Catalytic Mg2 Research Articles

The chromatin remodeler SMARCA5 binds to d-block metal supports: Characterization of affinities by IMAC chromatography and QM analysis.

The ISWI family protein SMARCA5 contains the ATP-binding pocket that coordinates the catalytic Mg2+ ion and water molecules for ATP hydrolysis. In this study, we demonstrate that SMARCA5 can also possess an alternative metal-binding ability. First, we isolated SMARCA5 on the cobalt column (IMAC) to near homogeneity. Examination of the interactions of SMARCA5 with metal-chelating supports showed that, apart from Co2+, it binds to Cu2+, Zn2+ and Ni2+. The efficiency of the binding to the last-listed metal was influenced by the chelating ligand, resulting in a strong preference for Ni-NTA over the Ni-CM-Asp equivalent. To gain insight in the preferential affinity for the Ni-NTA ligand, QM calculations were performed on model systems and metal-ligand complexes with a limited protein fragment of SMARCA5 containing the double-histidine (dHis) motif. The calculations correlated the observed affinity with the relative stability of the d-block metals to tetradentate ligand coordination over tridentate, as well as their overall octahedral coordination capacity. Likewise, binding free energies derived from model imidazole complexes mirrored the observed Ni-NTA/Ni-CM-Asp preferential affinity. Finally, similar calculations on complexes with a SMARCA5 peptide fragment derived from the AlphaFold structural prediction, captured almost accurately the expected relative stability of the TM complexes, and produced a large energetic separation (~10 kcal∙mol-1) between Ni-NTA and Ni-CM-Asp in favour of the former.

Human Immunodeficiency Viral Reverse Transcriptases: Analyses of the Active Sites of the Polymerase Domain and Drug-Resistant Mutants of the Reverse Transcriptases

It is well-known that human immunodeficiency viruses (HIVs) cause the chronic, potentially life-threatening condition known as human Acquired Immuno-Deficiency Syndrome (AIDS). As the lifecycle of HIVs heavily depends on the crucial enzyme, the reverse transcriptase (RT), it has been used as a potential therapeutic target to treat and control the spread of AIDS. The active site amino acids of the polymerase domain of the RTs and their anti-HIV drug-binding sites are analyzed. The catalytic region of HIV RTs and the E. coli DNA polymerase I showed very similar active site amino acids, suggesting that HIV RTs would have possibly evolved from the bacterial DNA polymerase. The catalytic proton abstractor is identified as a K and the nucleotide selection amino acid as an N. However, the regular template-binding pair –YG- is slightly modified to a –YXG- in HIV RTs. Three completely conserved Ds in HIV RTs are involved in binding to the catalytic Mg2+. The sensitive and resistant strains of HIV-1 for the HIV antiretroviral drug, azidothymidine (AZT), a nucleoside analogue of thymidine, show only a few non-isofunctional amino acid replacements and are located mainly in the palm and thumb subdomains of the RT polymerase, whereas the rest of the polymerase catalytic core and metal-binding sites are completely conserved in AZT-sensitive and -resistant HIV-1 strains. In the drug-resistant mutants of non-nucleoside RT inhibitors of HIV-2, the crucial mutations are located mainly near the -MDD- motif of the catalytic metal-binding region. Even though a large number of amino acid replacements are seen between the RTs of HIV-1 and HIV-2, the polymerase active sites are completely conserved in both. The HIV-1 and HIV-2 catalytic and metal-binding sites are completely conserved in simian immunodeficiency virus (SIV) as well. The absence of DEDD-superfamily of proofreading exonuclease domain in the HIV RTs, might cause the virus to evolve rapidly in patients. A possible mechanism of action for the HIV RTs is also proposed.

A Magnesium Binding Site And The Anomeric Effect Regulate The Abiotic Redox Chemistry Of Nicotinamide Nucleotides.

