Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor.

Rocaglamide A (RocA) typifies a class of protein synthesis inhibitors that selectively kill aneuploid tumour cells and repress translation of specific messenger RNAs. RocA targets eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase; its messenger RNA selectivity is proposed to reflect highly structured 5' untranslated regions that depend strongly on eIF4A-mediated unwinding. However, rocaglate treatment may not phenocopy the loss of eIF4A activity, as these drugs actually increase the affinity between eIF4A and RNA. Here we show that secondary structure in 5' untranslated regions is only a minor determinant for RocA selectivity and that RocA does not repress translation by reducing eIF4A availability. Rather, in vitro and in cells, RocA specifically clamps eIF4A onto polypurine sequences in an ATP-independent manner. This artificially clamped eIF4A blocks 43S scanning, leading to premature, upstream translation initiation and reducing protein expression from transcripts bearing the RocA-eIF4A target sequence. In elucidating the mechanism of selective translation repression by this lead anti-cancer compound, we provide an example of a drug stabilizing sequence-selective RNA-protein interactions

Dynamic control of selectivity in the ubiquitination pathway revealed by an ASP to GLU substitution in an intra-molecular salt-bridge network

Ubiquitination relies on a subtle balance between selectivity and promiscuity achieved through specific interactions between ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). Here, we report how a single aspartic to glutamic acid substitution acts as a dynamic switch to tip the selectivity balance of human E2s for interaction toward E3 RING-finger domains. By combining molecular dynamic simulations, experimental yeast-two-hybrid screen of E2-E3 (RING) interactions and mutagenesis, we reveal how the dynamics of an internal salt-bridge network at the rim of the E2-E3 interaction surface controls the balance between an “open”, binding competent, and a “closed”, binding incompetent state. The molecular dynamic simulations shed light on the fine mechanism of this molecular switch and allowed us to identify its components, namely an aspartate/glutamate pair, a lysine acting as the central switch and a remote aspartate. Perturbations of single residues in this network, both inside and outside the interaction surface, are sufficient to switch the global E2 interaction selectivity as demonstrated experimentally. Taken together, our results indicate a new mechanism to control E2-E3 interaction selectivity at an atomic level, highlighting how minimal changes in amino acid side-chain affecting the dynamics of intramolecular salt-bridges can be crucial for protein-protein interactions. These findings indicate that the widely accepted sequence-structure-function paradigm should be extended to sequence-structure-dynamics-function relationship and open new possibilities for control and fine-tuning of protein interaction selectivity

The p110 delta structure: mechanisms for selectivity and potency of new PI(3)K inhibitors.

Deregulation of the phosphoinositide-3-OH kinase (PI(3)K) pathway has been implicated in numerous pathologies including cancer, diabetes, thrombosis, rheumatoid arthritis and asthma. Recently, small-molecule and ATP-competitive PI(3)K inhibitors with a wide range of selectivities have entered clinical development. In order to understand the mechanisms underlying the isoform selectivity of these inhibitors, we developed a new expression strategy that enabled us to determine to our knowledge the first crystal structure of the catalytic subunit of the class IA PI(3)K p110 delta. Structures of this enzyme in complex with a broad panel of isoform- and pan-selective class I PI(3)K inhibitors reveal that selectivity toward p110 delta can be achieved by exploiting its conformational flexibility and the sequence diversity of active site residues that do not contact ATP. We have used these observations to rationalize and synthesize highly selective inhibitors for p110 delta with greatly improved potencies

Sterol 3β-glucosyltransferase biocatalysts with a range of selectivities, including selectivity for testosterone

The main objectives of this work were to characterise a range of purified recombinant sterol 3β-glucosyltransferases and show that rational sampling of the diversity that exists within sterol 3β-glucosyltransferase sequence space can result in a range of enzyme selectivities. In our study the catalytically active domain of the Saccharomyces cerevisiae 3β-glucosyltransferase was used to mine putative sterol 3β-glucosyltransferases from the databases. Selected diverse sequences were expressed in and purified from Escherichia coli and shown to have different selectivities for the 3β-hydroxysteroids ergosterol and cholesterol. Surprisingly, three enzymes were also selective for testosterone, a 17β-hydroxysteroid. This study therefore reports for the first time sterol 3β-glucosyltransferases with selectivity for both 3β- and 17β-hydroxysteroids and is also the first report of recombinant 3β-glucosyltransferases with selectivity for steroids with a hydroxyl group at positions other than C-3. These enzymes could therefore find utility in the pharmaceutical industry for the green synthesis of a range of glycosylated compounds of medicinal interest

Selectivity of interaction of spin-labelled lipids with peripheral proteins bound to dimyristoylphosphatidylglycerol bilayers, as determined by ESR spectroscopy.

