RESUMEN
The drug Rimantadine binds to two different sites in the M2 protein from influenza A, a peripheral site and a pore site that is the primary site of efficacy. It remained enigmatic that pore binding did not occur in certain detergent micelles, and in particular incomplete binding was observed in a mixture of lipids selected to match the viral membrane. Here we show that two effects are responsible, namely changes in the protein upon pore binding that prevented detergent solubilization, and slow binding kinetics in the lipid samples. Using 55-100 kHz magic-angle spinning NMR, we characterize kinetics of drug binding in three different lipid environments: DPhPC, DPhPC with cholesterol and viral mimetic membrane lipid bilayers. Slow pharmacological binding kinetics allowed the characterization of spectral changes associated with non-specific binding to the protein periphery in the kinetically trapped pore-apo state. Resonance assignments were determined from a set of proton-detected 3D spectra. Chemical shift changes associated with functional binding in the pore of M2 were tracked in real time in order to estimate the activation energy. The binding kinetics are affected by pH and the lipid environment and in particular cholesterol. We found that the imidazole-imidazole hydrogen bond at residue histidine 37 is a stable feature of the protein across several lipid compositions. Pore binding breaks the imidazole-imidazole hydrogen bond and limits solubilization in DHPC detergent.
RESUMEN
Small molecules that bind to oligomeric protein species such as membrane proteins and fibrils are of clinical interest for development of therapeutics and diagnostics. Definition of the binding site at atomic resolution via NMR is often challenging due to low binding stoichiometry of the small molecule. For fibrils and aggregation intermediates grown in the presence of lipids, we report atomic-resolution contacts to the small molecule at sub nm distance via solid-state NMR using dynamic nuclear polarization (DNP) and orthogonally labelled samples of the protein and the small molecule. We apply this approach to α-synuclein (αS) aggregates in complex with the small molecule anle138b, which is a clinical drug candidate for disease modifying therapy. The small central pyrazole moiety of anle138b is detected in close proximity to the protein backbone and differences in the contacts between fibrils and early intermediates are observed. For intermediate species, the 100 K condition for DNP helps to preserve the aggregation state, while for both fibrils and oligomers, the DNP enhancement is essential to obtain sufficient sensitivity.
Asunto(s)
Pirazoles , alfa-Sinucleína , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Pirazoles/química , Benzodioxoles/química , Espectroscopía de Resonancia Magnética , Agregado de ProteínasRESUMEN
The arrangement of histidine side chains in influenza A M2 tetramer determines their pKa values, which define pH-controlled proton conduction critical to the virus lifecycle. Both water-associated and hydrogen-bonded imidazole-imidazolium histidine quaternary structures have been proposed, based on crystal structures and NMR chemical shifts, respectively. Here we show, using the conduction domain construct of M2 in lipid bilayers, that the imidazole rings are hydrogen bonded even at a pH of 7.8 in the neutral charge state. An intermolecular 8.9 ± 0.3 Hz 2hJNN hydrogen bond is observed between H37 Nε and Nδ recorded in a fully protonated sample with 100 kHz magic-angle spinning. This interaction could not be detected in the drug-bound sample.
Asunto(s)
Histidina/química , Concentración de Iones de Hidrógeno , Imidazoles/química , Proteínas de la Matriz Viral/química , Enlace de HidrógenoRESUMEN
Bactofilins are a recently discovered class of cytoskeletal proteins of which no atomic-resolution structure has been reported thus far. The bacterial cytoskeleton plays an essential role in a wide range of processes, including morphogenesis, cell division, and motility. Among the cytoskeletal proteins, the bactofilins are bacteria-specific and do not have a eukaryotic counterpart. The bactofilin BacA of the species Caulobacter crescentus is not amenable to study by x-ray crystallography or solution nuclear magnetic resonance (NMR) because of its inherent noncrystallinity and insolubility. We present the atomic structure of BacA calculated from solid-state NMR-derived distance restraints. We show that the core domain of BacA forms a right-handed ß helix with six windings and a triangular hydrophobic core. The BacA structure was determined to 1.0 Å precision (heavy-atom root mean square deviation) on the basis of unambiguous restraints derived from four-dimensional (4D) HN-HN and 2D C-C NMR spectra.
RESUMEN
Structure-based drug design (SBDD) is a powerful and widely used approach to optimize affinity of drug candidates. With the recently introduced INPHARMA method, the binding mode of small molecules to their protein target can be characterized even if no spectroscopic information about the protein is known. Here, we show that the combination of the spin-diffusion-based NMR methods INPHARMA, trNOE, and STD results in an accurate scoring function for docking modes and therefore determination of protein-ligand complex structures. Applications are shown on the model system protein kinaseâ A and the drug targets glycogen phosphorylase and soluble epoxide hydrolase (sEH). Multiplexing of several ligands improves the reliability of the scoring function further. The new score allows in the case of sEH detecting two binding modes of the ligand in its binding site, which was corroborated by X-ray analysis.
Asunto(s)
Diseño de Fármacos , Ligandos , Proteínas/química , Sitios de Unión , Cristalografía por Rayos X , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Difusión , Epóxido Hidrolasas/antagonistas & inhibidores , Epóxido Hidrolasas/metabolismo , Glucógeno Fosforilasa/antagonistas & inhibidores , Glucógeno Fosforilasa/metabolismo , Espectroscopía de Resonancia Magnética , Simulación del Acoplamiento Molecular , Unión Proteica , Proteínas/metabolismoRESUMEN
Paramagnetic effects provide unique information about the structure and dynamics of biomolecules. We developed a method in which the lanthanoid tag is not directly attached to the protein of interest, but instead to a "reporter" protein, which binds and then transmits paramagnetic information to the target. The designed method allows access to a large number of paramagnetic restraints and residual dipolar couplings produced from independent molecular alignments in high-molecular-weight proteins with unknown 3D structure.