Nicotinamide adenine dinucleotide (NAD+) is a redox active molecule that is universally found in biology. Despite the importance and simplicity of this molecule, few reports exist that investigate which molecular features are important for the activity of this ribodinucleotide. By exploiting the nonenzymatic reduction and oxidation of NAD+ by pyruvate and methylene blue, respectively, we were able to identify key molecular features necessary for the intrinsic activity of NAD+ through kinetic analysis. Such features may explain how NAD+ could have been selected early during the emergence of life. Simpler molecules, such as nicotinamide, that lack an anomeric carbon are incapable of accepting electrons from pyruvate. The phosphate moiety inhibits activity in the absence of metal ions but facilitates activity at physiological pH and model prebiotic conditions by recruiting catalytic Mg2+. Reduction proceeds through consecutive single electron transfer events. Of the derivatives tested, including nicotinamide mononucleotide, nicotinamide riboside, 3-(aminocarbonyl)-1-(2,3-dihydroxypropyl)pyridinium, 1-methylnicotinamide, and nicotinamide, only NAD+ and nicotinamide mononucleotide would be capable of efficiently accepting and donating electrons within a nonenzymatic electron transport chain. The data are consistent with early metabolic chemistry exploiting NAD+ or nicotinamide mononucleotide and not simpler molecules.

Structural and biochemical characterization of the mitomycin C repair exonuclease MrfB.

Mitomycin C (MMC) repair factor A (mrfA) and factor B (mrfB), encode a conserved helicase and exonuclease that repair DNA damage in the soil-dwelling bacterium Bacillus subtilis. Here we have focused on the characterization of MrfB, a DEDDh exonuclease in the DnaQ superfamily. We solved the structure of the exonuclease core of MrfB to a resolution of 2.1 Å, in what appears to be an inactive state. In this conformation, a predicted α-helix containing the catalytic DEDDh residue Asp172 adopts a random coil, which moves Asp172 away from the active site and results in the occupancy of only one of the two catalytic Mg2+ ions. We propose that MrfB resides in this inactive state until it interacts with DNA to become activated. By comparing our structure to an AlphaFold prediction as well as other DnaQ-family structures, we located residues hypothesized to be important for exonuclease function. Using exonuclease assays we show that MrfB is a Mg2+-dependent 3'-5' DNA exonuclease. We show that Leu113 aids in coordinating the 3' end of the DNA substrate, and that a basic loop is important for substrate binding. This work provides insight into the function of a recently discovered bacterial exonuclease important for the repair of MMC-induced DNA adducts.

Catalytic Metal Ion-Substrate Coordination during Nonenzymatic RNA Primer Extension.

Nonenzymatic template-directed RNA copying requires catalysis by divalent metal ions. The primer extension reaction involves the attack of the primer 3'-hydroxyl on the adjacent phosphate of a 5'-5'-imidazolium-bridged dinucleotide substrate. However, the nature of the interaction of the catalytic metal ion with the reaction center remains unclear. To explore the coordination of the catalytic metal ion with the imidazolium-bridged dinucleotide substrate, we examined catalysis by oxophilic and thiophilic metal ions with both diastereomers of phosphorothioate-modified substrates. We show that Mg2+ and Cd2+ exhibit opposite preferences for the two phosphorothioate substrate diastereomers, indicating a stereospecific interaction of the divalent cation with one of the nonbridging phosphorus substituents. High-resolution X-ray crystal structures of the products of primer extension with phosphorothioate substrates reveal the absolute stereochemistry of this interaction and indicate that catalysis by Mg2+ involves inner-sphere coordination with the nonbridging phosphate oxygen in the pro-SP position, while thiophilic cadmium ions interact with sulfur in the same position, as in one of the two phosphorothioate substrates. These results collectively suggest that during nonenzymatic RNA primer extension with a 5'-5'-imidazolium-bridged dinucleotide substrate the interaction of the catalytic Mg2+ ion with the pro-SP oxygen of the reactive phosphate plays a crucial role in the metal-catalyzed SN2(P) reaction.

Assessing the mechanism of fast-cycling cancer-associated mutations of Rac1 small Rho GTPase.

Rho-GTPases proteins function as molecular switches alternating from an active to an inactive state upon Guanosine triphosphate (GTP) binding and hydrolysis to Guanosine diphosphate (GDP). Among them, Rac subfamily regulates cell dynamics, being overexpressed in distinct cancer types. Notably, these proteins are object of frequent cancer-associated mutations at Pro29 (P29S, P29L, and P29Q). To assess the impact of these mutations on Rac1 structure and function, we performed extensive all-atom molecular dynamics simulations on wild-type (wt) and oncogenic isoforms of this protein in GDP- and GTP-bound states. Our results unprecedentedly elucidate that P29Q/S-induced structural and dynamical perturbations of Rac1 core domain weaken the binding of the catalytic site Mg2+ ion, and reduce the GDP residence time within protein, enhancing the GDP/GTP exchange rate and Rac1 activity. This broadens our knowledge of the role of cancer-associated mutations on small GTPases mechanism supplying valuable information for future drug discovery efforts targeting specific Rac1 isoforms.