The selectivity of interaction between spin-labelled lipids and the peripheral proteins, apocytochrome c, cytochrome c, lysozyme and polylysine has been studied using ESR spectroscopy. Derivatives of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), diphosphatidylglycerol (CL) and diacylglycerol (DG) spin-labelled at the 5-C atom position of the sn-2 chain were used to study the association of these proteins with bilayers of dimyristoylphosphatidylglycero. Binding of the proteins increased the outer hyperfine splitting in the ESR spectra of the lipid spin labees to an extent which depended both on the spin-labelled lipid species involved and on the particular protein. The order of selectivity for apocytochrome c follows the sequence: PI−>CL−≈DG PS−>PC±>PG−>PE±. The selectivity pattern for cytochrome c is: PI−>PG−>CL−>DG PS−≈PC±>PE±; for lysozyme is: CL−>PG−>DG PE−>PC±PS−>PI−; and that for polylysine is: CL−>PS−⩾PG−>PI−>PC±>DG PE+-. The overall strength of interaction is in the order lysozyme>cytochrome c>apcoytochrome c, for equivalent binding, and the spread of the selectivity for the different proteins is in the reverse order. Assuming fast exchange for the ESR spectra of the 5-C atom labelled lipids, the relative association constants of the different labels with the different proteins have been estimated

Island Size Selectivity during 2D Ag Island Coarsening on Ag (111)

We report on early stages of submonolayer Ag island coarsening on Ag(111) surface at room temperature ($300$ K) carried out using realistic kinetic Monte Carlo (KMC) simulations. We find that during early stages, coarsening proceeds as a sequence of selected island sizes creating peaks and valleys in the island size distribution. We find that island-size selectivity is due to formation of kinetically stable islands for certain sizes because of adatom detachment/attachment processes and large activation barrier for kink detachment. In addition, we find that the ratio of number of adatom attachment to detachment processes to be independent of parameters of initial configuration and also on the initial shapes of the islands confirming that island-size selectivity is independent of initial conditions.These simulations were carried out using a very large database of processes identified by their local environment and whose activation barriers were calculated using the embedded-atom method

Exquisite Sequence Selectivity with Small Conditional RNAs

Dynamic RNA nanotechnology based on programmable hybridization cascades with small conditional RNAs (scRNAs) offers a promising conceptual framework for engineering programmable conditional regulation in vivo. While single-base substitution (SBS) somatic mutations and single-nucleotide polymorphisms (SNPs) are important markers and drivers of disease, it is unclear whether synthetic RNA signal transducers are sufficiently programmable to accept a cognate RNA input while rejecting single-nucleotide sequence variants. Here, we explore the limits of scRNA programmability, demonstrating isothermal, enzyme-free genotyping of RNA SBS cancer markers and SNPs using scRNAs that execute a conditional hybridization cascade in the presence of a cognate RNA target. Kinetic discrimination can be engineered on a time scale of choice from minutes to days. To discriminate even the most challenging single-nucleotide sequence variants, including those that lead to nearly isoenergetic RNA wobble pairs, competitive inhibition with an unstructured scavenger strand or with other scRNAs provides a simple and effective principle for achieving exquisite sequence selectivity

Imprinting Patterns of Neutral Atoms in an Optical Lattice using Magnetic Resonance Techniques

We prepare arbitrary patterns of neutral atoms in a one-dimensional (1D) optical lattice with single-site precision using microwave radiation in a magnetic field gradient. We give a detailed account of the current limitations and propose methods to overcome them. Our results have direct relevance for addressing of planes, strings or single atoms in higher dimensional optical lattices for quantum information processing or quantum simulations with standard methods in current experiments. Furthermore, our findings pave the way for arbitrary single qubit control with single site resolution.Comment: 9 pages, 7 figure