Catalytic Mg2 Research Articles

Molecular simulations of the pistol ribozyme: unifying the interpretation of experimental data and establishing functional links with the hammerhead ribozyme.

The pistol ribozyme (Psr) is among the most recently discovered RNA enzymes and has been the subject of experiments aimed at elucidating the mechanism. Recent biochemical studies have revealed exciting clues about catalytic interactions in the active site not apparent from available crystallographic data. The present work unifies the interpretation of the existing body of structural and functional data on Psr by providing a dynamical model for the catalytically active state in solution from molecular simulation. Our results suggest that a catalytic Mg2+ ion makes inner-sphere contact with G33:N7 and outer-sphere coordination to the pro-RP of the scissile phosphate, promoting electrostatic stabilization of the dianionic transition state and neutralization of the developing charge of the leaving group through a metal-coordinated water molecule that is made more acidic by a hydrogen bond donated from the 2′OH of P32. This model is consistent with experimental activity-pH and mutagenesis data, including sensitivity to G33(7cG) and phosphorothioate substitution/metal ion rescue. The model suggests several experimentally testable predictions, including the response of cleavage activity to mutations at G42 and P32 positions in the ribozyme, and thio substitutions of the substrate in the presence of different divalent metal ions. Further, the model identifies striking similarities of Psr to the hammerhead ribozyme (HHr), including similar global fold, organization of secondary structure around an active site three-way junction, catalytic metal ion binding mode, and guanine general base. However, the specific binding mode and role of the Mg2+ ion, as well as a conserved 2′-OH in the active site, are interrelated but subtly different between the ribozymes.

How Does the Spliceosome Catalyze Intron Lariat Formation? Insights from Quantum Mechanics/Molecular Mechanics Free-Energy Simulations.

The spliceosome catalyzes nuclear pre-mRNA splicing via formation of an intron lariat and is arguably the most complex macromolecular machine in eukaryotic cells. Intron lariat formation is a conservative feature of the splicing reaction for both spliceosomal and group II introns. Despite the importance of the lariat formation in pre-mRNA splicing, an atomic-level understanding of the reaction mechanism remains elusive. In this work, a quantum mechanics and molecular mechanics method with thermodynamic integration is used to calculate the free-energy profile along the reaction pathway. The results demonstrate that the catalytic Mg2+ ion does not act as a Lewis acid to activate the nucleophile and the intron lariat is formed via a dissociative transition state stabilized by electrostatic interactions with two catalytic Mg2+ ions. Proton transfer occurs from the nucleophile to the leaving oxygen group through the phosphate substrate after the transition state is reached, which is important for stabilization of the intron-lariat product and the efficiency of pre-mRNA splicing. The two-metal-ion mechanism proposed in this study provides a novel insight into understanding of the splicing reaction catalyzed by the spliceosome.

Use of Cryo-EM To Uncover Structural Bases of pH Effect and Cofactor Bispecificity of Ketol-Acid Reductoisomerase.

While cryo-EM is revolutionizing structural biology, its impact on enzymology is yet to be fully demonstrated. The ketol-acid reductoisomerase (KARI) catalyzes conversion of (2 S)-acetolactate or (2 S)-aceto-2-hydroxybutyrate to 2,3-dihydroxy-3-alkylbutyrate. We found that KARI from archaea Sulfolobus solfataricus (Sso-KARI) is unusual in being a dodecamer, bispecific to NADH and NADPH, and losing activity above pH 7.8. While crystals were obtainable only at pH 8.5, cryo-EM structures were solved at pH 7.5 and 8.5 for Sso-KARI:2Mg2+. The results showed that the distances of the two catalytic Mg2+ ions are lengthened in both structures at pH 8.5. We next solved cryo-EM structures of two Sso-KARI complexes, with NADH+inhibitor and NADPH+inhibitor at pH 7.5, which indicate that the bispecificity can be attributed to a unique asparagine at the cofactor binding loop. Unexpectedly, Sso-KARI also differs from other KARI enzymes in lacking "induced-fit", reflecting structural rigidity. Thus, cryo-EM is powerful for structural and mechanistic enzymology.