An Insight into the Active sites of the RNA Polymerase and Proofreading Exonuclease of the Human Respiratory Syncytial Virus

Human respiratory syncytial virus (hRSV) is one of the triple epidemic viruses that causes infections of the respiratory tract and lungs. For multiplication of the virus in human cells, its RNA polymerase is the crucial enzyme and it forms a part of a large protein (LP). The LP is a multicomponent and multifunctional protein harboring at least 3 different enzymes. The RNA polymerase belongs to RNA-dependent RNA polymerase (RdRp) (EC: 2.7.7.48) type and performs the synthesis of both mRNAs (transcription) and genomic RNA (gRNA) (replication). In addition to the RNA polymerase, the LP also harbours two more enzymes, viz. enzymes for cap addition and cap methylation of mRNAs. The polymerase domain of the LP is analyzed for its active site amino acids and its proofreading (PR) domain. Two polymerase active site regions and a DEDD-superfamily of 3’→5’ PR exonuclease active site domain are identified in the polymerase region. The signature metal-binding motifs, viz. -GDNQ- and –SDD- which are commonly found in the RdRps of all the (-) strand RNA viral pathogens are also found in the hRSV RNA polymerase. The two highly conserved polymerase catalytic core regions identified by sequence similarity are in close agreement with other DNA/RNA polymerases already reported and hence, proposed to function in the nucleotidyl transfer reactions. Presence of the two catalytic regions also suggest that the polymerase may function in a dual mode, one for transcription and the other one for replication, using the same invariant catalytic Mg2+-binding –GDNQ- and –SDD- motifs.

An insight into the active sites of the catalytic basic protein subunit PB1 of the RNA polymerase of human influenza viruses

RNA polymerase from human influenza viruses is a heterotrimeric enzyme and performs the crucial functions of both transcription and replication for multiplication of the viruses in human cells. The heterotrimeric enzyme is made up of two basic protein subunits (PB1 and PB2) and an acidic protein subunit (PA). All the three subunits perform well-defined function(s) in the transcription and replication processes in the human cells. The basic protein subunit PB1 is shown to possess the polymerization activity, whereas the PB2 and the PA subunits are found to be involved in the cap-snatching and proofreading (PR) activities, respectively. The polymerase activity in the catalytic subunit, PB1, is found to be an RNA-dependent RNA polymerase (RdRp). Multiple sequence alignment (MSA) analysis of the PB1 subunits from all the three human influenza viruses, A, B and C shows large number of highly conserved peptides, amino acid motifs and invariant amino acids. Site-directed mutagenesis (SDM) analysis and X-ray crystallographic data have shown that two completely conserved motifs, viz. –GDN- and –SDD-, are involved in binding to the catalytic Mg2+ ion. These data are in close agreement with the MSA analysis data of the polymerases from all the three human influenza viruses. Furthermore, two highly conserved polymerase catalytic regions are identified in the PB1 subunits by sequence similarity to other DNA/RNA polymerases and hence, are proposed to function in the nucleotidyl transfer activities. Presence of the two catalytic regions suggest that the polymerase may function in a dual mode, i.e., in phase I, in association with the cap-snatching subunit PB2, it could be involved in the synthesis of mRNAs (transcription mode) and once enough proteins are made from the mRNAs, in the second phase, in association with PR exonuclease subunit PA, it could switch to the replication mode to synthesize error-free, exact copies of the viral genome. For both the activities, it could use the same invariant catalytic Mg2+-binding –GDN- and –SDD- motifs.

Principles of target DNA cleavage and the role of Mg2+ in the catalysis of CRISPR-Cas9.

