A glimpse into the active site of a group II intron and maybe the spliceosome, too

The X-ray crystal structure of an excised group II self-splicing intron was recently solved by the Pyle group. Here we review some of the notable features of this structure and what they may tell us about the catalytic active site of the group II ribozyme and potentially the spliceosome. The new structure validates the central role of domain V in both the structure and catalytic function of the ribozyme and resolves several outstanding puzzles raised by previous biochemical, genetic and structural studies. While lacking both exons as well as the cleavage sites and nucleophiles, the structure reveals how a network of tertiary interactions can position two divalent metal ions in a configuration that is ideal for catalysis.     

Functional group requirements in the probable active site of the VS ribozyme

The VS ribozyme catalyses the site-specific cleavage of a phosphodiester linkage by a transesterification reaction that entails the attack of the neighbouring 2'-oxygen with departure of the 5'-oxygen. We have previously suggested that the A730 loop is an important component of the active site of the ribozyme, and that A756 is especially important in the cleavage reaction. Functional group modification experiments reported here indicate that the base of A756 is more important than its ribose for catalysis. A number of changes to the base, including complete ablation, lead to cleavage rates that are reduced 1000-fold, while removal of the 2'-hydroxyl group from the ribose results in tenfold slower cleavage. 2-Aminopurine fluorescence experiments indicate that this 2'-hydroxyl group is important for the structure of the A730 loop. Catalytic activity is especially sensitive to changes involving the exocyclic amine of A756; by contrast, the cleavage activity is only weakly sensitive to modification at the 7-position of the purine nucleus. These results suggest that the Watson-Crick edge of the adenine base is important in ribozyme function. We sought to test the possibility of a direct role of the nucleobase in the chemistry of the cleavage reaction. Addition of imidazole base in the medium failed to restore the activity of a ribozyme from which the nucleobase of A756 was removed. However, no restoration was obtained with exogenous adenine base either, indicating that the cavity that might result from ablation of the base was closed.     

RNA tertiary interactions mediate native collapse of a bacterial group I ribozyme

Large RNAs collapse into compact intermediates in the presence of counterions before folding to the native state. We previously found that collapse of a bacterial group I ribozyme correlates with the formation of helices within the ribozyme core, but occurs at Mg2+ concentrations too low to support stable tertiary structure and catalytic activity. Here, using small-angle X-ray scattering, we show that Mg2+-induced collapse is a cooperative folding transition that can be fit by a two-state model. The Mg2+ dependence of collapse is similar to the Mg2+ dependence of helix assembly measured by partial ribonuclease T1 digestion and of an unfolding transition measured by UV hypochromicity. The correspondence between multiple probes of RNA structure further supports a two-state model. A mutation that disrupts tertiary contacts between the L9 tetraloop and its helical receptor destabilized the compact state by 0.8 kcal/mol, while mutations in the central triplex were less destabilizing. These results show that native tertiary interactions stabilize the compact folding intermediates under conditions in which the RNA backbone remains accessible to solvent.     

A kinetic and thermodynamic framework for the Azoarcus group I ribozyme reaction

Determination of quantitative thermodynamic and kinetic frameworks for ribozymes derived from the Azoarcus group I intron and comparisons to their well-studied analogs from the Tetrahymena group I intron reveal similarities and differences between these RNAs. The guanosine (G) substrate binds to the Azoarcus and Tetrahymena ribozymes with similar equilibrium binding constants and similar very slow association rate constants. These and additional literature observations support a model in which the free ribozyme is not conformationally competent to bind G and in which the probability of assuming the binding-competent state is determined by tertiary interactions of peripheral elements. As proposed previously, the slow binding of guanosine may play a role in the specificity of group I intron self-splicing, and slow binding may be used analogously in other biological processes. The internal equilibrium between ribozyme-bound substrates and products is similar for these ribozymes, but the Azoarcus ribozyme does not display the coupling in the binding of substrates that is observed with the Tetrahymena ribozyme, suggesting that local preorganization of the active site and rearrangements within the active site upon substrate binding are different for these ribozymes. Our results also confirm the much greater tertiary binding energy of the 5′-splice site analog with the Azoarcus ribozyme, binding energy that presumably compensates for the fewer base-pairing interactions to allow the 5′-exon intermediate in self splicing to remain bound subsequent to 5′-exon cleavage and prior to exon ligation. Most generally, these frameworks provide a foundation for design and interpretation of experiments investigating fundamental properties of these and other structured RNAs.     

