Conformational states dynamically populated by a kinase determine its function

Abstract

Protein kinases intrinsically sample a number of conformational states with distinct catalytic and binding activities. We used nuclear magnetic resonance spectroscopy to describe in atomic-level detail how Abl kinase interconverts between an active and two discrete inactive structures. Extensive differences in key structural elements between the conformational states give rise to multiple intrinsic regulatory mechanisms. The findings explain how oncogenic mutants can counteract inhibitory mechanisms to constitutively activate the kinase. Energetic dissection revealed the contributions of the activation loop, the Asp-Phe-Gly (DFG) motif, the regulatory spine, and the gatekeeper residue to kinase regulation. Characterization of the transient conformation to which the drug imatinib binds enabled the elucidation of drug-resistance mechanisms. Structural insight into inactive states highlights how they can be leveraged for the design of selective inhibitors.

Fig. 1.. Characterization of the energetically excited conformational states in Abl kinase by NMR CEST experiments.

(A) Structure of the Abl kinase domain in the active conformation determined in the current work. Key structural features are highlighted. (B) Methyl groups in Abl indicating the presence of one (colored cyan) or two (colored magenta) excited conformational states in the NMR CEST experiments. (C) Representative ¹³C CEST profiles of the indicated methyl groups. The major dip corresponds to the major (ground) state whereas the minor dips correspond to the energetically excited conformational states E₁ and E₂. As explained in the main text, excited states E₁ and E₂ correspond to inactive states I₁ and I₂, respectively. (D) Energy landscape of the ground (G), E₁ and E₂ states denoting their populations and kinetics of interconversion as measured by fitting the CEST data. (E) Correlation of the CEST-derived chemical shift difference between the ground and E₁ states (Δ(E₁–G)_CEST) with the chemical shift difference between apo and PD173955-bound Abl. Methyls (marked with an asterisk) for which the dips corresponding to the ground and E₁ states are within ±0.3 p.p.m. cannot be resolved in the CEST profile and their chemical shift difference was extracted from ¹H-¹³C–correlated experiments (see materials and methods). Methyls that are close (within 6 Å) to PD173955 were excluded from the correlation because their chemical shifts are directly affected by the inhibitor. Val398^CG1 deviates from linearity because of the rearrangement of the nearby Phe401 side chain in the inhibitor complex compared to the E₁ state. (F) Correlation of the CEST-derived chemical shift difference between the ground and E₂ states (Δϖ(E₂–G)_CEST) with the chemical shift difference between apo and imatinib-bound Abl. Methyls that are close (within 6 Å) to imatinib were excluded from the correlation because their chemical shifts are directly affected by the inhibitor. Methyls colored blue deviate from linearity as discussed in the text and in fig. S8. Methyls (marked with an asterisk) for which the dips corresponding to the ground and E₂ states are within ±0.3 p.p.m. cannot be resolved in the CEST profile and their chemical shift difference was extracted from ¹H-¹³C–correlated experiments.

Fig. 2.. Structures of the ground (active), E₁ (I₁) and E₂ (I₂) states of Abl.

(A) NMR solution structure of the ground state of Abl shows that the Abl kinase domain inherently adopts the fully active state. The zoomed view shows the hydrophobic residues surrounding Phe401. (B) Structure of the Abl E₁ state (green) superimposed on the structure of the active state (blue). Because Abl E₁ adopts an inactive state is referred to as I₁. The zoomed views highlight similarities (e.g. αC helix) and differences (e.g. DFG motif and A-loop) in the disposition of key structural elements between the two states. (C) Structure of the Abl E₂ state (orange) superimposed on the structure of the active state (blue). Because Abl E₂ adopts an inactive state is referred to as I₂. The major structural rearrangement undergone by the αC helix, the A-loop and the P-loop between the two states is indicated by an arrow. (D) Superposition of the structures of the Abl I₂ state (orange) and the Abl–imatinib complex (cyan; PDB ID 1IEP). The zoomed view highlights the structural rearrangement upon imatinib binding in the residues lining the pocket. (E) ¹³C CEST profiles of Abl and Abl^E311K demonstrate that the E311K substitution destabilizes the Abl I₁ state but has no apparent effect on the I₂ state. A denotes active state. The E311K substitution abrogates the ion pair between Glu311 and Arg405 in the I₁ state (panel B).