At the core of the CRISPR-Cas9 genome-editing technology, the endonuclease Cas9 introduces site-specific breaks in DNA. However, precise mechanistic information to ameliorating Cas9 function is still missing. Here, multi-microsecond molecular dynamics, free-energy and multiscale simulations are combined with solution NMR and DNA cleavage experiments to resolve the catalytic mechanism of target DNA cleavage. We show that the conformation of an active HNH nuclease is tightly dependent on the catalytic Mg2+, unveiling its cardinal structural role. This activated Mg2+-bound HNH is consistently described through molecular simulations, solution NMR and DNA cleavage assays, revealing also that the protonation state of the catalytic H840 is strongly affected by active site mutations. Finally, ab-initio QM(DFT)/MM simulations and metadynamics establish the catalytic mechanism, showing that the catalysis is activated by H840 and completed by K866, rationalising DNA cleavage experiments. This information is critical to enhance the enzymatic function of CRISPR-Cas9 toward improved genome-editing.

Aptamer-based and sensitive label-free colorimetric sensing of phenylalanine via cascaded signal amplifications

The concentration variation of phenylalanine (Phe), an essential amino acid in humans, can cause metabolism disorders and even mental disability. Sensitive and convenient monitoring of Phe is therefore important for disease diagnosis. We describe here the establishment of a new aptamer-based, sensitive and label-free colorimetric Phe detection strategy by integrating catalytic hairpin assembly (CHA) and Mg2+-dependent DNAzyme amplification cascades. The target Phe coordinates with pentamethylcyclopentadienyl rhodium(III) chloride dimer [(Cp*RhCl2)2] to form a complex that has a high affinity to the corresponding aptamer sequence. Upon its binding to the aptamers in DNA duplex probes, ssDNA strands are released to trigger subsequent CHA reactions for the formation of many DNAzymes, which cleave the substrate signal probes to liberate lots of CHA initiation strands and free G-quadruplexes to realize the cascaded amplifications. Hemin further associates with the many G-quadruplexes to yield hemin/G-quadruplex mimicking peroxidases, which catalyze solution of substrate to exhibit highly enhanced UV–vis adsorption for detecting Phe at 0.19 μM level. At the meantime, the monitoring of Phe in diluted serums with high selectivity has also been demonstrated by the developed method, indicating its potential for simple diagnosis of Phe-related diseases.

Structure of the Repurposed Fungal Terpene Cyclase FlvF Implicated in the C-N Bond-Forming Reaction of Flavunoidine Biosynthesis.

The fungal species Aspergillus flavus produces an alkaloid terpenoid, flavunoidine, through a hybrid biosynthetic pathway combining both terpene cyclase and nonribosomal peptide synthetase enzymes. Flavunoidine consists of a tetracyclic, oxygenated sesquiterpene core decorated with dimethyl cadaverine and 5,5-dimethyl-l-pipecolate moieties. Unique to the flavunoidine biosynthetic pathway is FlvF, a putative enzyme implicated in stereospecific C-N bond formation as dimethyl cadaverine is linked to the sesquiterpene core to generate pre-flavunoidine. Here, we report the 2.6 Å resolution crystal structure of FlvF, which adopts the α-helical fold of a class I terpene synthase. However, FlvF is not a terpene synthase, as indicated by its lack of enzymatic activity with farnesyl diphosphate and its lack of signature metal ion binding motifs that would coordinate to catalytic Mg2+ ions. Thus, FlvF is the first example of a protein that adopts a terpene synthase fold but is not a terpene synthase. Two Bis-Tris molecules bind in the active site of FlvF, and the binding of these ligands guided the docking of pre-flavunoidine to generate a model of the enzyme-product complex. Phylogenetic analysis of FlvF and related fungal homologues reveals conservation of residues that interact with the tetracyclic sesquiterpene in this model, but less conservation of residues interacting with the pendant amino moiety. This may hint toward the possibility that alternative amino substrates can be linked to a common sesquiterpene core by FlvF homologues to generate flavunoidine congeners, such as the phospholipase C inhibitor hispidospermidin.

Structure-based thermodynamics of ion selectivity (Mg2+versus Ca2+ and K+versus Na+) in the active site of the eukaryotic lariat group II intron from algae Pylaiella littoralis.

