Identification of allosteric hotspots regulating the ribosomal RNA binding by antibiotic resistance-conferring Erm methyltransferases

Antibiotic resistance via epigenetic methylation of ribosomal RNA is one of the most prevalent strategies adopted by multidrug resistant pathogens. The erythromycin-resistance methyltransferase (Erm) methylates rRNA at the conserved A2058 position and imparts resistance to macrolides such as erythromycin. However, the precise mechanism adopted by Erm methyltransferases for locating the target base within a complicated rRNA scaffold remains unclear. Here, we show that a conserved RNA architecture, including specific bulge sites, present more than 15 Å from the reaction center, is key to methylation at the pathogenic site. Using a set of RNA sequences site-specifically labeled by fluorescent nucleotide surrogates, we show that base flipping is a prerequisite for effective methylation and that distal bases assist in the recognition and flipping at the reaction center. The Erm–RNA complex model revealed that intrinsically flipped-out bases in the RNA serve as a putative anchor point for the Erm. Molecular dynamic simulation studies demonstrated the RNA undergoes a substantial change in conformation to facilitate an effective protein–rRNA handshake. This study highlights the importance of unique architectural features exploited by RNA to impart fidelity to RNA methyltransferases via enabling allosteric crosstalk. Moreover, the distal trigger sites identified here serve as attractive hotspots for the development of combination drug therapy aimed at reversing resistance

Ultrafast Proton Transfer Reaction in Phenol–(Ammonia)n Clusters: An Ab-Initio Molecular Dynamics Investigation.

The ability of phenol to transfer the proton to surrounding ammonia molecules in a phenol-(ammonia)n cluster will depend on the relative orientation of the ammonia molecules and a critical field of about 285 MV cm-1 is essential along the O–H bond for the transfer process. Ab-initio MD simulations reveal that for a spontaneous proton transfer process, the phenol molecule must be embedded in a cluster consisting of at least eight ammonia molecules, even though several local minima with proton transferred can be observed for clusters consisting of 5-7 ammonia molecules. Further, phenol solvated in large clusters of ammonia, the proton transfer is spontaneous with the proton transfer event being instantaneous (about 20-120 fs). These simulations indicate that the rate-determining step for the proton transfer process is the reorganization of the solvent around the OH group and the proton transfer process in phenol-(ammonia)n clusters. The fluctuations in the solvent occur until a particular set of configurations projects the field in excess of critical electric field along the O–H bond which drives the proton transfer process with a respone time of about 70 fs. Further, the proton transfer process follows a curvilinear path which includes the O–H bond elongation and out-of-plane movement of the proton and can be referred to as a “Bend-to-Break” process

Molecular Origin of DNA Kinking by Transcription Factors

Binding of transcription factor (TF) proteins with DNA may cause severe kinks in the latter. Here, we investigate the molecular origin of the DNA kinks observed in the TF-DNA complexes using small molecule intercalation pathway, crystallographic analysis, and free energy calculations involving four different transcription factor (TF) protein–DNA complexes. We find that although protein binding may bend the DNA, bending alone is not sufficient to kink the DNA. We show that partial, not complete, intercalation is required to form the kink at a particular place in the DNA. It turns out that while amino acid alone can induce the desired kink through partial intercalation, protein provides thermodynamic stabilization of the kinked state in TF-DNA complexes

Urea Induced Unfolding Dynamics of Flavin Adenine Dinucleotide (FAD): Spectroscopic and Molecular Dynamics Simulation Studies from Femto-Second to Nanosecond Regime

Here, we investigate the effect of urea in the unfolding dynamics of flavin adenine dinucleotide (FAD), an important enzymatic cofactor, through steady state, time-resolved fluorescence spectroscopic and molecular dynamics (MD) simulation studies. Steady state results indicate the possibility of urea induced unfolding of FAD, inferred from increasing emission intensity of FAD with urea. The TCSPC and up-conversion results suggest that the stack–unstack dynamics of FAD severely gets affected in the presence of urea and leads to an increase in the unstack conformation population from 15% in pure water to 40% in 12 M urea. Molecular dynamics simulation was employed to understand the nature of the interaction between FAD and urea at the molecular level. Results depict that urea molecules replace many of the water molecules around adenine and isoalloxazine rings of FAD. However, the major driving force for the stability of this unstack conformations arises from the favorable stacking interaction of a significant fraction of the urea molecules with adenine and isoalloxazine rings of FAD, which overcomes the intramolecular stacking interaction between themselves observed in pure water