Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
1.
J Chem Inf Model ; 62(22): 5622-5633, 2022 11 28.
Artículo en Inglés | MEDLINE | ID: mdl-36351167

RESUMEN

The development of accurate transferable force fields is key to realizing the full potential of atomistic modeling in the study of biological processes such as protein-ligand binding for drug discovery. State-of-the-art transferable force fields, such as those produced by the Open Force Field Initiative, use modern software engineering and automation techniques to yield accuracy improvements. However, force field torsion parameters, which must account for many stereoelectronic and steric effects, are considered to be less transferable than other force field parameters and are therefore often targets for bespoke parametrization. Here, we present the Open Force Field QCSubmit and BespokeFit software packages that, when combined, facilitate the fitting of torsion parameters to quantum mechanical reference data at scale. We demonstrate the use of QCSubmit for simplifying the process of creating and archiving large numbers of quantum chemical calculations, by generating a dataset of 671 torsion scans for druglike fragments. We use BespokeFit to derive individual torsion parameters for each of these molecules, thereby reducing the root-mean-square error in the potential energy surface from 1.1 kcal/mol, using the original transferable force field, to 0.4 kcal/mol using the bespoke version. Furthermore, we employ the bespoke force fields to compute the relative binding free energies of a congeneric series of inhibitors of the TYK2 protein, and demonstrate further improvements in accuracy, compared to the base force field (MUE reduced from 0.560.390.77 to 0.420.280.59 kcal/mol and R2 correlation improved from 0.720.350.87 to 0.930.840.97).


Asunto(s)
Proteínas , Programas Informáticos , Ligandos , Proteínas/química , Entropía , Unión Proteica
2.
Biochim Biophys Acta Gen Subj ; 1862(4): 836-845, 2018 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-29339082

RESUMEN

BACKGROUND: Efflux pumps of the Resistance-Nodulation-cell Division superfamily confer multi-drug resistance to Gram-negative bacteria. The most-studied polyspecific transporter belonging to this class is the inner-membrane trimeric antiporter AcrB of Escherichia coli. In previous studies, a functional rotation mechanism was proposed for its functioning, according to which the three monomers undergo concerted conformational changes facilitating the extrusion of substrates. However, the molecular determinants and the energetics of this mechanism still remain unknown, so its feasibility must be proven mechanistically. METHODS: A computational protocol able to mimic the functional rotation mechanism in AcrB was developed. By using multi-bias molecular dynamics simulations we characterized the translocation of the substrate doxorubicin driven by conformational changes of the protein. In addition, we estimated for the first time the free energy profile associated to this process. RESULTS: We provided a molecular view of the process in agreement with experimental data. Moreover, we showed that the conformational changes occurring in AcrB enable the formation of a layer of structured waters on the internal surface of the transport channel. This water layer, in turn, allows for a fairly constant hydration of the substrate, facilitating its diffusion over a smooth free energy profile. CONCLUSIONS: Our findings reveal a new molecular mechanism of polyspecific transport whereby water contributes by screening potentially strong substrate-protein interactions. GENERAL SIGNIFICANCE: We provided a mechanistic understanding of a fundamental process related to multi-drug transport. Our results can help rationalizing the behavior of other polyspecific transporters and designing compounds avoiding extrusion or inhibitors of efflux pumps.


Asunto(s)
Proteínas de Escherichia coli/química , Simulación de Dinámica Molecular , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Agua/química , Transporte Biológico , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Termodinámica , Agua/metabolismo
3.
Virus Res ; 247: 61-70, 2018 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-29427597

RESUMEN

The multifunctional Ebola virus (EBOV) VP35 protein is a key determinant of virulence. VP35 is essential for EBOV replication, is a component of the viral RNA polymerase and participates in nucleocapsid formation. Furthermore, VP35 contributes to EBOV evasion of the host innate immune response by suppressing RNA silencing and blocking RIG-I like receptors' pathways that lead to type I interferon (IFN) production. VP35 homo-oligomerization has been reported to be critical for its replicative function and to increase its IFN-antagonism properties. Moreover, homo-oligomerization is mediated by a predicted coiled-coil (CC) domain located within its N-terminal region. Here we report the homo-oligomerization profile of full-length recombinant EBOV VP35 (rVP35) assessed by size-exclusion chromatography and native polyacrylamide gel electrophoresis. Based on our biochemical results and in agreement with previous experimental observations, we have built an in silico 3D model of the so-far structurally unsolved EBOV VP35 CC domain and performed self-assembly homo-oligomerization simulations by means of molecular dynamics. Our model advances the understanding of how VP35 may associate in different homo-oligomeric species, a crucial process for EBOV replication and pathogenicity.


