RESUMEN
Gramicidin A, a topical antibiotic made from alternating L and D amino acids, is characterized by its wide central pore; upon insertion into membranes, it forms channels that disrupts ion gradients. We present helical peptidomimetics with this characteristic wide central pore that have been designed to mimic gramicidin A channels. Mimetics were designed using molecular modeling focused on oligomers of heterochiral dipeptides of proline analogs, in particular azaproline (AzPro). Molecular Dynamics simulations in water confirmed the stability of the designed helices. A sixteen-residue Formyl-(AzPro-Pro)8-NHCH2CH2OH helix was synthesized as well as a full thirty-two residue Cbz-(AzPro-Pro)16-OtBu channels. No liposomal lysis activity was observed suggesting lack of channel formation, possibly due to inappropriate hydrogen-bonding interactions in the membrane. These peptidomimetics also did not hemolyze red blood cells, unlike gramicidin A.
Asunto(s)
Antibacterianos/síntesis química , Diseño de Fármacos , Hemólisis/efectos de los fármacos , Peptidomiméticos/síntesis química , Antibacterianos/farmacología , Dipéptidos/química , Modelos Moleculares , Simulación de Dinámica Molecular , Peptidomiméticos/química , Peptidomiméticos/farmacología , Prolina/análogos & derivados , Prolina/química , Estructura Secundaria de ProteínaRESUMEN
Allosteric drugs, which bind to proteins in regions other than their main ligand-binding or active sites, make it possible to target proteins considered "undruggable" and to develop new therapies that circumvent existing resistance. Despite growing interest in allosteric drug discovery, rational design is limited by a lack of sufficient structural information about alternative binding sites in proteins. Previously, we used Markov State Models (MSMs) to identify such "cryptic pockets," and here we describe a method for identifying compounds that bind in these cryptic pockets and modulate enzyme activity. Experimental tests validate our approach by revealing both an inhibitor and two activators of TEM ß-lactamase (TEM). To identify hits, a library of compounds is first virtually screened against either the crystal structure of a known cryptic pocket or an ensemble of structures containing the same cryptic pocket that is extracted from an MSM. Hit compounds are then screened experimentally and characterized kinetically in individual assays. We identify three hits, one inhibitor and two activators, demonstrating that screening for binding to allosteric sites can result in both positive and negative modulation. The hit compounds have modest effects on TEM activity, but all have higher affinities than previously identified inhibitors, which bind the same cryptic pocket but were found, by chance, via a computational screen targeting the active site. Site-directed mutagenesis of key contact residues predicted by the docking models is used to confirm that the compounds bind in the cryptic pocket as intended. Because hit compounds are identified from docking against both the crystal structure and structures from the MSM, this platform should prove suitable for many proteins, particularly targets whose crystal structures lack obvious druggable pockets, and for identifying both inhibitory and activating small-molecule modulators.
Asunto(s)
Proteínas/metabolismo , Bibliotecas de Moléculas Pequeñas , Regulación Alostérica , Simulación del Acoplamiento Molecular , Unión Proteica , beta-Lactamasas/metabolismoRESUMEN
Relative to the apolipoprotein E (apoE) E3 allele of the APOE gene, apoE4 strongly increases the risk for the development of late-onset Alzheimer's disease. However, apoE4 differs from apoE3 by only a single amino acid at position 112, which is arginine in apoE4 and cysteine in apoE3. It remains unclear why apoE3 and apoE4 are functionally different. Described here is a proposal for understanding the functional differences between these two isoforms with respect to lipid binding. A mechanism is proposed that is based on the full-length monomeric structure of the protein, on hydrogen-deuterium exchange mass spectrometry data, and on the role of intrinsically disordered regions to control protein motions. It is proposed that lipid binds between the N-terminal and C-terminal domains and that separation of the two domains, along with the presence of intrinsically disordered regions, controls this process. The mechanism explains why apoE3 differs from apoE4 with respect to different lipid-binding specificities, why lipid increases the binding of apoE to its receptor, and why specific residues are conserved.
Asunto(s)
Apolipoproteína E3/química , Apolipoproteína E3/metabolismo , Apolipoproteína E4/química , Apolipoproteína E4/metabolismo , Metabolismo de los Lípidos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Apolipoproteína E3/genética , Apolipoproteína E4/genética , Sitios de Unión/genética , Fenómenos Biofísicos , Secuencia Conservada , Medición de Intercambio de Deuterio , Humanos , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Espectrometría de Masas , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
The physical organization of DNA enzymes at a replication fork enables efficient copying of two antiparallel DNA strands, yet dynamic protein interactions within the replication complex complicate replisome structural studies. We employed a combination of crystallographic, native mass spectrometry and small-angle X-ray scattering experiments to capture alternative structures of a model replication system encoded by bacteriophage T7. Two molecules of DNA polymerase bind the ring-shaped primase-helicase in a conserved orientation and provide structural insight into how the acidic C-terminal tail of the primase-helicase contacts the DNA polymerase to facilitate loading of the polymerase onto DNA. A third DNA polymerase binds the ring in an offset manner that may enable polymerase exchange during replication. Alternative polymerase binding modes are also detected by small-angle X-ray scattering with DNA substrates present. Our collective results unveil complex motions within T7 replisome higher-order structures that are underpinned by multivalent protein-protein interactions with functional implications.
