No abstract

N6A methylation is the most abundant RNA modification occurring within messenger RNA. Impairment of methylase or demethylase functions are associated with severe phenotypes and diseases in several organisms. Beside writer and eraser enzymes of this dynamic RNA epigenetic modification, reader proteins that recognize this modification are involved in numerous cellular processes. Although the precise characterization of these reader proteins remains unknown, preliminary data showed that most potential reader proteins contained a conserved YT521-B homology (YTH) domain. Here we define the YTH domain of rat YT521-B as a N6-methylated adenosine reader domain and report its solution structure in complex with a N6-methylated RNA. The structure reveals a binding preference for NGANNN RNA hexamer and a deep hydrophobic cleft for m6A recognition. These findings establish a molecular function for YTH domains as m6A reader domains and should guide further studies into the biological functions of YTH-containing proteins in m6A recognition.

Cyclization and selected backbone N-methylations are found to be often necessary but not sufficient conditions for peptidic drugs to have a good bioavailability. Thus, the design of cyclic peptides with good passive membrane permeability and good solubility remains a challenge. The backbone scaffold of a recently published series of cyclic decapeptides with six selected backbone N-methylations was designed to favor the adoption of a closed conformation with β-turns and four transannular hydrogen bonds. Although this conformation was indeed adopted by the peptides as determined by NMR measurements, substantial differences in the membrane permeability were observed. In this work, we aim to rationalize the impact of discrete side chain modifications on membrane permeability for six of these cyclic decapeptides. The thermodynamic and kinetic properties were investigated using molecular dynamics simulations and Markov state modeling in water and chloroform. The study highlights the influence that side-chain modifications can have on the backbone conformation. Peptides with a d-proline in the β-turns were more likely to adopt, even in water, the closed conformation with transannular hydrogen bonds, which facilitates transition through the membrane. The population of the closed conformation in water was found to correlate positively with PAMPA log P e.

Cyclic peptides have regained interest as potential inhibitors of challenging targets but have often a low bioavailability. The natural product cyclosporine A (CsA) is the textbook exception. Despite its size and polar backbone, it is able to passively cross membranes. This ability is hypothesized to be due to a conformational change from the low-energy conformation in water to a "congruent" conformation that is populated both in water and inside the membrane. Here, we use a combination of NMR measurements and kinetic models based on molecular dynamics simulations to rationalize the difference in the membrane permeability of cyclosporine E (CsE) and CsA. The structure of CsE differs only in a backbone methylation, but its membrane permeability is one order of magnitude lower. The most striking difference is found in the interconversion rates between the conformational states favored in water and in chloroform, which are up to one order of magnitude slower for CsE compared to CsA.

RNA recognition motif (RRM) proteins represent an abundant class of proteins playing key roles in RNA biology. We present a joint atomistic molecular dynamics (MD) and experimental study of two RRM-containing proteins bound with their single-stranded target RNAs, namely the Fox-1 and SRSF1 complexes. The simulations are used in conjunction with NMR spectroscopy to interpret and expand the available structural data. We accumulate more than 50 μs of simulations and show that the MD method is robust enough to reliably describe the structural dynamics of the RRM–RNA complexes. The simulations predict unanticipated specific participation of Arg142 at the protein–RNA interface of the SRFS1 complex, which is subsequently confirmed by NMR and ITC measurements. Several segments of the protein–RNA interface may involve competition between dynamical local substates rather than firmly formed interactions, which is indirectly consistent with the primary NMR data. We demonstrate that the simulations can be used to interpret the NMR atomistic models and can provide qualified predictions. Finally, we propose a protocol for ‘MD-adapted structure ensemble’ as a way to integrate the simulation predictions and expand upon the deposited NMR structures. Unbiased μs-scale atomistic MD could become a technique routinely complementing the NMR measurements of protein–RNA complexes.