RESUMEN
WW domains are small domains present in many human proteins with a wide array of functions and acting through the recognition of proline-rich sequences. The WW domain belonging to polyglutamine tract-binding protein 1 (PQBP1) is of particular interest due to its direct involvement in several X chromosome-linked intellectual disabilities, including Golabi-Ito-Hall (GIH) syndrome, where a single point mutation (Y65C) correlates with the development of the disease. The mutant cannot bind to its natural ligand WBP11, which regulates mRNA processing. In this work we use high-field high-resolution NMR and enhanced sampling molecular dynamics simulations to gain insight into the molecular causes the disease. We find that the wild type protein is partially unfolded exchanging among multiple beta-strand-like conformations in solution. The Y65C mutation further destabilizes the residual fold and primes the protein for the formation of a disulphide bridge, which could be at the origin of the loss of function.
Asunto(s)
Proteínas Portadoras/genética , Parálisis Cerebral/genética , Proteínas de Unión al ADN/genética , Discapacidad Intelectual/genética , Discapacidad Intelectual Ligada al Cromosoma X/genética , Proteínas Nucleares/genética , Parálisis Cerebral/patología , Proteínas de Unión al ADN/química , Humanos , Discapacidad Intelectual/patología , Discapacidad Intelectual Ligada al Cromosoma X/patología , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Proteínas Nucleares/química , Mutación Puntual/genética , Unión Proteica , Conformación Proteica en Lámina beta , Pliegue de Proteína , Factores de Empalme de ARN , ARN Mensajero/química , ARN Mensajero/genética , Dominios WW/genéticaRESUMEN
Phosphorylation of the activation loop is a fundamental step in the activation of most protein kinases. In the case of the Src tyrosine kinase, a prototypical kinase due to its role in cancer and its historic importance, phosphorylation of tyrosine 416 in the activation loop is known to rigidify the structure and contribute to the switch from the inactive to a fully active form. However, whether or not phosphorylation is able per-se to induce a fully active conformation, that efficiently binds ATP and phosphorylates the substrate, is less clear. Here we employ a combination of solution NMR and enhanced-sampling molecular dynamics simulations to fully map the effects of phosphorylation and ATP/ADP cofactor loading on the conformational landscape of Src tyrosine kinase. We find that both phosphorylation and cofactor binding are needed to induce a fully active conformation. What is more, we find a complex interplay between the A-loop and the hinge motion where the phosphorylation of the activation-loop has a significant allosteric effect on the dynamics of the C-lobe.
Asunto(s)
Adenosina Trifosfato/metabolismo , Familia-src Quinasas/metabolismo , Regulación Alostérica , Sitios de Unión , Dominio Catalítico , Humanos , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Fosforilación , Termodinámica , Tirosina/metabolismo , Familia-src Quinasas/químicaRESUMEN
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.
Asunto(s)
Antineoplásicos/química , Mesilato de Imatinib/química , Familia-src Quinasas/química , Proteína Tirosina Quinasa CSK , Ligandos , Imagen por Resonancia Magnética , Conformación Molecular , Simulación de Dinámica Molecular , Unión Proteica , Resonancia por Plasmón de SuperficieRESUMEN
Due to its inhibition of the Abl kinase domain in the BCR-ABL fusion protein, imatinib is strikingly effective in the initial stage of chronic myeloid leukemia with more than 90% of the patients showing complete remission. However, as in the case of most targeted anti-cancer therapies, the emergence of drug resistance is a serious concern. Several drug-resistant mutations affecting the catalytic domain of Abl and other tyrosine kinases are now known. But, despite their importance and the adverse effect that they have on the prognosis of the cancer patients harboring them, the molecular mechanism of these mutations is still debated. Here by using long molecular dynamics simulations and large-scale free energy calculations complemented by in vitro mutagenesis and microcalorimetry experiments, we model the effect of several widespread drug-resistant mutations of Abl. By comparing the conformational free energy landscape of the mutants with those of the wild-type tyrosine kinases we clarify their mode of action. It involves significant and complex changes in the inactive-to-active dynamics and entropy/enthalpy balance of two functional elements: the activation-loop and the conserved DFG motif. What is more the T315I gatekeeper mutant has a significant impact on the binding mechanism itself and on the binding kinetics.