Fig. 3.. Structural and energetic dissection of imatinib-resistance Abl variants.

(A) Structures of the three Abl conformational states and binding of imatinib to the I₂ state highlighting the disposition of key structural elements. (B) Patient-derived imatinib-resistance mutants studied here shown on the structure of Abl in complex with imatinib and on the structure of the Abl I₂ state. (C) Change in affinity for imatinib by the Abl variants relative to wild type Abl as determined by ITC (fig. S9). Error bars are standard deviation determined from a triplicate. (D) Effect of the H415P substitution on the population of the Abl states measured by ¹³C CEST experiments. The changes in the populations are indicated in a free energy diagram. A zoomed view of the mutation site suggests how the Pro substitution may destabilize the Abl I₂ state. (E) Effect of the Y272H substitution on the population of the Abl states measured by ¹³C CEST using the wild type Abl as reference (top) and by ¹H-¹³C–correlated experiments using the Abl^T408Y variant, which populates Abl I₂ state at 70%, as reference (bottom). A zoomed view of the mutation site indicates that substitution of Tyr272 will disrupt the hydrogen bond to Glu305 thereby destabilizing the Abl I₂ state. (F) Effect of the E274V substitution on the population of the Abl states measured by ¹H¹³C–correlated spectra using the Abl^I2M variant as reference. A zoomed view of the mutation site indicates that substitution of Glu274 will disrupt the hydrogen bond to Lys293 thereby destabilizing the Abl I₂ state. (G) Effect of the F378V substitution on the population of the Abl states measured by ¹³C CEST using the wild type Abl as reference (top) and by ¹H-¹³C–correlated spectra using the Abl^T408Y variant as reference (bottom). For additional data and discussion see fig. S10.

Fig. 4.. Quantitative dissection of the effect of the regulatory spine, the gatekeeper, the DFG motif, and phosphorylation on the Abl conformational ensemble.

(A) Structure of the active state of Abl highlighting the regulatory spine, made up by Met309, Leu320, His380, and Phe401. The disposition of the regulatory spine is also shown for Abl states I₁ and I₂. (B) Effect of amino acid substitutions at the regulatory spine on the populations of the Abl conformational states as measured by NMR. Because the k_ex rates of the transitions (Fig. 1D) are slow on the NMR chemical shift time scale the resonances of the individual conformational states can be seen in ¹H-¹³C–correlated spectra if their population is above ~5%. Thus, in addition to ¹³C CEST, ¹H-¹³C–correlated experiments were also used to quantitate the populations, especially for Abl variants that are not stable over the period of time required for CEST experiments (materials and methods). There is excellent agreement in the population measurements by CEST and ¹H-¹³C–correlated experiments. The labeling scheme of the Abl variants is as shown in fig. S1. Determination of the kinase activity (fig. S11) shows that the Abl^M309L/L320I variant inhibits Abl. (C) Energy contribution to the active and I₂ states of Abl by the indicated variants. Negative values of ΔΔG indicate increased stability of the Abl I₂ state whereas positive values indicate increased stability of the Abl active state. A graph is included where the populations of the active and I₂ states are plotted as a function of the associated free energy, ΔG/RT, where R is the gas constant and T is the temperature. A 0.6 kcal mol⁻¹ change in ΔG corresponds to a change by 1 unit in ΔG/RT at room temperature (77). The populations of the two states for Abl (isolated kinase domain) and full-kinase Abl^FK are shown to help assess the effect that each one of the variants has on the activation or inhibition of Abl. (D) Effect of the Thr334 to Ile substitution at the gatekeeper position on the populations of the Abl states. Given that Abl exists predominantly in the active state (p_A ~88%), to measure the full effect we used the Abl^I2M variant (Abl^{G269E/M309L/T408Y}), which populates predominantly the I₂ state (p_I2 ~90%). (E) Effect of amino acid substitutions of the Phe residue in the DFG motif on the populations of the Abl states. Val or Tyr substitution results in an Abl kinase that adopts primarily the I₂ state. The low kinase activity of Abl^F401V is consistent with the NMR findings. (F) Effect of Tyr412 phosphorylation in the A-loop on the populations of the Abl states. Tyr412 phosphorylation eliminates both I₁ and I₂ states and stabilizes the active state as evidenced by ¹³C CEST experiments. To measure the full effect, we used the Abl^I2M variant as discussed in (D). The zoomed views of the superposition of the structure of each of the inactive states on the structure of the active state provides mechanistic insight into the effect.