Asunto(s)
Ebolavirus/genética , Nucleoproteínas/química , Proteínas del Núcleo Viral/química , Factores de Virulencia/química , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Ebolavirus/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Simulación de Dinámica Molecular , Proteínas de la Nucleocápside , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología Estructural de Proteína , Termodinámica , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo
4.
Res Microbiol ; 169(7-8): 384-392, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29407044

RESUMEN

The putative mechanism by which bacterial RND-type multidrug efflux pumps recognize and transport their substrates is a complex and fascinating enigma of structural biology. How a single protein can recognize a huge number of unrelated compounds and transport them through one or just a few mechanisms is an amazing feature not yet completely unveiled. The appearance of cooperativity further complicates the understanding of structure-dynamics-activity relationships in these complex machineries. Experimental techniques may have limited access to the molecular determinants and to the energetics of key processes regulating the activity of these pumps. Computer simulations are a complementary approach that can help unveil these features and inspire new experiments. Here we review recent computational studies that addressed the various molecular processes regulating the activity of RND efflux pumps.


Asunto(s)
Antibacterianos/metabolismo , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Simulación por Computador , Proteínas de Transporte de Membrana/metabolismo , Bacterias/química , Bacterias/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética
5.
J Mol Biol ; 430(9): 1368-1385, 2018 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-29530612

RESUMEN

Secondary multidrug (Mdr) transporters utilize ion concentration gradients to actively remove antibiotics and other toxic compounds from cells. The model Mdr transporter MdfA from Escherichia coli exchanges dissimilar drugs for protons. The transporter should open at the cytoplasmic side to enable access of drugs into the Mdr recognition pocket. Here we show that the cytoplasmic rim around the Mdr recognition pocket represents a previously overlooked important regulatory determinant in MdfA. We demonstrate that increasing the positive charge of the electrically asymmetric rim dramatically inhibits MdfA activity and sometimes even leads to influx of planar, positively charged compounds, resulting in drug sensitivity. Our results suggest that unlike the mutants with the electrically modified rim, the membrane-embedded wild-type MdfA exhibits a significant probability of an inward-closed conformation, which is further increased by drug binding. Since MdfA binds drugs from its inward-facing environment, these results are intriguing and raise the possibility that the transporter has a sensitive, drug-induced conformational switch, which favors an inward-closed state.


Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Mutación , Sitios de Unión , Cristalografía por Rayos X , Citoplasma/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Modelos Moleculares , Simulación del Acoplamiento Molecular , Unión Proteica , Estructura Secundaria de Proteína , Especificidad por Sustrato
6.
Essays Biochem ; 61(1): 141-156, 2017 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-28258237

RESUMEN

Antimicrobial resistance is based on the multifarious strategies that bacteria adopt to face antibiotic therapies, making it a key public health concern of our era. Among these strategies, efflux pumps (EPs) contribute significantly to increase the levels and profiles of resistance by expelling a broad range of unrelated compounds - buying time for the organisms to develop specific resistance. In Gram-negative bacteria, many of these chromosomally encoded transporters form multicomponent 'pumps' that span both inner and outer membranes and are driven energetically by a primary or secondary transporter component.One of the strategies to reinvigorate the efficacy of antimicrobials is by joint administration with EP inhibitors (EPI), which either block the substrate binding and/or hinder any of the transport-dependent steps of the pump. In this review, we provide an overview of multidrug-resistance EPs, their inhibition strategies and the relevant findings from the various computational simulation studies reported to date with respect to deciphering the mechanism of action of inhibitors with the purpose of improving their rational design.


Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Simulación por Computador , Antiinfecciosos/química , Antiinfecciosos/farmacología , Farmacorresistencia Bacteriana Múltiple , Modelos Moleculares
7.
Sci Rep ; 7(1): 8075, 2017 08 14.
Artículo en Inglés | MEDLINE | ID: mdl-28808284

RESUMEN

Resistance-Nodulation-cell Division (RND) transporters AcrB and AcrD of Escherichia coli expel a wide range of substrates out of the cell in conjunction with AcrA and TolC, contributing to the onset of bacterial multidrug resistance. Despite sharing an overall sequence identity of ~66% (similarity ~80%), these RND transporters feature distinct substrate specificity patterns whose underlying basis remains elusive. We performed exhaustive comparative analyses of the putative substrate binding pockets considering crystal structures, homology models and conformations extracted from multi-copy µs-long molecular dynamics simulations of both AcrB and AcrD. The impact of physicochemical and topographical properties (volume, shape, lipophilicity, electrostatic potential, hydration and distribution of multi-functional sites) within the pockets on their substrate specificities was quantitatively assessed. Differences in the lipophilic and electrostatic potentials among the pockets were identified. In particular, the deep pocket of AcrB showed the largest lipophilicity convincingly pointing out its possible role as a lipophilicity-based selectivity filter. Furthermore, we identified dynamic features (not inferable from sequence analysis or static structures) such as different flexibilities of specific protein loops that could potentially influence the substrate recognition and transport profile. Our findings can be valuable for drawing structure (dynamics)-activity relationship to be employed in drug design.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Farmacorresistencia Bacteriana Múltiple/fisiología , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , División Celular/fisiología , Diseño de Fármacos , Farmacorresistencia Bacteriana/fisiología , Escherichia coli/metabolismo , Simulación de Dinámica Molecular , Unión Proteica/fisiología , Conformación Proteica , Especificidad por Sustrato
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA