Controlled sulfur-based engineering confers mouldability to phosphorothioate antisense oligonucleotides.

Phosphorothioates (PS) have proven their effectiveness in the area of therapeutic oligonucleotides with applications spanning from cancer treatment to neurodegenerative disorders. Initially, PS substitution was introduced for the antisense oligonucleotides (PS ASOs) because it confers an increased nuclease resistance meanwhile ameliorates cellular uptake and in-vivo bioavailability. Thus, PS oligonucleotides have been elevated to a fundamental asset in the realm of gene silencing therapeutic methodologies. But, despite their wide use, little is known on the possibly different structural changes PS-substitutions may provoke in DNA·RNA hybrids. Additionally, scarce information and significant controversy exists on the role of phosphorothioate chirality in modulating PS properties. Here, through comprehensive computational investigations and experimental measurements, we shed light on the impact of PS chirality in DNA-based antisense oligonucleotides; how the different phosphorothioate diastereomers impact DNA topology, stability and flexibility to ultimately disclose pro-Sp S and pro-Rp S roles at the catalytic core of DNA Exonuclease and Human Ribonuclease H; two major obstacles in ASOs-based therapies. Altogether, our results provide full-atom and mechanistic insights on the structural aberrations PS-substitutions provoke and explain the origin of nuclease resistance PS-linkages confer to DNA·RNA hybrids; crucial information to improve current ASOs-based therapies.

Palladium-mediated enzyme activation suggests multiphase initiation of glycogenesis.

Biosynthesis of glycogen, the essential glucose (and hence energy) storage molecule in humans, animals and fungi1, is initiated by the glycosyltransferase enzyme, glycogenin (GYG). Deficiencies in glycogen formation cause neurodegenerative and metabolic disease2-4, and mouse knockout5 and inherited human mutations6 of GYG impair glycogen synthesis. GYG acts as a 'seed core' for the formation of the glycogen particle by catalysing its own stepwise autoglucosylation to form a covalently bound gluco-oligosaccharide chain at initiation site Tyr 195. Precise mechanistic studies have so far been prevented by an inability to access homogeneous glycoforms of this protein, which unusually acts as both catalyst and substrate. Here we show that unprecedented direct access to different, homogeneously glucosylated states of GYG can be accomplished through a palladium-mediated enzyme activation 'shunt' process using on-protein C-C bond formation. Careful mimicry of GYG intermediates recapitulates catalytic activity at distinct stages, which in turn allows discovery of triphasic kinetics and substrate plasticity in GYG's use of sugar substrates. This reveals a tolerant but 'proof-read' mechanism that underlies the precision of this metabolic process. The present demonstration of direct, chemically controlled access to intermediate states of active enzymes suggests that such ligation-dependent activation could be a powerful tool in the study of mechanism.

Brave new surfactant world revisited by thermoalkalophilic lipases: computational insights into the role of SDS as a substrate analog.

Non-ionic surfactants were shown to stabilize the active conformation of thermoalkalophilic lipases by mimicking the lipid substrate while the catalytic interactions formed by anionic surfactants have not been well characterized. In this study, we combined µs-scale molecular dynamics (MD) simulations and lipase activity assays to analyze the effect of ionic surfactant, sodium dodecyl sulfate (SDS), on the structure and activity of thermoalkalophilic lipases. Both the open and closed lipase conformations that differ in geometry were recruited to the MD analysis to provide a broader understanding of the molecular effect of SDS on the lipase structure. Simulations at 298 K showed the potential of SDS for maintaining the active lipase through binding to the sn-1 acyl-chain binding pocket in the open conformation or transforming the closed conformation to an open-like state. Consistent with MD findings, experimental analysis showed increased lipase activity upon SDS incubation at ambient temperature. Notably, the lipase cores stayed intact throughout 2 µs regardless of an increase in the simulation temperature or SDS concentration. However, the surface structures were unfolded in the presence of SDS and at elevated temperature for both conformations. Simulations of the dimeric lipase were also carried out and showed reduced flexibility of the surface structures which were unfolded in the monomer, indicating the insulating role of dimer interactions against SDS. Taken together, this study provides insights into the possible substrate mimicry by the ionic surfactant SDS for the thermoalkalophilic lipases without temperature elevation, underscoring SDS's potential for interfacial activation at ambient temperatures.

Enzyme Conformation Influences the Performance of Lipase-powered Nanomotors.

