No abstract

Lassa virus protects its viral genome through the formation of a ribonucleoprotein complex in which the nucleoprotein (NP) encapsidates the single-stranded RNA genome. Crystal structures provide evidence that a conformational change must occur to allow for RNA binding. In this study, the mechanism by which NP binds to RNA and how the conformational changes in NP are achieved was investigated with molecular-dynamics simulations. NP was structurally characterized in an open configuration when bound to RNA and in a closed form in the absence of RNA. Our results show that when NP is bound to RNA, the protein is highly dynamic and the system undergoes spontaneous deviations away from the open-state configuration. The equilibrium simulations are supported by free-energy calculations that quantify the influence of RNA on the free-energy surface, which governs NP dynamics. We predict that the globally stable states are qualitatively in agreement with the observed crystal structures, but that both open and closed conformations are thermally accessible in the presence of RNA. The free-energy calculations also provide a prediction of the location of the transition state for RNA binding and identify an intermediate metastable state that exhibits correlated motions that could promote RNA binding.

Flock House virus (FHV) is a well-characterized model system to study infection mechanisms in non-enveloped viruses. A key stage of the infection cycle is the disruption of the endosomal membrane by a component of the FHV capsid, the membrane active γ peptide. In this study, we perform all-atom molecular dynamics simulations of the 21 N-terminal residues of the γ peptide interacting with membranes of differing compositions. We carry out umbrella sampling calculations to study the folding of the peptide to a helical state in homogenous and heterogeneous membranes consisting of neutral and anionic lipids. From the trajectory data, we evaluate folding energetics and dissect the mechanism of folding in the different membrane environments. We conclude the study by analyzing the extent of configurational sampling by performing time-lagged independent component analysis.

Many years ago, the natural secondary metabolite SF2312, produced by the actinomycete Micromonospora, was reported to display broad spectrum antibacterial properties against both Gram-positive and Gram-negative bacteria. Recent studies have revealed that SF2312, a natural phosphonic acid, functions as a potent inhibitor of human enolase. The mechanism of SF2312 inhibition of bacterial enolase and its role in bacterial growth and reproduction, however, have remained elusive. In this work, we detail a structural analysis of E. coli enolase bound to both SF2312 and its oxidized imide-form. Our studies support a model in which SF2312 acts as an analog of a high energy intermediate formed during the catalytic process. Biochemical, biophysical, computational and kinetic characterization of these compounds confirm that altering features characteristic of a putative carbanion (enolate) intermediate significantly reduces the potency of enzyme inhibition. When SF2312 is combined with fosfomycin in the presence of glucose-6 phosphate, significant synergy is observed. This suggests the two agents could be used as a potent combination, targeting distinct cellular mechanism for the treatment of bacterial infections. Together, our studies rationalize the structure-activity relationships for these phosphonates and validate enolase as a promising target for antibiotic discovery.

The enzyme phosphodiesterase 6 (PDE6) is a critical component of the visual signaling pathway, and functions to convert cGMP to GMP. The ability of PDE6 to affect cellular cGMP levels leads to deactivation of cGMP-gated ion channels in both rod and cone cells. PDE6 has been difficult to structurally characterize experimentally, though the structures of the closely related PDE5 and a PDE5/6 chimera have been determined by X-ray crystallography. In this work, we employ a computational approach to study the dynamics of the catalytic domains of PDE6, PDE5 and the PDE5/6 chimera. Through equilibrium molecular dynamics (MD) simulations we identify a region of PDE6 (α12) to be correlated to distal regions of the enzyme (H- and M-loops) which surround the catalytic center. These correlations are not observed for PDE5 and we speculate that these unique motions in PDE6 may relate to the high catalytic efficiency of PDE6 and the requirement for an endogenous inhibitory subunit (Pγ). We further investigate the ligand binding pathways and energetics by enhanced sampling simulations (metadynamics) using the inhibitor sildenafil and GMP. The energetics and pathways of ligand binding are consistent with the high efficiency of PDE6 and further implicate the α12 region as an important regulatory element for PDE6 functional dynamics.