Structures of ATP-bound DNA ligase D in a closed domain conformation reveal a network of amino acid and metal contacts to the ATP phosphates

DNA ligases are the sine qua non of genome integrity and essential for DNA replication and repair in all organisms. DNA ligases join 3'-OH and 5'-PO4 ends via a series of three nucleotidyl transfer steps. In step 1, ligase reacts with ATP or NAD+ to form a covalent ligase-(lysyl-Nζ)-AMP intermediate and release pyrophosphate (PPi) or nicotinamide mononucleotide. In step 2, AMP is transferred from ligase-adenylate to the 5'-PO4 DNA end to form a DNA-adenylate intermediate (AppDNA). In step 3, ligase catalyzes attack by a DNA 3'-OH on the DNA-adenylate to seal the two ends via a phosphodiester bond and release AMP. Eukaryal, archaeal, and many bacterial and viral DNA ligases are ATP-dependent. The catalytic core of ATP-dependent DNA ligases consists of an N-terminal nucleotidyltransferase domain fused to a C-terminal OB domain. Here we report crystal structures at 1.4-1.8 Å resolution of Mycobacterium tuberculosis LigD, an ATP-dependent DNA ligase dedicated to nonhomologous end joining, in complexes with ATP that highlight large movements of the OB domain (∼50 Å), from a closed conformation in the ATP complex to an open conformation in the covalent ligase-AMP intermediate. The LigD·ATP structures revealed a network of amino acid contacts to the ATP phosphates that stabilize the transition state and orient the PPi leaving group. A complex with ATP and magnesium suggested a two-metal mechanism of lysine adenylylation driven by a catalytic Mg2+ that engages the ATP α phosphate and a second metal that bridges the ATP β and γ phosphates.

Moraxella catarrhalis NucM is an entry nuclease involved in extracellular DNA and RNA degradation, cell competence and biofilm scaffolding

Moraxella catarrhalis is a host-adapted bacterial pathogen that causes otitis media and exacerbations of chronic obstructive pulmonary disease. This study characterises the conserved M. catarrhalis extracellular nuclease, a member of the ββα metal finger family of nucleases, that we have named NucM. NucM shares conserved sequence motifs from the ββα nuclease family, including the DRGH catalytic core and Mg2+ co-ordination site, but otherwise shares little primary sequence identity with other family members, such as the Serratia Nuc and pneumococcal EndA nucleases. NucM is secreted from the cell and digests linear and circular nucleic acid. However, it appears that a proportion of NucM is also associated with the cell membrane and acts as an entry nuclease, facilitating transformation of M. catarrhalis cells. This is the first example of a ββα nuclease in a Gram negative bacteria that acts as an entry nuclease. In addition to its role in competence, NucM affects cell aggregation and biofilm formation by M. catarrhalis, with ΔnucM mutants having increased biofilm biomass. NucM is likely to increase the ability of cells to survive and persist in vivo, increasing the virulence of M. catarrhalis and potentially affecting the behaviour of other pathogens that co-colonise the otorhinolaryngological niche.

Template-Directed Catalysis of a Multistep Reaction Pathway for Nonenzymatic RNA Primer Extension.

Before the advent of polymerase enzymes, the copying of genetic material during the origin of life may have involved the nonenzymatic polymerization of RNA monomers that are more reactive than the biological nucleoside triphosphates. Activated RNA monomers such as nucleotide 5′-phosphoro-2-aminoimidazolides spontaneously form an imidazolium-bridged dinucleotide intermediate that undergoes rapid nonenzymatic template-directed primer extension. However, it is unknown whether the intermediate can form on the template or only in solution and whether the intermediate is prone to hydrolysis when bound to the template or reacts preferentially with the primer. Here we show that an activated monomer can first bind the template and then form an imidazolium-bridged intermediate by reacting with a 2-aminoimidazole-activated downstream oligonucleotide. We have also characterized the partition of the template-bound intermediate between hydrolysis and primer extension. In the presence of the catalytic metal ion Mg2+, >90% of the template-bound intermediate reacts with the adjacent primer to generate the primer extension product while less than 10% reacts with competing water. Our results indicate that an RNA template can catalyze a multistep phosphodiester bond formation pathway while minimizing hydrolysis with a specificity reminiscent of an enzyme-catalyzed reaction.