Enzyme-powered micro/nanomotors have myriads of potential applications in various areas. To efficiently reach those applications, it is necessary and critical to understand the fundamental aspects affecting the motion dynamics. Herein, we explored the impact of enzyme orientation on the performance of lipase-powered nanomotors by tuning the lipase immobilization strategies. The influence of the lipase orientation and lid conformation on substrate binding and catalysis was analyzed using molecular dynamics simulations. Besides, the motion performance indicates that the hydrophobic binding (via OTES) represents the best orienting strategy, providing 48.4 % and 95.4 % increase in diffusion coefficient compared to hydrophilic binding (via APTES) and Brownian motion (no fuel), respectively (with C[triacetin] of 100 mm). This work provides vital evidence for the importance of immobilization strategy and corresponding enzyme orientation for the catalytic activity and in turn, the motion performance of nanomotors, and is thus helpful to future applications.

Deciphering the Allosterically Driven Conformational Ensemble in Tryptophan Synthase Evolution.

Multimeric enzyme complexes are ubiquitous in nature and catalyze a broad range of useful biological transformations. They are often characterized by a tight allosteric coupling between subunits, making them highly inefficient when isolated. A good example is Tryptophan synthase (TrpS), an allosteric heterodimeric enzyme in the form of an αßßα complex that catalyzes the biosynthesis of L-tryptophan. In this study, we decipher the allosteric regulation existing in TrpS from Pyrococcus furiosus (PfTrpS), and how the allosteric conformational ensemble is recovered in laboratory-evolved stand-alone ß-subunit variants. We find that recovering the conformational ensemble of a subdomain of TrpS affecting the relative stabilities of open, partially closed, and closed conformations is a prerequisite for enhancing the catalytic efficiency of the ß-subunit in the absence of its binding partner. The distal mutations resuscitate the allosterically driven conformational regulation and alter the populations and rates of exchange between these multiple conformational states, which are essential for the multistep reaction pathway of the enzyme. Interestingly, these distal mutations can be a priori predicted by careful analysis of the conformational ensemble of the TrpS enzyme through computational methods. Our study provides the enzyme design field with a rational approach for evolving allosteric enzymes toward improved stand-alone function for biosynthetic applications.

Multivalent Ligands with Tailor-Made Anion Binding Motif as Stabilizers of Protein-Protein Interactions.

Modulation of protein-protein interactions (PPIs) is essential for understanding and tuning biologically relevant processes. Although inhibitors for PPIs are widely used, the field still lacks the targeted design of stabilizers. Here, we report unnatural stabilizers based on the combination of multivalency effects and the artificial building block guanidiniocarbonylpyrrol (GCP), an arginine mimetic. Unlike other GCP-based ligands that modulate PPIs in different protein targets, only a tetrameric design shows potent activity as stabilizer of the 14-3-3ζ/C-Raf and 14-3-3ζ/Tau complexes in the low-micromolar range. This evidences the role of multivalency for achieving higher specificity in the modulation of PPIs.

A front-face 'SNi synthase' engineered from a retaining 'double-SN2' hydrolase.

SNi-like mechanisms, which involve front-face leaving group departure and nucleophile approach, have been observed experimentally and computationally in chemical and enzymatic substitution at α-glycosyl electrophiles. Since SNi-like, SN1 and SN2 substitution pathways can be energetically comparable, engineered switching could be feasible. Here, engineering of Sulfolobus solfataricus ß-glycosidase, which originally catalyzed double SN2 substitution, changed its mode to SNi-like. Destruction of the first SN2 nucleophile through E387Y mutation created a ß-stereoselective catalyst for glycoside synthesis from activated substrates, despite lacking a nucleophile. The pH profile, kinetic and mutational analyses, mechanism-based inactivators, X-ray structure and subsequent metadynamics simulations together suggest recruitment of substrates by π-sugar interaction and reveal a quantum mechanics-molecular mechanics (QM/MM) free-energy landscape for the substitution reaction that is similar to those of natural, SNi-like glycosyltransferases. This observation of a front-face mechanism in a ß-glycosyltransfer enzyme highlights that SNi-like pathways may be engineered in catalysts with suitable environments and suggests that 'ß-SNi' mechanisms may be feasible for natural glycosyltransfer enzymes.

Hidden Conformations in Aspergillus niger Monoamine Oxidase are Key for Catalytic Efficiency.