Identification of the catalytic subdomain of the VS ribozyme and evidence for remarkable sequence tolerance in the active site loop

We show here that the ribozyme domain of the Neurospora VS ribozyme consists of separable upper and lower subdomains. Deletion analysis demonstrates that the entire upper subdomain (helices III/IV/V) is dispensable for site-specific cleavage activity, providing experimental evidence that the active site is contained within the lower subdomain and within the substrate itself. We demonstrate an important role in cleavage activity for a region of helix VI called the 730 loop. Surprisingly, several loop sequences, sizes, and structures at this position can support site-specific cleavage, suggesting that a variety of non-Watson-Crick structures, rather than a specific loop structure, in this region of the ribozyme can contribute to formation of the active site.     

Terbium-mediated footprinting probes a catalytic conformational switch in the antigenomic hepatitis delta virus ribozyme

The two forms of the hepatitis delta virus ribozyme are derived from the genomic and antigenomic RNA strands of the human hepatitis delta virus (HDV), where they serve a crucial role in pathogen replication by catalyzing site-specific self-cleavage reactions. The HDV ribozyme requires divalent metal ions for formation of its tertiary structure, consisting of a tight double-nested pseudoknot, and for efficient self- (or cis-) cleavage. Comparison of recently solved crystal structures of the cleavage precursor and 3' product indicates that a significant conformational switch is required for catalysis by the genomic HDV ribozyme. Here, we have used the lanthanide metal ion terbium(III) to footprint the precursor and product solution structures of the cis-acting antigenomic HDV ribozyme. Inhibitory Tb(3+) binds with high affinity to similar sites on RNA as Mg(2+) and subsequently promotes slow backbone scission. We find subtle, yet significant differences in the terbium(III) footprinting pattern between the precursor and product forms of the antigenomic HDV ribozyme, consistent with differences in conformation as observed in the crystal structures of the genomic ribozyme. In addition, UV melting profiles provide evidence for a less tight tertiary structure in the precursor. In both the precursor and product we observe high-affinity terbium(III) binding sites in joining sequence J4/2 (Tb(1/2) approximately 4 microM) and loop L3, which are key structural components forming the catalytic core of the HDV ribozyme, as well as in several single-stranded regions such as J1/2 and the L4 tetraloop (Tb(1/2) approximately 50 microM). Sensitized luminescence spectroscopy confirms that there are at least two affinity classes of Tb(3+) binding sites. Our results thus demonstrate that a significant conformational change accompanies catalysis in the antigenomic HDV ribozyme in solution, similar to the catalytic conformational switch observed in crystals of the genomic form, and that structural and perhaps catalytic metal ions bind close to the catalytic core.     

Protonated 2'-aminoguanosine as a probe of the electrostatic environment of the active site of the Tetrahymena group I ribozyme.

We have probed the electrostatic environment of the active site of the Tetrahymena group I ribozyme (E) using protonated 2'-aminoguanosine (), in which the 2'-OH of the guanosine nucleophile (G) is replaced by an group. At low concentrations of divalent metal ion (2 mM Mg(2+)), binds at least 200-fold stronger than G or G(NH)()2, with a dissociation constant of 相似文献   

An unconventional origin of metal-ion rescue and inhibition in the Tetrahymena group I ribozyme reaction