Group II introns are metalloenzymes that can catalyze self-splicing. Recently, the crystal structures of the eukaryotic group IIB lariat intron from the brown algae Pylaiella littoralis have been reported for two intermediate states (pre-hydrolytic (2s) and post-hydrolytic) along the self-splicing pathway. Three characteristic metal-ion binding sites (M1 and M2 sites for catalytic Mg2+ ions, and K1 site for K+) in the catalytic pocket of the lariat intron have been identified and proposed to be crucial for self-splicing. Using the X-ray structures as a template, we quantitatively estimated the energetics of divalent (Mg2+versus Ca2+) and monovalent (K+versus Na+) ion selectivity and established a direct link between the energetics and structures of this lariat intron (bound to cognate and near-cognate metal ions). Molecular dynamics (MD) free energy simulations showed that the lariat intron was strongly selective between divalent metal ions. The strength of divalent metal-ion selectivity was noticeably high in the post-hydrolytic state (ΔΔG ≈ 20 kcal mol-1) relative to its pre-hydrolytic (2s) state (ΔΔG ≈ 13 kcal mol-1). Quantum chemical calculations ensured that the sign of the estimated divalent metal-ion selectivity was correct. The M1-binding pocket was less solvent-exposed in the case of the post-hydrolytic state relative to the pre-hydrolytic (2s) state, which boosted the metal-ion selectivity of the former. Surprisingly, in contrast to the bacterial linear group II intron, the lariat intron was found to be non-selective between monovalent ions (K+versus Na+). The interaction network in the first coordination shell of Ca2+ in the M1-binding pocket was different relative to Mg2+. Mg2+ substitution by Ca2+ resulted in the substitution of a single M1-RNA interaction by the M1-water interaction. In the pre-hydrolytic (2s) state, Ca2+ substitution completely disrupted the M1⋯5'-exon interaction; thus, the nature of the divalent metal ion is critical for catalysis. The interaction network in the M2 site was independent of the nature of the divalent metal ions (Mg2+ or Ca2+). The monovalent ion was loosely bound in the wet binding pocket (K1 site) of the lariat intron; thus, the substitution of K+ by Na+ could not significantly alter the free energy of the complex. The metal ion selectivity was dependent on the solvent accessibility of the metal-ion-binding-pocket, dry pocket enhanced the selectivity.

Exploring the dNTP -binding site of HIV-1 reverse transcriptase for inhibitor design.

HIV-1 reverse transcriptase (RT) plays a central role in the viral life cycle, and roughly half of the FDA-approved anti-HIV drugs are targeting RT. Nucleoside analogs (NRTIs) require cellular phosphorylation for binding to RT, and to bypass this rate-limiting path, we designed a new series of acyclic nucleoside phosphonate analogs as nucleoside triphosphate mimics, aiming at the chelation of the catalytic Mg2+ ions via a phosphonate and/or a carboxylic acid group. Novel synthetic procedures were developed to access these nucleoside phosphonate analogs. X-ray structures in complex with HIV-1 RT/dsDNA demonstrated that their binding modes are distinct from that of our previously reported compound series. The impact of chain length, chirality and linker atom have been discussed. The detailed structural understanding of these new compounds provides opportunities for designing new class of HIV-1 RT inhibitors.

An isothermal, non-enzymatic, and dual-amplified fluorescent sensor for highly sensitive DNA detection

Abstract Sensitive DNA assays are of importance in life science and biomedical engineering, but they are heavily dependent on thermal cycling programs or enzyme-assisted schemes, which require the utilization of bulky devices and costly reagents. To circumvent such requirements, we developed an isothermal enzyme-free DNA sensing method with dual-stage signal amplification ability based on the coupling use of catalytic hairpin assembly (CHA) and Mg2+-dependent deoxyribozyme (DNAzyme). In this study, the sensing system involves a set of hairpin DNA probes for CHA (ensuring the first stage of signal amplification) as well as ribonucleobase-modified molecular beacons that serve as activatable substrates for DNAzymes (warranting the second stage of signal amplification). An experimentally determined detection limit of about 0.5 pM is achieved with a good linear range from 0.5 to 10 pM. The results from spiked fetal bovine serum samples further confirm the reliability for practical applications. The non-thermal cycling, enzyme-free, and dual-amplified features make it a powerful sensing tool for effective nucleic acid assay in a variety of biomedical applications.

An amplifying DNA circuit coupled with Mg2+-dependent DNAzyme for bisphenol A detection in milk samples

As bisphenol A (BPA) is harmful to human health, it is of great significance to develop a new method for BPA detection. Herein, we designed a BPA biosensor by integrating an amplifying DNA circuit with Mg2+-dependent DNAzyme into the sensing system. The BPA-aptamer binding activated a DNA circuit for signal amplification based on toehold-mediated strand displacement. A catalytic Mg2+-dependent DNAzyme was formed through synergistically DNA hybridization, which can cleave the dual-labeled substrate DNA into two segments. The separation of the fluorophore and quencher produces a high fluorescence response for BPA detection. This biosensor exhibited a superior sensitivity with a detection limit of 50 fM. The method is selective and robust, which can work even in milk samples with satisfactory accuracy. The biosensor analytical results were also verified by liquid chromatography coupled with mass spectrometry (LC–MS) and no obvious difference existed between the two methods.