Enzymes exist as an ensemble of conformational states, whose populations can be shifted by substrate binding, allosteric interactions, but also by introducing mutations to their sequence. Tuning the populations of the enzyme conformational states through mutation enables evolution towards novel activity. Herein, Markov state models are used to unveil hidden conformational states of monoamine oxidase from Aspergillus niger (MAO-N). These hidden conformations, not previously observed by any other technique, play a crucial role in substrate binding and enzyme activity. This reveals how distal mutations regulate MAO-N activity by stabilizing these hidden, catalytically important conformational states, but also by modulating the communication pathway between both MAO-N subunits.

A Multiperspective Approach to Solvent Regulation of Enzymatic Activity: HMG-CoA Reductase.

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase was investigated in different organic cosolvents by means of kinetic and calorimetric measurements, molecular dynamics simulations, and small-angle X-ray scattering. The combined experimental and theoretical techniques were essential to complement each other's limitations in the investigation of the complex interaction pattern between the enzyme, different solvent types, and concentrations. In this way, the underlying mechanisms for the loss of enzyme activity in different water-miscible solvents could be elucidated. These include direct inhibitory effects onto the active center and structural distortions.

Membrane Phase-Dependent Occlusion of Intramolecular GLUT1 Cavities Demonstrated by Simulations.

Experimental evidence has shown a close correlation between the composition and physical state of the membrane bilayer and glucose transport activity via the glucose transporter GLUT1. Cooling alters the membrane lipids from the fluid to gel phase, and also causes a large decrease in the net glucose transport rate. The goal of this study is to investigate how the physical phase of the membrane alters glucose transporter structural dynamics using molecular-dynamics simulations. Simulations from an initial fluid to gel phase reduce the size of the cavities and tunnels traversing the protein and connecting the external regions of the transporter and the central binding site. These effects can be ascribed solely to membrane structural changes since in silico cooling of the membrane alone, while maintaining the higher protein temperature, shows protein structural and dynamic changes very similar to those observed with uniform cooling. These results demonstrate that the protein structure is sensitive to the membrane phase, and have implications for how transmembrane protein structures respond to their physical environment.

The reaction mechanism of retaining glycosyltransferases.

The catalytic mechanism of retaining glycosyltransferases (ret-GTs) remains a controversial issue in glycobiology. By analogy to the well-established mechanism of retaining glycosidases, it was first suggested that ret-GTs follow a double-displacement mechanism. However, only family 6 GTs exhibit a putative nucleophile protein residue properly located in the active site to participate in catalysis, prompting some authors to suggest an unusual single-displacement mechanism [named as front-face or SNi (substitution nucleophilic internal)-like]. This mechanism has now received strong support, from both experiment and theory, for several GT families except family 6, for which a double-displacement reaction is predicted. In the last few years, we have uncovered the molecular mechanisms of several retaining GTs by means of quantum mechanics/molecular mechanics (QM/MM) metadynamics simulations, which we overview in the present work.

Surfactin at the Water/Air Interface and in Solution.

The lipopeptide surfactin produced by certain strains of Bacillus subtillis is a potent biosurfactant with high amphiphilicity and a strong tendency for self-aggregation. Surfactin possesses a number of valuable biological properties such as antiviral, antibacterial, antifungal, and hemolytic activities. Owing to these properties, in addition to the general advantages of biosurfactants over synthetic surfactants, surfactin has potential biotechnological and biomedical applications. Here, the aggregation properties of surfactin in solution together with its behavior at the water/air interface were studied using classical molecular dynamics simulations (MD) at three different pH values. Validation of the MD structural data was performed by comparing neutron reflectivity and volume fraction profiles computed from the simulations with their experimental counterparts. Analysis of the MD trajectories supported conclusions about the distribution, conformations, and interactions of surfactin in solution and at the water-air interface. Considering altogether, the work presented provides atomistic models for the rationalization of some of the structural and dynamic characteristics as well as the modes of action of surfactin at different pH values.

Evidence for a boat conformation at the transition state of GH76 α-1,6-mannanases--key enzymes in bacterial and fungal mannoprotein metabolism.