The presence of catalytic metal ions in RNA active sites has often been inferred from metal-ion rescue of modified substrates and sometimes from inhibitory effects of alternative metal ions. Herein we report that, in the Tetrahymena group I ribozyme reaction, the deleterious effect of a thio substitution at the pro-Sp position of the reactive phosphoryl group is rescued by Mn2+. However, analysis of the reaction of this thio substrate and of substrates with other modifications strongly suggest that this rescue does not stem from a direct Mn2+ interaction with the Sp sulfur. Instead, the apparent rescue arises from a Mn2+ ion interacting with the residue immediately 3' of the cleavage site, A(+1), that stabilizes the tertiary interactions between the oligonucleotide substrate (S) and the active site. This metal site is referred to as site D herein. We also present evidence that a previously observed Ca2+ ion that inhibits the chemical step binds to metal site D. These and other observations suggest that, whereas the interactions of Mn2+ at site D are favorable for the chemical reaction, the Ca2+ at site D exerts its inhibitory effect by disrupting the alignment of the substrates within the active site. These results emphasize the vigilance necessary in the design and interpretation of metal-ion rescue and inhibition experiments. Conversely, in-depth mechanistic analysis of the effects of site-specific substrate modifications can allow the effects of specific metal ion-RNA interactions to be revealed and the properties of individual metal-ion sites to be probed, even within the sea of metal ions bound to RNA.     

X-ray crystallographic analysis of the structural basis for the interactions of pokeweed antiviral protein with its active site inhibitor and ribosomal RNA substrate analogs.

The pokeweed antiviral protein (PAP) belongs to a family of ribosome-inactivating proteins (RIP), which depurinate ribosomal RNA through their site-specific N-glycosidase activity. We report low temperature, three-dimensional structures of PAP co-crystallized with adenyl-guanosine (ApG) and adenyl-cytosine-cytosine (ApCpC). Crystal structures of 2.0-2.1 A resolution revealed that both ApG or ApCpC nucleotides are cleaved by PAP, leaving only the adenine base clearly visible in the active site pocket of PAP. ApCpC does not resemble any known natural substrate for any ribosome-inactivating proteins and its cleavage by PAP provides unprecedented evidence for a broad spectrum N-glycosidase activity of PAP toward adenine-containing single stranded RNA. We also report the analysis of a 2.1 A crystal structure of PAP complexed with the RIP inhibitor pteoric acid. The pterin ring is strongly bound in the active site, forming four hydrogen bonds with active site residues and one hydrogen bond with the coordinated water molecule. The second 180 degrees rotation conformation of pterin ring can form only three hydrogen bonds in the active site and is less energetically favorable. The benzoate moiety is parallel to the protein surface of PAP and forms only one hydrogen bond with the guanido group of Arg135.     

Selection of the 3′-splice site in group I introns

A model for selection of 3′-splice sites in splicing of RNA precursors containing group I introns is presented. The key feature of this model is a newly identified tertiary interaction between the catalytic core of the intron and the 3′-splice site. This tertiary pairing would bring the 3′-splice site into the core of the intron, which is known to contain RNA sequences and structures essential for catalyzing the splicing reactions. The proposed tertiary interaction can coexist with P10, a pairing between 3′-exon sequences and the ‘internal guide sequence’ near the 5′-end of the intron. The model predicts that three RNA-RNA interactions are important in selection of 3′-splice sites: (i) binding of intron sequences with the core; (ii) pairing of exon sequences with the internal guide sequence; and (iii) binding of the terminal guanosine to an unknown site within the core.     

Ionogenic groups in the active site of lysostaphin. Kinetic and thermodynamic data compared with X-ray crystallographic data

The active site of lysostaphin is shown to contain a residue of glutamic acid. As judged by a pK value of 9.2 (with pentaglycine bridges in peptidoglycan of staphylococci as a substrate), another ionogenic residue could be the epsilon-amino group of a lysine. However, the pH value near a negatively charged cell is supposed to be strongly shifted to acidity as compared to the pH of the solution volume. This shifts the enzyme pH dependence curve in solution to alkalinity. Therefore, the other group might be histidine, which is consistent with the X-ray crystallographic data. A similar shift is likely to occur for lysozyme in the case of Micrococcus lysodeikticus cells. Determination of pK of ionogenic groups in the active sites of alkaline enzymes responsible for lysis of negatively charged bacterial cells gives their apparent values because the "pericellular" and "voluminous" values of pH are not coincident.     

Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site.

Two mutant forms of fumarase C from E. coli have been made using PCR and recombinant DNA. The recombinant form of the protein included a histidine arm on the C-terminal facilitating purification. Based on earlier studies, two different carboxylic acid binding sites, labeled A- and B-, were observed in crystal structures of the wild type and inhibited forms of the enzyme. A histidine at each of the sites was mutated to an asparagine. H188N at the A-site resulted in a large decrease in specific activity, while the H129N mutation at the B-site had essentially no effect. From the results, we conclude that the A-site is indeed the active site, and a dual role for H188 as a potential catalytic base is proposed. Crystal structures of the two mutant proteins produced some unexpected results. Both mutations reduced the affinity for the carboxylic acids at their respective sites. The H129N mutant should be particularly useful in future kinetic studies because it sterically blocks the B-site with the carboxyamide of asparagine assuming the position of the ligand's carboxylate. In the H188N mutation at the active site, the new asparagine side chain still interacts with an active site water that appears to have moved slightly as a result of the mutation.     

Leakage and slow allostery limit performance of single drug-sensing aptazyme molecules based on the hammerhead ribozyme

Engineered “aptazymes” fuse in vitro selected aptamers with ribozymes to create allosteric enzymes as biosensing components and artificial gene regulatory switches through ligand-induced conformational rearrangement and activation. By contrast, activating ligand is employed as an enzymatic cofactor in the only known natural aptazyme, the glmS ribozyme, which is devoid of any detectable conformational rearrangements. To better understand this difference in biosensing strategy, we monitored by single molecule fluorescence resonance energy transfer (FRET) and 2-aminopurine (AP) fluorescence the global conformational dynamics and local base (un)stacking, respectively, of a prototypical drug-sensing aptazyme, built from a theophylline aptamer and the hammerhead ribozyme. Single molecule FRET reveals that a catalytically active state with distal Stems I and III of the hammerhead ribozyme is accessed both in the theophylline-bound and, if less frequently, in the ligand-free state. The resultant residual activity (leakage) in the absence of theophylline contributes to a limited dynamic range of the aptazyme. In addition, site-specific AP labeling shows that rapid local theophylline binding to the aptamer domain leads to only slow allosteric signal transduction into the ribozyme core. Our findings allow us to rationalize the suboptimal biosensing performance of the engineered compared to the natural aptazyme and to suggest improvement strategies. Our single molecule FRET approach also monitors in real time the previously elusive equilibrium docking dynamics of the hammerhead ribozyme between several inactive conformations and the active, long-lived, Y-shaped conformer.     

Molecular mechanics simulations of a conformational rearrangement of D-xylose in the active site of D-xylose isomerase.

A proposed reaction mechanism for the enzyme D-xylose isomerase involves the ring opening of the cyclic substrate with a subsequent conformational rearrangement to an extended open-chain form. Restrained energy minimization was used to simulate the rearrangement. In the ring-opening step, the substrate energy function was gradually altered from a cyclic to an open-chain form, with energy minimization after each change. The protein/sugar contact energy did not increase significantly during the process, showing that there was no steric hindrance to ring opening. The conformational rearrangement involves an alteration in the coordination of the substrate to metal ion [1], which was induced by gradually changing restraints on metal/ligand distances. By allowing varying amounts of flexibility in the protein and examining a simplified model system, the interactions of the sugar with metal ion [1] and its immediate ligands were found to be the most important contributors to the energy barrier for the change. Only small changes in the positions of protein atoms were required. The energy barrier to the rearrangement was estimated to be less than the Arrhenius activation energy for the enzymatic reaction. This is in accordance with experimental indications that the isomerization step is rate determining.     

