Dataset Information

Structural mechanism of a drug-binding process involving a large conformational change of the protein target

ABSTRACT: Structural biology studies indicate that proteins often undergo large conformational changes when binding small molecules, but atomic-level descriptions of binding events involving such changes have been elusive. A prominent example of binding accompanied by a large conformational change is the binding of Abl kinase to the cancer drug imatinib, a detailed understanding of which could potentially inform future drug-discovery efforts targeting kinases. Here, we report unguided molecular dynamics simulations of Abl-imatinib binding that start from an unbound state and ultimately reach a bound state that is highly consistent with known crystal structures of the Abl- imatinib complex. In the course of this process, we observed that imatinib first selectively engages Abl kinase in its autoinhibitory conformation. Consistent with inferences drawn from previous experimental studies, imatinib then induces a large conformational change of the protein, and this motion is captured in atomic detail by the simulations. Moreover, the simulations reveal a surprising local structural instability in the C-terminal lobe of Abl kinase during binding and, to a lesser degree, in the bound state. The unstable region, which is distal to the imatinib-binding site, includes a number of residues that, when mutated, confer resistance to imatinib therapy by an unknown mechanism. Using NMR, thermostability, and hydrogen-deuterium exchange (HDX) measurements, along with energetic estimates, we determined that these mutations likely destabilize the Abl kinase structure. These findings, along with the simulations, suggest that these mutations confer imatinib resistance by a previously undescribed mechanism in which they exacerbate structural instability in the C-terminal lobe to the degree that the imatinib-bound state is energetically unfavorable.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: John R. Engen

LAB HEAD: John R. Engen

PROVIDER: PXD034008 | Pride | 2023-04-07

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	1-032017_ABL_KD_ctrl1.raw.zip	Raw
	1-032017_ABL_KD_ctrl2.raw.zip	Raw
	1-032017_ABL_KD_ctrl3.raw.zip	Raw
	1-032017_ABL_KD_ctrl4.raw.zip	Raw
	1-040417_ABL_WT_10minD2O.raw.zip	Raw

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Structural mechanism of a drug-binding process involving a large conformational change of the protein target.

Ayaz Pelin P Lyczek Agatha A Paung YiTing Y Mingione Victoria R VR Iacob Roxana E RE de Waal Parker W PW Engen John R JR Seeliger Markus A MA Shan Yibing Y Shaw David E DE

Nature communications 20230405 1

Proteins often undergo large conformational changes when binding small molecules, but atomic-level descriptions of such events have been elusive. Here, we report unguided molecular dynamics simulations of Abl kinase binding to the cancer drug imatinib. In the simulations, imatinib first selectively engages Abl kinase in its autoinhibitory conformation. Consistent with inferences drawn from previous experimental studies, imatinib then induces a large conformational change of the protein to reach ...[more]

PMID: 37019905

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Project description:To directly observe the conformational changes in Akt1 3P elicited by PIP 3 binding, we performed HDX-MS analysis of Akt1 3P in the presence of plasma membrane-mimicking liposomes (20% cholesterol, 30% phosphatidylcholine, 15% phosphatidylserine, 35% phosphatidylethanolamine) that contained either 0% or 5% PIP 3 . The sequence coverage of Akt1 was excellent, with 160 peptides spanning ~97% of all exchangeable amides (Supplementary Table 2). With 5% PIP 3 liposomes there was extensive protection of the PH domain, similar to what had been previously observed for non-phosphorylated Akt1. In addition to protection of the PH domain elicited by PIP 3 binding, Akt1 3P exhibited significant deprotection of the C-lobe of the kinase domain, including the activation loop, that corresponds to the interface between the PH and kinase domains observed in our structure of autoinhibited Akt1. We also carriedout HDX-MS experiments in the presence and absence of ATPS, for which we observed no significant differences. The likely binding pocket for the hydrophobic motif is the so-called PDK1- interacting fragment (PIF) pocket in Akt that binds the phosphorylated hydrophobic motif in the active conformation (21, 22) . Previous hydrogen- deuterium exchange mass spectrometry (HDX-MS) analysis of Akt1 DrLink indicated small, but significant exposure of the PIF pocket upon PI(3,4,5)P 3 binding (26) (Figure 5D, deuterium incorporation plots reproduced with permission). We confirmed this observation by comparing the deuterium incorporation rates for Akt1 DrLink and Akt1 ΔC in solution. Sequence coverage of the truncated Akt1 ΔC comprised 85 peptides spanning ~94% of all exchangeable amides (Supplementary Table 2). Two peptides in Akt1 ΔC , corresponding to β3-αB in the N-lobe and the catalytic loop in the C-lobe of the kinase domain exhibited a modest, but significant, 6% increase in deuterium incorporation (Figure 5E). These changes indicate exposure of the PIF pocket and consequent local disordering of the N-lobe in the absence of the hydrophobic motif. These observationsis areis consistent with the lack of electron density observed for theC helix and activation loop and overall higher temperature factors for the N- lobe of the kinase domain (Supplementary Figure S5CD). In order to test whether the unphosphorylated hydrophobic motif indeed binds to the PIF pocket in the inactive conformation, we measured the binding affinity of a tail peptide 19 containing the missing C-terminal tail residues in Akt1 ΔC (residues 457-480 of human Akt1) by fluorescence anisotropy. The binding constant was estimated to be approximately 0.5 mM from three independent titrations (Figure 5F), although it was not possible to reach saturation due to limiting Akt1 ΔC concentration. Whilst this is a relatively weak interaction, it is sufficient in the context of an intramolecular interaction to sequester the hydrophobic motif more than 99% of the time at equilibrium due to the almost infinite local concentration.