Asunto(s)
Bacteriófago T7/enzimología , ADN Primasa/química , ADN Primasa/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Bacteriófago T7/química , Sitios de Unión , Cristalografía por Rayos X , ADN Viral/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
TEM ß-lactamase confers bacteria with resistance to many antibiotics and rapidly evolves activity against new drugs. However, functional changes are not easily explained by differences in crystal structures. We employ Markov state models to identify hidden conformations and explore their role in determining TEM's specificity. We integrate these models with existing drug-design tools to create a new technique, called Boltzmann docking, which better predicts TEM specificity by accounting for conformational heterogeneity. Using our MSMs, we identify hidden states whose populations correlate with activity against cefotaxime. To experimentally detect our predicted hidden states, we use rapid mass spectrometric footprinting and confirm our models' prediction that increased cefotaxime activity correlates with reduced Ω-loop flexibility. Finally, we design novel variants to stabilize the hidden cefotaximase states, and find their populations predict activity against cefotaxime in vitro and in vivo. Therefore, we expect this framework to have numerous applications in drug and protein design.
Asunto(s)
Antibacterianos/química , Farmacorresistencia Bacteriana , Escherichia coli/enzimología , beta-Lactamasas/química , Algoritmos , Cefotaxima/química , Cristalografía por Rayos X , Diseño de Fármacos , Cinética , Cadenas de Markov , Espectrometría de Masas , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Conformación Proteica , Sensibilidad y Especificidad , SolventesRESUMEN
Reversible post-translational modification by poly(ADP-ribose) (PAR) regulates chromatin structure, DNA repair and cell fate in response to genotoxic stress. PAR glycohydrolase (PARG) removes PAR chains from poly ADP-ribosylated proteins to restore protein function and release oligo(ADP-ribose) chains to signal damage. Here we report crystal structures of mammalian PARG and its complex with a substrate mimic that reveal an open substrate-binding site and a unique 'tyrosine clasp' enabling endoglycosidic cleavage of branched PAR chains.
Asunto(s)
Glicósido Hidrolasas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Simulación por Computador , Cristalografía por Rayos X , Glicósido Hidrolasas/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Ratas , Alineación de SecuenciaRESUMEN
Examples of reverse turns as recognition motifs in biological systems can be found in high-resolution crystal structures of antibody-peptide complexes. Development of peptidomimetics is often based on replacing the amide backbone of peptides by sugar rings, steroids, benzodiazepines, or other hetero- and carbocycles. In this approach, the chemical scaffold of the peptide backbone can be replaced while retaining activity as long as the pharmacophoric groups of the peptide side chains stay in relatively the same place; in other words, similar functional groups must overlap in space for interaction with critical receptor sites. This study evaluates the potential of metal complexes of chiral pentaazacrowns (PAC) derived by reduction of cyclic pentapeptides as beta-turn mimetics. Due to the limited flexibility of the pendant chiral side groups in these metal complexes, one can potentially elicit information about the receptor-bound conformation from their binding affinities. 11 PAC crystal structures with different substitution patterns complexed with 3 different metals (Mn, Fe, Cd) as a prototypical database of potential side-chain orientations. Complexation with different metals induces subtle differences in the conformations of a particular azacrown scaffold. The lack of parameterization of transition metals for force field calculations precludes a thorough theoretical study. Thus, this study utilizes a simple geometrical comparison between the experimental data for crystalline PAC complexes and the side-chain orientations seen in classic beta-turns. The FOUNDATION program was used to overlap the Calpha-Cbeta vectors of the corresponding ideal beta-turn side-chains to all possible leaving groups of the PAC complexes. When comparing the relative orientations of the chiral side chains, a strong overlap of the bonds (between about 0.1 A to about 0.5 A RMS for 3 residues and up to about 1 A RMS for 4 residues) was observed for many of the molecules. Such metal complexes may lack complete peptidomimetic activity due to the lack of spatial overlap of all four side-chain residues, however, if only three peptide side chains are needed for receptor recognition and/or binding, the metal complexes should show biological activity.