Conservation of helical structure contributes to functional metal ion interactions in the catalytic domain of ribonuclease P RNA

Like protein enzymes, catalytic RNAs contain conserved structure motifs important for function. A universal feature of the catalytic domain of ribonuclease P RNA is a bulged-helix motif within the P1-P4 helix junction. Here, we show that changes in bulged nucleotide identity and position within helix P4 affect both catalysis and substrate binding, while a subset of the mutations resulted only in catalytic defects. We find that the proximity of the bulge to sites of metal ion coordination in P4 is important for catalysis; moving the bulge distal to these sites and deleting it had similarly large effects, while moving it proximal to these sites had only a moderate effect on catalysis. To test whether the effects of the mutations are linked to metal ion interactions, we used terbium-dependent cleavage of the phosphate backbone to probe metal ion-binding sites in the wild-type and mutant ribozymes. We detect cleavages at specific sites within the catalytic domain, including helix P4 and J3/4, which have previously been shown to participate directly in metal ion interactions. Mutations introduced into P4 cause local changes in the terbium cleavage pattern due to alternate metal ion-binding configurations with the helix. In addition, a bulge deletion mutation results in a 100-fold decrease in the single turnover cleavage rate constant at saturating magnesium levels, and a reduced affinity for magnesium ions important for catalysis. In light of the alternate terbium cleavage pattern in P4 caused by bulge deletion, this decreased ability to utilize magnesium ions for catalysis appears to be due to localized structural changes in the ribozyme's catalytic core that weaken metal ion interactions in P4 and J3/4. The information reported here, therefore, provides evidence that the universal conservation of the P4 structure is based in part on optimization of metal ion interactions important for catalysis.     

Influence of decreased solvent permittivity on the structure and magnesium(II)-binding properties of the catalytic domain 5 of a group II intron ribozyme

Although it is well known that the so-called “equivalent solution” or “effective” solvent permittivity (dielectric constant) in proteins and nucleic acids is lower than in bulk water, this fact is commonly neglected in (bioinorganic) studies of such compounds. Using domain 5 of the group II intron ribozyme Sc.ai5γ, we describe here the influence of 1,4-dioxane-d₈ on the structure and magnesium(II)-binding properties of this catalytic domain. Applying one- and two-dimensional NMR, we observe distinct structural changes in the functionally important bulge region following a decrease in solvent permittivity. Concomitantly, an increase by a factor of 1.5 in the affinity of Mg²⁺ towards the individual-binding sites in the catalytic core domain is observed upon addition of 1,4-dioxane-d₈. This has led to the detection of a new metal ion coordination site near the GU wobble pair in the catalytic triad. Our results show that solvent permittivity is an important factor in the formation of intrinsic RNA structures and affects their metal ion-binding properties. Hence, solvent permittivity should be taken into account in future studies.     

Structural details at active site of hen egg white lysozyme with di- and trivalent metal ions

Metal binding to lysozyme has received wide interest. In particular, it is interesting that Ni2+, Mn2+, Co2+, and Yb3+ chloride salts induce an increase in the solubility of the tetragonal form in crystals of hen egg white lysozyme at high salt concentration, but that Mg2+ and Ca2+ chloride salts do not. To investigate the interactions of the di- and trivalent metal ions with the active site of lysozyme and compare the effects of the di- and trivalent metal ions on molecular conformation of lysozyme based on the structural analysis, the crystal structures of hen egg white lysozyme grown at pH 4.6, in the presence of 0.5 M MgCl2, CaCl2, NiCl2, MnCl2, CoCl2, and YbCl3, have been determined by X-ray crystallography at 1.58 A resolution. The crystals grown in these salts have an identical space group, P4(3)2(1)2. The molecules show no conformational changes, irrespective of the salts used. Ni2+ and Co2+ binding to the Odelta atom of Asp52 in the active site at 1.98 and 2.02 A, respectively, and Yb3+ binding to both the Odelta atom of Asp52 and the Odelta1 atom of Asn46 at 2.25 A have been identified. The binding sites of Mn2+, Mg2+, and Ca2+ have not been found from different Fourier electron density maps. The Ni2+ and Co2+ ions bind to the Odelta atom of Asp52 at almost the same position, while the Yb3+ ion takes a different position from the Ni2+ and Co2+ ions. On the other hand, the anion Cl-, interacting with the Oeta atom of Tyr23 at a site of about 2.90 A, has also been determined for each crystal.     