Structure and Dynamics of Cas9 HNH Domain Catalytic State

The bacterial CRISPR-Cas9 immune system has been harnessed as a powerful and versatile genome-editing tool and holds immense promise for future therapeutic applications. Despite recent advances in understanding Cas9 structures and its functional mechanism, little is known about the catalytic state of the Cas9 HNH nuclease domain, and identifying how the divalent metal ions affect the HNH domain conformational transition remains elusive. A deeper understanding of Cas9 activation and its cleavage mechanism can enable further optimization of Cas9-based genome-editing specificity and efficiency. Using two distinct molecular dynamics simulation techniques, we have obtained a cross-validated catalytically active state of Cas9 HNH domain primed for cutting the target DNA strand. Moreover, herein we demonstrate the essential roles of the catalytic Mg2+ for the active state formation and stability. Importantly, we suggest that the derived catalytic conformation of the HNH domain can be exploited for rational engineering of Cas9 variants with enhanced specificity.

A three-dimensional model of RNase P in the hyperthermophilic archaeon Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is an endoribonuclease involved in maturation of the 5′-end of tRNA. We found previously that RNase P in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 consists of a catalytic RNase P RNA (PhopRNA) and five protein cofactors designated PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38. The crystal structures of the five proteins have been determined, a three-dimensional (3-D) model of PhopRNA has been constructed, and biochemical data, including protein-RNA interaction sites, have become available. Here, this information was combined to orient the crystallographic structures of the proteins relative to their RNA binding sites in the PhopRNA model. Some alterations were made to the PhopRNA model to improve the fit. In the resulting structure, a heterotetramer composed of PhoPop5 and PhoRpp30 bridges helices P3 and P16 in the PhopRNA C-domain, thereby probably stabilizing a double-stranded RNA structure (helix P4) containing catalytic Mg2+ ions, while a heterodimer of PhoRpp21 and PhoRpp29 locates on a single-stranded loop connecting helices P11 and P12 in the specificity domain (S-domain) in PhopRNA, probably forming an appropriate conformation of the precursor tRNA (pre-tRNA) binding site. The fifth protein PhoRpp38 binds each kink-turn (K-turn) motif in helices P12.1, P12.2, and P16 in PhopRNA. Comparison of the structure of the resulting 3-D model with that of bacterial RNase P suggests transition from RNA-RNA interactions in bacterial RNase P to protein-RNA interactions in archaeal RNase P. The proposed 3-D model of P. horikoshii RNase P will serve as a framework for further structural and functional studies on archaeal, as well as eukaryotic, RNase Ps.

Timing and Reset Mechanism of GTP Hydrolysis-Driven Conformational Changes of Atlastin

The endoplasmic reticulum (ER) forms a branched, dynamic membrane tubule network that is vital for cellular function. Branching arises from membrane fusion facilitated by the GTPase atlastin (ATL). Many metazoan genomes encode for three ATL isoforms that appear to fulfill partially redundant function despite differences in their intrinsic GTPase activity and localization within the ER; however, the underlying mechanistic differences between the isoforms are poorly understood. Here, we identify discrete temporal steps in the catalytic cycle for thetwo most dissimilar isoforms, ATL1 and ATL3, revealing an overall conserved progression of molecular events from nucleotide binding and hydrolysis to ATL dimerization and phosphate release. A crystal structure of ATL3 suggests a mechanism for the displacement of the catalytic Mg2+ ion following guanosine triphosphate (GTP) hydrolysis. Together, the data extend the mechanistic framework for howGTP hydrolysis drives conformational changes in ATL and how the cycle is reset for subsequent rounds of catalysis.

