Abstract
Understanding the conformational changes associated with the binding of small ligands to their biological targets is a fascinating and meaningful question in chemistry, biology and drug discovery. One of the most studied and important is the so-called “DFG-flip” of tyrosine kinases. The conserved three amino-acid DFG motif undergoes an “in to out” movement resulting in a particular inactive conformation to which “type II” kinase inhibitors, such as the anti-cancer drug Imatinib, bind. Despite many studies, the details of this prototypical conformational change are still debated. Here we combine various NMR experiments and surface plasmon resonance with enhanced sampling molecular dynamics simulations to shed light into the conformational dynamics associated with the binding of Imatinib to the proto-oncogene c-Src. We find that both conformational selection and induced fit play a role in the binding mechanism, reconciling opposing views held in the literature. Moreover, an external binding pose and local unfolding (cracking) of the aG helix are observed.
Highlights
Molecular recognition plays a fundamental role in all biological processes
Most of the signals corresponding to the activation loop (A-loop) (Figs 1 and 2), the αC helix and some of those corresponding to the αG helix are missing
As the solvent exchange rate is highly dependent on pH, its extent can be inferred by acquiring the spectrum at different pH28
Summary
Molecular recognition plays a fundamental role in all biological processes. Understanding it in atomic detail is of great importance as it could lead to more effective and less toxic drugs. The drug has a 2300-fold lower inhibitory power towards the c-Src tyrosine kinase (TK)[7] relative to its specific target, the c-Abl tyrosine kinase[8,9], despite the high sequence identity (47%) Both conformational selection and induced fit effects have been invoked to justify such a dramatic difference. Using state-of-the-art free energy methods, we have proposed that the most important contribution to the binding free energy difference of Imatinib to c-Src and c-Abl is, the stability of the DFG-out conformation[15], according to a “conformational selection” mechanism. In the case of Abl and its drug-resistant mutants, we recently proposed a binding mechanism in which two different conformational changes play a role, providing a consistent interpretation to the recent experimental and computational findings[25]. Distant structural elements such as the A-loop, the αC, αG and αD helices are involved in coordinated movements thanks to a network of electrostatic interactions involving the conserved residues Glu[310], Arg[409], Asp[404] and Lys[295] What is more, both simulation and experiments agree on a significant role of local unfolding, supporting predictions based on energy landscape theory[26,27]
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