Jpx RNA regulates CTCF anchor site selection and formation of chromosome loops

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An essential role of active site arginine residue in iodide binding and histidine residue in electron transfer for iodide oxidation by horseradish peroxidase

The objective of the present study is to delineate the role of active site arginine and histidine residues of horseradish peroxidase (HRP) in controlling iodide oxidation using chemical modification technique. The arginine specific reagent, phenylglyoxal (PGO) irreversibly blocks iodide oxidation following pseudofirst order kinetics with second order rate constant of 25.12 min^-1 M^-1. Radiolabelled PGO incorporation studies indicate an essential role of a single arginine residue in enzyme inactivation. The enzyme can be protected both by iodide and an aromatic donor such as guaiacol. Moreover, guaiacol-protected enzyme can oxidise iodide and iodide-protected enzyme can oxidise guaiacol suggesting the regulatory role of the same active site arginine residue in both iodide and guaiacol binding. The protection constant (K_p) for iodide and guaiacol are 500 and 10 M respectively indicating higher affinity of guaiacol than iodide at this site. Donor binding studies indicate that guaiacol competitively inhibits iodide binding suggesting their interaction at the same binding site. Arginine-modified enzyme shows significant loss of iodide binding as shown by increased K_d value to 571 mM from the native enzyme (K_d = 150 mM). Although arginine-modified enzyme reacts with H₂O₂ to form compound II presumably at a slow rate, the latter is not reduced by iodide presumably due to low affinity binding.The role of the active site histidine residue in iodide oxidation was also studied after disubstitution reaction of the histidine imidazole nitrogens with diethylpyrocarbonate (DEPC), a histidine specific reagent. DEPC blocks iodide oxidation following pseudofirst order kinetics with second order rate constant of 0.66 min^-1 M^-1. Both the nitrogens (, ) of histidine imidazole were modified as evidenced by the characteristic peak at 222 nm. The enzyme is not protected by iodide suggesting that imidazolium ion is not involved in iodide binding. Moreover, DEPC-modified enzyme binds iodide similar to the native enzyme. However, the modified enzyme does not form compound II but forms compound I only with higher concentration of H₂O₂ suggesting the catalytic role of this histidine in the formation and autoreduction of compound I. Interestingly, compound I thus formed is not reduced by iodide indicating block of electron transport from the donor to the compound I. We suggest that an active site arginine residue regulates iodide binding while the histidine residue controls the electron transfer to the heme ferryl group during oxidation.     

Dynamics of the active site loops in catalyzing aminoacylation reaction in seryl and histidyl tRNA synthetases

Aminoacylation reaction is the first step of protein biosynthesis. The catalytic reorganization at the active site of aminoacyl tRNA synthetases (aaRSs) is driven by the loop motions. There remain lacunae of understanding concerning the catalytic loop dynamics in aaRSs. We analyzed the functional loop dynamics in seryl tRNA synthetase from Methanopyrus kandleri (^mkSerRS) and histidyl tRNA synthetases from Thermus thermophilus (^ttHisRS), respectively, using molecular dynamics. Results confirm that the motif 2 loop and other active site loops are flexible spots within the catalytic domain. Catalytic residues of the loops form a network of interaction with the substrates to form a reactive state. The loops undergo transitions between closed state and open state and the relaxation of the constituent residues occurs in femtosecond to nanosecond time scale. Order parameters are higher for constituent catalytic residues which form a specific network of interaction with the substrates to form a reactive state compared to the Gly residues within the loop. The development of interaction is supported from mutation studies where the catalytic domain with mutated loop exhibits unfavorable binding energy with the substrates. During the open-close motion of the loops, the catalytic residues make relaxation by ultrafast librational motion as well as fast diffusive motion and subsequently relax rather slowly via slower diffusive motion. The Gly residues act as a hinge to facilitate the loop closing and opening by their faster relaxation behavior. The role of bound water is analyzed by comparing implicit solvent-based and explicit solvent-based simulations. Loops fail to form catalytically competent geometry in absence of water. The present result, for the first time reveals the nature of the active site loop dynamics in aaRS and their influence on catalysis.