RESUMEN
Computer-aided drug discovery methods play a major role in the development of therapeutically important small molecules, but their performance needs to be improved. Molecular dynamics simulations in mixed solvents are useful in understanding protein-ligand recognition and improving molecular docking predictions. In this work, we used ethanol as a cosolvent to find relevant interactions for ligands toward protein kinase G, an essential protein of Mycobacterium tuberculosis (Mtb). We validated the hot spots by screening a database of fragment-like compounds and another one of known kinase inhibitors. Next, we performed a pharmacophore-guided docking simulation and found three low micromolar inhibitors, including one with a novel chemical scaffold that we expanded to four derivative compounds. Binding affinities were characterized by intrinsic fluorescence quenching assays, isothermal titration calorimetry, and the analysis of melting curves. The predicted binding mode was confirmed by X-ray crystallography. Finally, the compounds significantly inhibited the viability of Mtb in infected THP-1 macrophages.
Asunto(s)
Mycobacterium tuberculosis , Sitios de Unión , Proteínas Quinasas Dependientes de GMP Cíclico , Ligandos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Mycobacterium tuberculosis (Mtb), the causative agent of Tuberculosis, has 11 eukaryotic-like serine/threonine protein kinases, which play essential roles in cell growth, signal transduction, and pathogenesis. Protein kinase G (PknG) regulates the carbon and nitrogen metabolism by phosphorylation of the glycogen accumulation regulator (GarA) protein at Thr21. Protein kinase B (PknB) is involved in cell wall synthesis and cell shape, as well as phosphorylates GarA but at Thr22. While PknG seems to be constitutively activated and recognition of GarA requires phosphorylation in its unstructured tail, PknB activation is triggered by phosphorylation of its activation loop, which allows binding of the forkhead-associated domain of GarA. In the present work, we used molecular dynamics and quantum-mechanics/molecular mechanics simulations of the catalytically competent complex and kinase activity assays to understand PknG/PknB specificity and reactivity toward GarA. Two hydrophobic residues in GarA, Val24 and Phe25, seem essential for PknG binding and allow specificity for Thr21 phosphorylation. On the other hand, phosphorylated residues in PknB bind Arg26 in GarA and regulate its specificity for Thr22. We also provide a detailed analysis of the free energy profile for the phospho-transfer reaction and show why PknG has a constitutively active conformation not requiring priming phosphorylation in contrast to PknB. Our results provide new insights into these two key enzymes relevant for Mtb and the mechanisms of serine/threonine phosphorylation in bacteria.
Asunto(s)
Mycobacterium tuberculosis , Proteínas Bacterianas/química , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina , Treonina/metabolismoRESUMEN
All biological processes rely on the formation of protein-ligand, protein-peptide and protein-protein complexes. Studying the affinity, kinetics and thermodynamics of binding between these pairs is critical for understanding basic cellular mechanisms. Many different technologies have been designed for probing interactions between biomolecules, each based on measuring different signals (fluorescence, heat, thermophoresis, scattering and interference, among others). Evaluation of the data from binding experiments and their fitting is an essential step towards the quantification of binding affinities. Here, user-friendly online tools to analyze biophysical data from steady-state fluorescence spectroscopy, microscale thermophoresis and differential scanning fluorimetry experiments are presented. The modules of the data-analysis platform (https://spc.embl-hamburg.de/) contain classical thermodynamic models and clear user guidelines for the determination of equilibrium dissociation constants (Kd) and thermal unfolding parameters such as melting temperatures (Tm).
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/química , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Fluorescencia , Mycobacterium tuberculosis/metabolismo , Sistemas en Línea , Temperatura , Termodinámica , Cinética , Ligandos , Unión Proteica , Espectrometría de FluorescenciaRESUMEN
Histidine kinases (HK) of bacterial two-component systems represent a hallmark of allosterism in proteins, being able to detect a signal through the sensor domain and transmit this information through the protein matrix to the kinase domain which, once active, autophosphorylates a specific histidine residue. Inactive-to-active transition results in a large conformational change that moves the kinase on top of the histidine. In the present work, we use several molecular simulation techniques (Molecular Dynamics, Hybrid QM/MM, and constant pH molecular dynamics) to study the activation and autophosphorylation reactions in L. plantarum WalK, a cis-acting HK. In agreement with previous results, we show that the chemical step requires tight coupling with the conformational step in order to maintain the histidine phosphoacceptor in the correct tautomeric state, with a reactive δ-nitrogen. During the conformational transition, the kinase domain is never released and walks along the HK helix axis, breaking and forming several conserved residue-based contacts. The phosphate transfer reaction is concerted in the transition state region and is catalyzed through the stabilization of the negative developing charge of transferring phosphate along the reaction.
Asunto(s)
Histidina Quinasa/química , Histidina Quinasa/metabolismo , Simulación de Dinámica Molecular , Teoría Cuántica , Concentración de Iones de Hidrógeno , Lactobacillus plantarum/enzimología , Fosforilación , Conformación Proteica , TermodinámicaRESUMEN
Protein kinases (PKs) are allosteric enzymes that play an essential role in signal transduction by regulating a variety of key cellular processes. Most PKs suffer conformational rearrangements upon phosphorylation that strongly enhance the catalytic activity. Generally, it involves the movement of the phosphorylated loop toward the active site and the rotation of the whole C-terminal lobe. However, not all kinases undergo such a large configurational change: The MAPK extracellular signal-regulated protein kinases ERK1 and ERK2 achieve a 50â¯000 fold increase in kinase activity with only a small motion of the C-terminal region. In the present work, we used a combination of molecular simulation tools to characterize the conformational landscape of ERK2 in the active (phosphorylated) and inactive (unphosphorylated) states in solution in agreement with NMR experiments. We show that the chemical reaction barrier is strongly dependent on ATP conformation and that the "active" low-barrier configuration is subtly regulated by phosphorylation, which stabilizes a key salt bridge between the conserved Lys52 and Glu69 belonging to helix-C and promotes binding of a second Mg ion. Our study highlights that the on-off switch embedded in the kinase fold can be regulated by small, medium, and large conformational changes.
Asunto(s)
Proteína Quinasa 1 Activada por Mitógenos/química , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Secuencia Conservada , Disulfuros/química , Activación Enzimática , Simulación de Dinámica Molecular , Fosforilación , Conformación ProteicaRESUMEN
Sensor histidine kinases (SHKs) are an integral component of the molecular machinery that permits bacteria to adapt to widely changing environmental conditions. CpxA, an extensively studied SHK, is a multidomain homodimeric protein with each subunit consisting of a periplasmic sensor domain, a transmembrane domain, a signal-transducing HAMP domain, a dimerization and histidine phospho-acceptor sub-domain (DHp) and a catalytic and ATP-binding subdomain (CA). The key activation event involves the rearrangement of the HAMP-DHp helical core and translation of the CA towards the acceptor histidine, which presumably results in an autokinase-competent complex. In the present work we integrate coarse-grained, all-atom, and hybrid QM-MM computer simulations to probe the large-scale conformational reorganization that takes place from the inactive to the autokinase-competent state (conformational step), and evaluate its relation to the autokinase reaction itself (chemical step). Our results highlight a tight coupling between conformational and chemical steps, underscoring the advantage of CA walking along the DHp core, to favor a reactive tautomeric state of the phospho-acceptor histidine. The results not only represent an example of multiscale modelling, but also show how protein dynamics can promote catalysis.