α-Mannosidases and α-mannanases have attracted attention for the insight they provide into nucleophilic substitution at the hindered anomeric center of α-mannosides, and the potential of mannosidase inhibitors as cellular probes and therapeutic agents. We report the conformational itinerary of the family GH76 α-mannanases studied through structural analysis of the Michaelis complex and synthesis and evaluation of novel aza/imino sugar inhibitors. A Michaelis complex in an (O) S2 conformation, coupled with distortion of an azasugar in an inhibitor complex to a high energy B2,5 conformation are rationalized through ab initio QM/MM metadynamics that show how the enzyme surface restricts the conformational landscape of the substrate, rendering the B2,5 conformation the most energetically stable on-enzyme. We conclude that GH76 enzymes perform catalysis using an itinerary that passes through (O) S2 and B2,5 (≠) conformations, information that should inspire the development of new antifungal agents.

Combined inhibitor free-energy landscape and structural analysis reports on the mannosidase conformational coordinate.

Mannosidases catalyze the hydrolysis of a diverse range of polysaccharides and glycoconjugates, and the various sequence-based mannosidase families have evolved ingenious strategies to overcome the stereoelectronic challenges of mannoside chemistry. Using a combination of computational chemistry, inhibitor design and synthesis, and X-ray crystallography of inhibitor/enzyme complexes, it is demonstrated that mannoimidazole-type inhibitors are energetically poised to report faithfully on mannosidase transition-state conformation, and provide direct evidence for the conformational itinerary used by diverse mannosidases, including ß-mannanases from families GH26 and GH113. Isofagomine-type inhibitors are poor mimics of transition-state conformation, owing to the high energy barriers that must be crossed to attain mechanistically relevant conformations, however, these sugar-shaped heterocycles allow the acquisition of ternary complexes that span the active site, thus providing valuable insight into active-site residues involved in substrate recognition.

Substrate-guided front-face reaction revealed by combined structural snapshots and metadynamics for the polypeptide N-acetylgalactosaminyltransferase†2.

The retaining glycosyltransferase GalNAc-T2 is a member of a large family of human polypeptide GalNAc-transferases that is responsible for the post-translational modification of many cell-surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum-mechanics/molecular-mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the atomic-electronic level of detail. Our study provides a detailed structural rationale for an ordered bi-bi kinetic mechanism and reveals critical aspects of substrate recognition, which dictate the specificity for acceptor Thr versus Ser residues and enforce a front-face SN i-type reaction in which the substrate N-acetyl sugar substituent coordinates efficient glycosyl transfer.

Nucleic acids in modern molecular therapies: A realm of opportunities for strategic drug design.

RNA vaccines have made evident to society what was already known by the scientific community: nucleic acids will be the "drugs of the future." By modifying the genome, interfering in transcription or translation, and by introducing new catalysts into the cell or by mimicking antibody effects, nucleic acids can generate therapeutic activities that are not accessible by any other therapeutic agents. There are, however, challenges that need to be solved in the next few years to make nucleic acids usable in a wide range of therapeutic scenarios. This review illustrates how simulation methods can help achieve this goal.

Structural analysis and insights into the glycon specificity of the rice GH1 Os7BGlu26 ß-D-mannosidase.

Rice Os7BGlu26 is a GH1 family glycoside hydrolase with a threefold higher kcat/Km value for 4-nitrophenyl ß-D-mannoside (4NPMan) compared with 4-nitrophenyl ß-D-glucoside (4NPGlc). To investigate its selectivity for ß-D-mannoside and ß-D-glucoside substrates, the structures of apo Os7BGlu26 at a resolution of 2.20 Šand of Os7BGlu26 with mannose at a resolution of 2.45 Šwere elucidated from isomorphous crystals in space group P212121. The (ß/α)8-barrel structure is similar to other GH1 family structures, but with a narrower active-site cleft. The Os7BGlu26 structure with D-mannose corresponds to a product complex, with ß-D-mannose in the (1)S5 skew-boat conformation. Docking of the (1)S3, (1)S5, (2)SO and (3)S1 pyranose-ring conformations of 4NPMan and 4NPGlc substrates into the active site of Os7BGlu26 indicated that the lowest energies were in the (1)S5 and (1)S3 skew-boat conformations. Comparison of these docked conformers with other rice GH1 structures revealed differences in the residues interacting with the catalytic acid/base between enzymes with and without ß-D-mannosidase activity. The mutation of Tyr134 to Trp in Os7BGlu26 resulted in similar kcat/Km values for 4NPMan and 4NPGlc, while mutation of Tyr134 to Phe resulted in a 37-fold higher kcat/Km for 4NPMan than 4NPGlc. Mutation of Cys182 to Thr decreased both the activity and the selectivity for ß-D-mannoside. It was concluded that interactions with the catalytic acid/base play a significant role in glycon selection.