Does Cas9-Catalyzed DNA Cleavage Generate Blunt Ends or Staggered Ends? Insight from Molecular Dynamic Simulations

The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has merged as a versatile toolbox for genome editing. Despite recent advances in the mechanistic studies on spCas9-sgRNA-mediated target double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg2+ ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the target DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2+ bound. The simulation results show that binding of two Mg2+ ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we argue that Cas9-catalyzed target DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.

Highly effective molecule converting strategy based on enzyme-free dual recycling amplification for ultrasensitive electrochemical detection of ATP.

An enzyme-free and highly effective molecule converting strategy based on target-driven catalytic hairpin assembly and Mg2+-dependent DNAzyme recycling dual-amplification was proposed to construct an ultrasensitive electrochemical biosensor for adenosine triphosphate (ATP) detection.

Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations.

The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg2+ ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2+ bound. The simulation results showed that binding of two Mg2+ ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.

Cryo-EM structure of the spliceosome immediately after branching.

Pre-mRNA splicing proceeds by two consecutive trans-esterification reactions via a lariat-intron intermediate. We present the 3.8Å cryoEM structure of the spliceosome immediately after lariat formation. The 5’-splice site is cleaved but remains close to the catalytic Mg2+ site in the U2/U6 snRNA triplex, and the 5’-phosphate of the intron nucleotide G(+1) is linked to the branch adenosine 2’OH. The 5’-exon is held between the Prp8 N-terminal and Linker domains, and base-pairs with U5 snRNA loop 1. Non-Watson-Crick interactions between the branch helix and 5’-splice site dock the branch adenosine into the active site, while intron nucleotides +3 to +6 base-pair with the U6 snRNA ACAGAGA sequence. Isy1 and the step one factors Yju2 and Cwc25 stabilise docking of the branch helix. The intron downstream of the branch site emerges between the Prp8 RT and Linker domains and extends towards Prp16 helicase, suggesting a plausible mechanism of remodelling before exon ligation.

A new autoinhibited kinase conformation reveals a salt-bridge switch in kinase activation.

In the structure of autoinhibited EphA2 tyrosine kinase reported herein, we have captured the entire activation segment, revealing a previously unknown role of the conserved Arg762 in kinase autoinhibition by interacting with the essential Mg2+-chelating Asp757. While it is well known that this Arg residue is involved in an electrostatic interaction with the phospho-residue of the activation loop to stabilize the active conformation, our structure determination revealed a new role for the Arg, acting as a switch between the autoinhibited and activated conformations. Mutation of Arg762 to Ala in EphA2 sensitized Mg2+ response, resulting in enhanced kinase catalytic activity and Mg2+ cooperativity. Furthermore, mutation of the corresponding Arg/Lys to Ala in PKA and p38MAPK also exhibited similar behavior. This new salt bridge-mediated switch may thus be an important mechanism of activation on a broader scope for kinases which utilize autophosphorylation.

A targeted DNA substrate mechanism for the inhibition of HIV-1 integrase by inhibitors with antiretroviral activity.

We recently reported that viral DNA could be the primary target of raltegravir (RAL), an efficient anti‐HIV‐1 drug, which acts by inhibiting integrase. To elucidate this mechanism, we conducted a comparative analysis of RAL and TB11, a diketoacid abandoned as an anti‐HIV‐1 drug for its weak efficiency and marked toxicity, and tested the effects of the catalytic cofactor Mg2+ (5 mm) on drug‐binding properties. We used circular dichroism and fluorescence to determine drug affinities for viral DNA long terminal repeats (LTRs) and peptides derived from the integrase active site and DNA retardation assays to assess drug intercalation into DNA base pairs. We found that RAL bound more tightly to LTR ends than did TB11 (a diketo acid bearing an azido group) and that Mg2+ significantly increased the affinity of both RAL and TB11. We also observed a good relationship between drug binding with processed LTR and strand transfer inhibition. This unusual type of inhibition was caused by Mg2+‐assisted binding of drugs to DNA substrate, rather than to enzyme. Notably, while RAL bound exclusively to the cleavable/cleaved site, TB11 further intercalated into DNA base pairs and interacted with the integrase‐derived peptides. These unwanted binding sites explain the weaker bioavailability and higher toxicity of TB11 compared with the more effective RAL.

