RESUMEN
In optimal cases, bivalent ligands can bind with exceptionally high affinity to their protein targets. However, designing optimised linkers, that orient the two binding groups perfectly, is challenging, and yet crucial in both fragment-based ligand design and in the discovery of bisubstrate enzyme inhibitors. To further our understanding of linker design, a series of novel bivalent S-adenosylmethionine (SAM) analogues were designed with the aim of interacting with the MetJ dimer in a bivalent sense (1:1 ligand/MetJ dimer). A range of ligands was synthesised and analyzed for ability to promote binding of the Escherichia coli methionine repressor, MetJ, to its operator DNA. Binding of bivalent SAM analogues to the MetJ homodimer in the presence of operator DNA was evaluated by fluorescence anisotropy and the effect of linker length and structure was investigated. The most effective bivalent ligand identified had a flexible linker, and promoted the DNA-protein interaction at 21-times lower concentration than the corresponding monovalent control compound.
Asunto(s)
S-Adenosilmetionina/química , Anisotropía , Proteínas Bacterianas/química , Química Farmacéutica/métodos , ADN/química , Dimerización , Diseño de Fármacos , Escherichia coli/metabolismo , Ligandos , Modelos Químicos , Conformación Molecular , Unión Proteica , Proteínas Represoras/química , S-Adenosilmetionina/análogos & derivados , Espectrometría de Fluorescencia/métodosRESUMEN
ToxR-based transcriptional reporter assays allow the strength of transmembrane helix interactions in biological membranes to be measured. Previously, these assays have only been used to study single-pass transmembrane systems. To facilitate investigation of polytopic transmembrane domain (TMD) oligomerization, we applied the ToxR methodology to the study of multi-pass TMD oligomerization to give 'Multi-Tox'. Association propensities of the viral oncoprotein, latent membrane protein-1 (LMP-1), and the E. coli membrane-integral diacylglycerol kinase (DAGK) were studied by Multi-Tox, highlighting residues of particular mechanistic importance. Both homo- and hetero-oligomerizations were studied.
Asunto(s)
Proteínas Bacterianas/genética , Biopolímeros/metabolismo , Proteínas de Unión al ADN/genética , Genes Reporteros , Proteínas de la Membrana/metabolismo , Factores de Transcripción/genética , Transcripción Genética , Diacilglicerol Quinasa/metabolismo , Escherichia coli/genéticaRESUMEN
The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven. In many cases membrane-spanning proteins have evolved highly sophisticated mechanisms of action. One way in which membrane proteins can modulate their structures and functions is by direct and specific contact of hydrophobic helices, forming structured transmembrane oligomers. Much recent work has focused on the distribution of amino acids preferentially found in the membrane environment in comparison to aqueous solution and the different intermolecular forces that drive protein association. Nevertheless, studies of molecular recognition at the transmembrane domain of proteins still lags behind those of water-soluble regions. A major hurdle remains: despite the remarkable specificity and affinity that transmembrane oligomerization can achieve, direct measurement of their association is challenging. Traditional methodologies applied to the study of integral membrane protein function can be hampered by the inherent insolubility of the sequences under examination. Biophysical insights gained from studying synthetic peptides representing transmembrane domains can provide useful structural insight. However, the biological relevance of the detergent micellar or liposome systems used in these studies to mimic cellular membranes is often questioned; do peptides adopt a native-like structure under these conditions and does their functional behaviour truly reflect the mode of action within a native membrane? In order to study the interactions of transmembrane sequences in natural phospholipid bilayers, the Langosch lab developed ToxR transcriptional reporter assays. The transmembrane domain of interest is expressed as a chimeric protein with maltose binding protein for location to the periplasm and ToxR to provide a report of the level of oligomerization (Figure 1). In the last decade, several other groups (e.g. Engelman, DeGrado, Shai) further optimized and applied this ToxR reporter assay. The various ToxR assays have become a gold standard to test protein-protein interactions in cell membranes. We herein demonstrate a typical experimental operation conducted in our laboratory that primarily follows protocols developed by Langosch. This generally applicable method is useful for the analysis of transmembrane domain self-association in E. coli, where ß-galactosidase production is used to assess the TMD oligomerization propensity. Upon TMD-induced dimerization, ToxR binds to the ctx promoter causing up-regulation of the LacZ gene for ß-galactosidase. A colorimetric readout is obtained by addition of ONPG to lyzed cells. Hydrolytic cleavage of ONPG by ß-galactosidase results in the production of the light absorbing species o-nitrophenolate (ONP) (Figure 2).