MEP pathway products allosterically promote monomerization of deoxy-D-xylulose-5-phosphate synthase to feedback-regulate their supply.

Isoprenoids are a very large and diverse family of metabolites required by all living organisms. All isoprenoids derive from the double-bond isomers isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are produced by the methylerythritol 4-phosphate (MEP) pathway in bacteria and plant plastids. It has been reported that IPP and DMAPP feedback-regulate the activity of deoxyxylulose 5-phosphate synthase (DXS), a dimeric enzyme that catalyzes the main flux-controlling step of the MEP pathway. Here we provide experimental insights into the underlying mechanism. Isothermal titration calorimetry and dynamic light scattering approaches showed that IPP and DMAPP can allosterically bind to DXS in vitro, causing a size shift. In silico ligand binding site analysis and docking calculations identified a potential allosteric site in the contact region between the two monomers of the active DXS dimer. Modulation of IPP and DMAPP contents in vivo followed by immunoblot analyses confirmed that high IPP/DMAPP levels resulted in monomerization and eventual aggregation of the enzyme in bacterial and plant cells. Loss of the enzymatically active dimeric conformation allows a fast and reversible reduction of DXS activity in response to a sudden increase or decrease in IPP/DMAPP supply, whereas aggregation and subsequent removal of monomers that would otherwise be available for dimerization appears to be a more drastic response in the case of persistent IPP/DMAPP overabundance (e.g., by a blockage in their conversion to downstream isoprenoids). Our results represent an important step toward understanding the regulation of the MEP pathway and rational design of biotechnological endeavors aimed at increasing isoprenoid contents in microbial and plant systems.

Structural basis of a redox-dependent conformational switch that regulates the stress kinase p38α.

Many functional aspects of the protein kinase p38α have been illustrated by more than three hundred structures determined in the presence of reducing agents. These structures correspond to free forms and complexes with activators, substrates, and inhibitors. Here we report the conformation of an oxidized state with an intramolecular disulfide bond between Cys119 and Cys162 that is conserved in vertebrates. The structure of the oxidized state does not affect the conformation of the catalytic site, but alters the docking groove by partially unwinding and displacing the short αD helix due to the movement of Cys119 towards Cys162. The transition between oxidized and reduced conformations provides a mechanism for fine-tuning p38α activity as a function of redox conditions, beyond its activation loop phosphorylation. Moreover, the conformational equilibrium between these redox forms reveals an unexplored cleft for p38α inhibitor design that we describe in detail.

Designing Efficient Enzymes: Eight Predicted Mutations Convert a Hydroxynitrile Lyase into an Efficient Esterase.

Hydroxynitrile lyase from rubber tree (HbHNL) shares 45% identical amino acid residues with the homologous esterase from tobacco, SABP2, but the two enzymes catalyze different reactions. The x-ray structures reveal a serine-histidine-aspartate catalytic triad in both enzymes along with several differing amino acid residues within the active site. Previous exchange of three amino acid residues in the active site of HbHNL with the corresponding amino acid residue in SABP2 (T11G-E79H-K236M) created variant HNL3, which showed low esterase activity toward p-nitrophenyl acetate. Further structure comparison reveals additional differences surrounding the active site. HbHNL contains an improperly positioned oxyanion hole residue and differing solvation of the catalytic aspartate. We hypothesized that correcting these structural differences would impart good esterase activity on the corresponding HbHNL variant. To predict the amino acid substitutions needed to correct the structure, we calculated shortest path maps for both HbHNL and SABP2, which reveal correlated movements of amino acids in the two enzymes. Replacing four amino acid residues (C81L-N104T-V106F-G176S) whose movements are connected to the movements of the catalytic residues yielded variant HNL7TV (stabilizing substitution H103V was also added), which showed an esterase catalytic efficiency comparable to that of SABP2. The x-ray structure of an intermediate variant, HNL6V, showed an altered solvation of the catalytic aspartate and a partially corrected oxyanion hole. This dramatic increase in catalytic efficiency demonstrates the ability of shortest path maps to predict which residues outside the active site contribute to catalytic activity.