Synthesis of dihydropyrimidine α,γ-diketobutanoic acid derivatives targeting HIV integrase

The synthesis and antiviral evaluation of a series of dihydropyrimidinone and thiopyrimidine derivatives bearing aryl α,γ-diketobutanoic acid moiety are described using the Biginelli multicomponent reaction as key step. The most active among 20 synthesized novel compounds were 4c, 4d and 5b, which possess nanomolar HIV-1 integrase (IN) stand transfer (ST) inhibition activities. In order to understand their mode of interactions within the IN active site, we docked all the compounds into the previously reported X-ray crystal structure of IN. We observed that compounds 4c, 4d and 5b occupied an area close to the two catalytic Mg2+ ions surrounded by their chelating triad (E221, D128 and D185), DC16, Y212 and the β-diketo acid moiety of 4c, 4d and 5b chelating Mg2+. As those compounds lack anti-HIV activities in cell, their prodrugs were synthetized. The prodrug 4c′ exhibited an anti-HIV activity of 0.19 μM in primary human lymphocytes with some cytotoxicity. All together, these results indicate that the new analogs potentially interact within the catalytic site with highly conserved residues important for IN catalytic activity.

Protonation-Dependent Base Flipping at Neutral pH in the Catalytic Triad of a Self-Splicing Bacterial Group II Intron

NMR spectroscopy has revealed pH-dependent structural changes in the highly conserved catalytic domain 5 of a bacterial group II intron. Two adenines with pKa values close to neutral pH were identified in the catalytic triad and the bulge. Protonation of the adenine opposite to the catalytic triad is stabilized within a G(syn)–AH+(anti) base pair. The pH-dependent anti-to-syn flipping of this G in the catalytic triad modulates the known interaction with the linker region between domains 2 and 3 (J23) and simultaneously the binding of the catalytic Mg2+ ion to its backbone. Hence, this here identified shifted pKa value controls the conformational change between the two steps of splicing.

Thermodynamic Examination of the U6 ISL RNA Bulge

Spliceosome is large complex made up of five snRNA and nearly 70 proteins that assembles on pre‐mRNA to excise the introns. The U6 subunit of the spliceosome is required for spliceosome assembly and activation. Spliceosome undergoes number of structural rearrangements that disrupt U4/U6 complex and form U2/U6 complex to become activated. The association of U2/U6 complex results in the formation of a highly conserved internal stem loop (ISL) in U6 RNA. The formation of a 1x2 nucleotide bulge that binds catalytically active Mg2+ at U80 position is essential in the RNA cleavage. The 1x2 bulge contains an A79•C67 non‐canonical base pair, which forms a stronger A+•C base pair in lower pH conditions. Upon the A+•C base pair formation, the ISL adopts a conformation closer to an A‐form helix and the unbound U80 in the bulge is forced out into the major groove. This conformation is mutually antagonistic to the binding of the catalytic Mg2+ ion, and therefore prevents splicing activity. Thermal denaturation experiments were performed on RNA constructs derived to mimic 1x2 bulge of the U6 RNA. We tested constructs containing the conserved bulged nucleotides as well as those with modifications that disrupt spliceosomal function. All constructs were examined in 1 M KCl and in varying Mg2+ concentrations at pH of 5.5 and 7.5. The thermodynamic contributions of the bulged RNA were compared to those of the helical RNA and DNA constructs. Thermodynamics of RNA with modification in and around the 1x2 bulge loop will be presented and influence of pH and ionic conditions will be discussed.

