unresolved resonances in NMR spectrum.

Figure 7

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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 KD-N and between SH2 and αI-helix resulting from this mobility are shown by red dots, the related clash-induced forces F_KD-N,SH3 and F_αI,SH2 by red triangles. Color coding: SH3 (green), SH2 (yellow), KD (blue), αI-helix (cyan), A-loop (inactive: magenta, active: green), P-loop (orange), tyrosine substrate (brown), R479-E528 salt bridge (orange). (B) Model of imatinib-induced Abl core disassembly. The color code follows panel A. Allo: allosteric inhibitor. (C) Detailed model of the interaction between the αI-helix and the SH2 domain for the various investigated Abl αI-helix constructs and inhibitor-bound conformations. The observed, respective Abl activity increases in the direction of the arrow at the bottom. The color code follows panel A.

Tables

Table 1

Michaelis-Menten parameters^* for the various Abl helix constructs.

	Abl^83-534	Abl^83-519	Abl^83-517	Abl^83-515	Abl^83-534•asciminib	Abl^83-534,E528K
v_max†	89.4±6.7	79.5±15.4	56.8±13.3	3.2±0.2	61.9±11.8	201.7±16.0
K_M‡	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

*

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.
†

in nmol P_i min^–1μmol^–1, where P_i is the transferred phosphate.
‡

in μM.
§

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 Abl^83-534	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