Fig. 5.. Quantitative dissection of the effect of variants on the conformational ensemble of the full-length Abl kinase.

(A) Effect of the SH3-SH2 regulatory module, the addition of the allosteric inhibitor GNF5, and the H415P substitution as measured by NMR. Abl^FK corresponds to the SH3-SH2-KD Abl fragment. Because Abl^FK is not sufficiently stable for ¹³C CEST experiments, we used ¹H-¹³C–correlated spectra to quantitate the populations. For the isolated Abl kinase domain we know from CEST experiments that the population of the I₁ state is 6%. However, while we can conclude from the ¹H-¹³C–correlated spectra that the population of the I₁ state does not increase in Abl^FK and its variants, we cannot conclude if it is depleted. Thus, the populations reported for Abl^FK variants are only for the active and the I₂ states. (B) Schematic of Abl^FK showing the equilibrium between a disassembled conformation, wherein the regulatory module is not bound to the kinase domain, and an assembled conformation, wherein the regulatory module is docked onto the back of the kinase domain. In the disassembled conformation the kinase adopts the active state whereas in the assembled one it adopts the I₂ state. The populations of the active and I₂ states in the Abl variants studied are indicated. (C) Schematic of Abl^FK summarizing the effect of the gatekeeper T334I substitution, which forces Abl to adopt the active state even when the regulatory module is docked onto the kinase. ¹H-¹³C–correlated spectra showing the M362 methyl resonance. M362 is located at the interface between the SH2 and the kinase domain and provides a sensitive probe for the assembled conformation. (D) Superposition of the structures of the Abl I₂ state and the Abl^T334I–axitinib complex (PDB ID 4TWP) highlights the steric clash between the bound inhibitor and the A-loop in the I₂ state, which explains the higher affinity of the inhibitor for the T334I variant. (E) Effect of Tyr412 phosphorylation on the populations of the active and I₂ states of Abl^FK in complex with the allosteric inhibitor GNF5 measured by NMR. (F) Effect of the extended conformation, wherein the SH2 domain docks on the top of the N-lobe, on the populations of the active and I₂ states of Abl measured by NMR. The structure of the Abl^FKΔSH3 fragment (magenta; PDB ID 4XEY) is superimposed on the structure of the Abl I₂ state (orange).

Fig. 6.. pH effect on the Abl conformational ensemble and activation energy of the DFG transition.

(A to C) Close-up view of the DFG motif conformation in the (A) active state, (B) the I₁ state and (C) the I₂ state. The positioning of Asp400 of the DFG motif differs among the various conformational states. In the active state Asp400 is exposed to the solvent whereas Phe401 is buried inside a hydrophobic pocket. The DFG motif flips 180° as it transitions from the active to the I₁ state with the two residues swapping positions: in the I₁ state Asp400 is now positioned inside the hydrophobic pocket while Phe401 is exposed to the solvent. Although the DFG motif remains in the “out” conformation in the I₂ state, the A-loop undergoes a major rearrangement and as a result Asp400 is found in a polar environment, similarly to the active state, and Phe401 is buried inside the hydrophobic pocket located in the nucleotide binding site. (D) ¹³C CEST profiles of representative residues as a function of pH. The results show that lower pH (pH 6.5) stabilizes selectively the I₁ state while higher pH (pH 7.7) stabilizes selectively the I₂ state. (E) ¹H-¹³C–correlated spectra of Abl^M309L and Abl^M309L/H415P variants as a function of pH. In Abl^M309L/H415P at pH 7.7 there is no detectable inactive state because the H415P substitution eliminates the I₂ state and the higher pH depletes the I₁ state. (F) Populations of the I₁ and I₂ states as a function of pH. The relative populations of the I₁ and I₂ states fluctuate antagonistically but the ratio of populations between the active state and the inactive states collectively is not affected by changes in pH within this range. (G) ¹³C CEST profiles of Abl^H415P as a function of temperature. (H) Arrhenius plot of the k_ex measured by the CEST experiments in (G) for the three temperatures indicated used to determine the activation energy of the DFG transition from the active to the I₁ inactive conformation. (I) Energy diagram indicating the activation enthalpy (~36.4 kcal mol⁻¹) of the DFG flip from DFG-in to DFG-out.