Residue Specific Radiation Damage of Protein Structures using High-Resolution Cryo-Electron Microscopy

High-resolution structures of proteins and protein complexes are currently determined using either X-ray crystallography, NMR spectroscopy, or now also cryo-electron microscopy (cryo-EM). The highest resolutions achieved by cryo-EM have been typically restricted to large, well-ordered entities such as helical or icosahedral assemblies or 2D crystals. However, we show that emerging methods in single-particle cryo-EM now allow structure determination at near-atomic resolution, even for much smaller protein complexes with low symmetry. We solved the structure of the ∼465-kDa Escherichia coli β-galactosidase at ∼3.2-Å resolution using single-particle cryo-EM. At this resolution, the majority of the side-chains, the N-termini, and the geometry of the active sites, including a catalytic Mg2+-ion, can be clearly discerned in the density map. Inspection of the map reveals that while densities for residues with positively charged and neutral side-chains are well resolved, systematically weaker densities are observed for residues with negatively charged side-chains. The negatively charged glutamate and aspartate show on average 30% less density than the similarly sized neutral glutamine and asparagine. This observation is independent of the exposure of these residues to solvent. Analysis of other high-resolution cryo-EM structures reveals similar weaker densities for these types of residues. Radiation damage in X-ray crystallography has been linked to decarboxylation of glutamate and aspartate residues, breakage of disulfide bonds, loss of hydroxyl-groups from tyrosine and methylthio-group of methionine. We now show that negatively charged residues exhibit more pronounced effects of radiation damage in structures solved by cryo-EM. We determined that the degree of damage is dose dependent by comparison of density maps obtained using electron doses ranging from 10-30 electrons/Å2. In summary, we establish the feasibility of determining structures at near-atomic resolution and provide a measure of the effects of radiation damage in high-resolution cryo-electron microscopy.

Thio Effects and an Unconventional Metal Ion Rescue in the Genomic Hepatitis Delta Virus Ribozyme

Metal ion and nucleobase catalysis are important for ribozyme mechanism, but the extent to which they cooperate is unclear. A crystal structure of the hepatitis delta virus (HDV) ribozyme suggested that the pro-RP oxygen at the scissile phosphate directly coordinates a catalytic Mg(2+) ion and is within hydrogen bonding distance of the amine of the general acid C75. Prior studies of the genomic HDV ribozyme, however, showed neither a thio effect nor metal ion rescue using Mn(2+). Here, we combine experiment and theory to explore phosphorothioate substitutions at the scissile phosphate. We report significant thio effects at the scissile phosphate and metal ion rescue with Cd(2+). Reaction profiles with an SP-phosphorothioate substitution are indistinguishable from those of the unmodified substrate in the presence of Mg(2+) or Cd(2+), supporting the idea that the pro-SP oxygen does not coordinate metal ions. The RP-phosphorothioate substitution, however, exhibits biphasic kinetics, with the fast-reacting phase displaying a thio effect of up to 5-fold and the slow-reacting phase displaying a thio effect of ~1000-fold. Moreover, the fast- and slow-reacting phases give metal ion rescues in Cd(2+) of up to 10- and 330-fold, respectively. The metal ion rescues are unconventional in that they arise from Cd(2+) inhibiting the oxo substrate but not the RP substrate. This metal ion rescue suggests a direct interaction of the catalytic metal ion with the pro-RP oxygen, in line with experiments with the antigenomic HDV ribozyme. Experiments without divalent ions, with a double mutant that interferes with Mg(2+) binding, or with C75 deleted suggest that the pro-RP oxygen plays at most a redundant role in positioning C75. Quantum mechanical/molecular mechanical (QM/MM) studies indicate that the metal ion contributes to catalysis by interacting with both the pro-RP oxygen and the nucleophilic 2'-hydroxyl, supporting the experimental findings.