Asunto(s)
Proteínas Bacterianas/análisis , Colorimetría/métodos , Proteínas de Unión al ADN/análisis , Proteínas de la Membrana/análisis , Factores de Transcripción/análisis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/enzimología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Oligonucleótidos/química , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Estructura Terciaria de Proteína , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/metabolismoRESUMEN
Epstein-Barr virus (EBV), a human γ-herpesvirus, establishes lifelong infection by targeting the adaptive immune system of the host through memory B cells. Although normally benign, EBV contributes to lymphoid malignancies and lymphoproliferative syndromes in immunocompromised individuals. The viral oncoprotein latent membrane protein 1 (LMP-1) is essential for B lymphocyte immortalization by EBV. The constitutive signaling activity of LMP-1 is dependent on homo-oligomerization of its six-spanning hydrophobic transmembrane domain (TMD). However, the mechanism driving LMP-1 intermolecular interaction is poorly understood. Here, we show that the fifth transmembrane helix (TM5) of LMP-1 strongly self-associates, forming a homotrimeric complex mediated by a polar residue embedded in the membrane, D150. Replacement of this aspartic acid residue with alanine disrupts TM5 self-association in detergent micelles and bacterial cell membranes. A full-length LMP-1 variant harboring the D150A substitution is deficient in NFκB activation, supporting the key role of the fifth transmembrane helix in constitutive activation of signaling by this oncoprotein.
Asunto(s)
Biopolímeros/metabolismo , Péptidos/metabolismo , Proteínas de la Matriz Viral/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Western Blotting , Dicroismo Circular , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutación Puntual , Homología de Secuencia de Aminoácido , Transducción de Señal , Ultracentrifugación , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genéticaRESUMEN
Increasing numbers of target protein structures available for computational studies makes the structure-based screening paradigm more attractive for initial hit indentification. We have developed a novel in silico screening methodology incorporating Molecular Mechanics (MM)/implicit solvent methods to evaluate binding free energies and applied this technology to the identification of inhibitors of the TLR4/MD-2 interaction.
Asunto(s)
Diseño de Fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Antígeno 96 de los Linfocitos/antagonistas & inhibidores , Antígeno 96 de los Linfocitos/metabolismo , Mapeo de Interacción de Proteínas/métodos , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/metabolismo , Animales , Línea Celular , Humanos , Lipopolisacáridos/inmunología , Antígeno 96 de los Linfocitos/química , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Receptor Toll-Like 4/química , Receptor Toll-Like 4/inmunologíaRESUMEN
The efficient synthesis of a range of stable SAM mimetics, and their ability to promote the binding of the E. coli methionine repressor (MetJ) to its operator DNA, is described.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN/metabolismo , Proteínas Represoras/metabolismo , S-Adenosilmetionina/análogos & derivados , Proteínas de Escherichia coli , Ligandos , Unión Proteica/efectos de los fármacos , S-Adenosilmetionina/síntesis químicaRESUMEN
The bisindolylmaleimides are selective protein kinase inhibitors that can adopt two limiting diastereomeric (syn and anti) conformations. The configurational stability of a range of substituted and macrocyclic bisindolylmaleimides was investigated by using appropriate techniques. With unconstrained bisindolylmaleimides, the size of the 2-indolyl substituents was found to affect configurational stability, though not sufficiently to allow atropisomeric bisindolylmaleimides to be obtained. However, with a tether between the two indole nitrogen atoms in place, the steric effect of 2-indolyl substituents was greatly exaggerated, leading to large differences in configurational stability. The rate of interconversion of the syn and anti conformers varied by over twenty orders of magnitude through substitution of a bisindolylmaleimide ring system, which was constrained within a macrocyclic ring. Indeed, the first examples of configurationally stable atropisomeric bisindolylmaleimides are reported; the half-life for epimerisation of these compounds at room temperature was estimated to be >10(7) years.