Kinetic and thermodynamic analysis defines roles for two metal ions in DNA polymerase specificity and catalysis

Magnesium ions play a critical role in catalysis by many enzymes and contribute to the fidelity of DNA polymerases through a two-metal ion mechanism. However, specificity is a kinetic phenomenon and the roles of Mg2+ ions in each step in the catalysis have not been resolved. We first examined the roles of Mg2+ by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase. We show that Mg.dNTP binding induces an enzyme conformational change at a rate that is independent of free Mg2+ concentration. Subsequently, the second Mg2+ binds to the closed state of the enzyme–DNA–Mg.dNTP complex (Kd = 3.7 mM) to facilitate catalysis. Weak binding of the catalytic Mg2+ contributes to fidelity by sampling the correctly aligned substrate without perturbing the equilibrium for nucleotide binding at physiological Mg2+ concentrations. An increase of the Mg2+ concentration from 0.25 to 10 mM increases nucleotide specificity (kcat/Km) 12-fold largely by increasing the rate of the chemistry relative to the rate of nucleotide release. Mg2+ binds very weakly (Kd ≤ 37 mM) to the open state of the enzyme. Analysis of published crystal structures showed that HIV reverse transcriptase binds only two metal ions prior to incorporation of a correct base pair. Molecular dynamics simulations support the two-metal ion mechanism and the kinetic data indicating weak binding of the catalytic Mg2+. Molecular dynamics simulations also revealed the importance of the divalent cation cloud surrounding exposed phosphates on the DNA. These results enlighten the roles of the two metal ions in the specificity of DNA polymerases.

The Filament Forming Mechanism of SgrAI Endonuclease‐Structural and Kinetic Analysis

Filament formation by enzymes has recently emerged as a new paradigm of enzyme regulation 1.Wide‐spread reversible self‐assembly of enzymes in cells has also been shown to occur in all branches of life and diverse metabolic and biological pathways. We use the model system, SgrAI, to investigate the filament forming enzyme mechanism.Filaments formed by SgrAI have been structurally characterized using cryo‐electron microscopy 2 and the full reaction pathway including all rate constants has been determined using single turnover enzyme reactions, FRET, and global kinetic modeling 3– 4.The structural studies uncover the mechanism of activation of enzyme activity in SgrAI by filament formation, as well as the origin of the unusual expansion of substrate specificity in SgrAI within the filament. A conformational change in the SgrAI dimer, stabilized by protein‐protein and protein‐DNA contacts in the filament, leads directly to a shift in the active site, hypothesized to lead to stronger binding of a second catalytic Mg2+ ion. DNA structure and energetics is hypothesized to control substrate specificity and filament formation by SgrAI such that the secondary class of DNA sequences are cleaved by SgrAI only upon joining a filament formed by SgrAI bound to a primary class of DNA sequences. The full kinetic model showed a slow second order step in filament formation which is shown to control enzyme activity such that only invading phage DNA is cleaved, leaving the host genome untouched. This model also shows that the filament forming mechanism is superior in fast activation of enzyme activity upon initial recognition of primary DNA sequences.Using the SgrAI endonuclease as a model system to study filament forming enzymes, we have discovered that the enzyme is activated by stabilization of an activated conformation by contacts made within the filament. Control of the equilibrium between a low activity, non‐filamentous state and the high activity, filamentous state by DNA structural energetics gives rise to the unusual apparent expansion of DNA sequence specificity of SgrAI by filament formation. Simulations using the full kinetic pathway reveal an advantage in the filament mechanism over other non‐filamentous mechanisms in the speed of activation in response to invading phage DNA, giving strong evolutionary advantage to this filament forming mechanism.Support or Funding InformationThis work was supported by the National Science Foundation under Grant No. MCB‐1410355.

Establishing Trypanosoma cruzi farnesyl pyrophosphate synthase as a viable target for biosensor driven fragment-based lead discovery.

Procedures for producing and exploring Trypanosoma cruzi farnesyl pyrophosphate synthase (tcFPPS) for surface plasmon resonance (SPR) biosensor‐driven fragment‐based discovery have been established. The method requires functional sensor surfaces with high sensitivity for extended times and appropriate controls. Initial problems with protein stability and lack of useful reference compounds motivated optimization of experimental procedures and conditions. The improved methods enabled the production of pure, folded and dimeric protein, and identified procedures for storage and handling. A new coupled enzymatic assay, using luciferase for detection of pyrophosphate, was developed and used to confirm that the purified enzyme was active after purification and storage. It also confirmed that sensor surfaces prepared with structurally intact protein was active. An SPR‐biosensor assay for fragment library screening and hit confirmation was developed. A thermal shift assay was used in parallel. A library of 90 fragments was efficiently screened by both assays at a single concentration in the presence and absence of the catalytic cofactor Mg2+. Hits were selected on the basis of response levels or ΔT m > 1°C and selectivity for tcFPPS in the presence of Mg2+. Characterization of hits by SPR showed that all had low affinities and the relationships between steady‐state responses and concentrations were not sufficiently hyperbolic for determination of KD‐values. Instead, ranking could be performed from the slope of the linear relationship at low concentrations. This pilot screen confirms that the procedures developed herein enables SPR‐biosensor driven fragment‐based discovery of leads targeting tcFPPS, despite the lack of a reference compound.Significance StatementTo enable the discovery of drugs, it is essential to have access to relevant forms of the target protein and valid biochemical methods for studying the protein and effects of compounds that may be evolved into drugs. We have established methods for the discovery of drugs for treatment of American Trypanosomiasis (Chagas disease), using farnesyl pyrophosphate synthase from Trypanosoma cruzi as a target.

Factor-specific effects of mutations in the active site of RNA polymerase on RNA cleavage

Bacterial RNA polymerase (RNAP) relies on the same active site for RNA synthesis and co-transcriptional RNA proofreading. The intrinsic RNA proofreading activity of RNAP can be greatly stimulated by Gre factors, which bind within the secondary channel and directly participate in the RNA cleavage reaction in the active site of RNAP. Here, we characterize mutations in Escherichia coli RNAP that differentially affect intrinsic and Gre-stimulated RNA cleavage. Substitution of a highly conserved arginine residue that contacts nascent RNA upstream of the active site strongly impairs intrinsic and GreA-dependent cleavage, without reducing GreA affinity or catalytic Mg2+ binding. In contrast, substitutions of several nonconserved residues at the Gre-interacting interface in the secondary channel primarily affect GreB-dependent cleavage, by decreasing both the catalytic rate and GreB affinity. The results suggest that RNAP residues not directly involved in contacts with the reacting RNA groups or catalytic ions play essential roles in RNA cleavage and can modulate its regulation by transcription factors.

Escapement mechanisms: Efficient free energy transduction by reciprocally-coupled gating.

Conversion of the free energy of NTP hydrolysis efficiently into mechanical work and/or information by transducing enzymes sustains living systems far from equilibrium, and so has been of interest for many decades. Detailed molecular mechanisms, however, remain puzzling and incomplete. We previously reported that catalysis of tryptophan activation by tryptophanyl-tRNA synthetase, TrpRS, requires relative domain motion to re-position the catalytic Mg2+ ion, noting the analogy between that conditional hydrolysis of ATP and the escapement mechanism of a mechanical clock. The escapement allows the time-keeping mechanism to advance discretely, one gear at a time, if and only if the pendulum swings, thereby converting energy from the weight driving the pendulum into rotation of the hands. Coupling of catalysis to domain motion, however, mimics only half of the escapement mechanism, suggesting that domain motion may also be reciprocally coupled to catalysis, completing the escapement metaphor. Computational studies of the free energy surface restraining the domain motion later confirmed that reciprocal coupling: the catalytic domain motion is thermodynamically unfavorable unless the PPi product is released from the active site. These two conditional phenomena-demonstrated together only for the TrpRS mechanism-function as reciprocally-coupled gates. As we and others have noted, such an escapement mechanism is essential to the efficient transduction of NTP hydrolysis free energy into other useful forms of mechanical or chemical work and/or information. Some implementation of both gating mechanisms-catalysis by domain motion and domain motion by catalysis-will thus likely be found in many other systems.