Divalent Metal Ion Binding Sites Research Articles

Simultaneous Localization of Two High Affinity Divalent Metal Ion Binding Sites in the Tetracycline RNA Aptamer with Mn2+-Based Pulsed Dipolar EPR Spectroscopy.

Mg2+ ions play an essential part in stabilizing the tertiary structure of nucleic acids. While the importance of these ions is well documented, their localization and elucidation of their role in the structure and dynamics of nucleic acids are often challenging. In this work, pulsed electron-electron double resonance spectroscopy (PELDOR, also known as DEER) was used to localize two high affinity divalent metal ion binding sites in the tetracycline RNA aptamer with high accuracy. For this purpose, the aptamer was labeled at different positions with a semirigid nitroxide spin label and diamagnetic Mg2+ was replaced with paramagnetic Mn2+, which did not alter the folding process or ligand binding. Out of the several divalent metal ion binding sites that are known from the crystal structure, two binding sites with high affinity were detected: one that is located at the ligand binding center and another at the J1/2 junction of the RNA.

Mechanics of divalent ion sensitivity of the manganese-sensing riboswitch measured by combined high-resolution optical tweezers and single-molecule fluorescence.

Riboswitches are structured non-coding RNA typically found in the 5′ untranslated regions of bacterial mRNAs. They modulate gene expression through the binding of specific ligands that induce changes in the riboswitch structure, most often at the level of transcription and translation. The Mn2+-sensing yybP-ykoY RNA motif is one of the most widespread riboswitches across bacteria, including human and plant pathogens. It contains two divalent metal ion binding sites: one is specific for Mn2+ whereas the other is nonspecific for a divalent metal ion, including Mg2+. Previous crystal structures have shown that Mn2+ binds in between the two distal helical legs of a four-way junction and induces a large “docking” conformational change. It is unclear how competition between Mn2+ and Mg2+ for the two binding sites and the associated RNA conformational changes give rise to Mn2+ detection function and sensitivity. Therefore, we explored the global structural dynamics of the L. lactis Mn2+-sensing riboswitch in the presence of competing divalent cations using high-resolution optical tweezers simultaneous with confocal Förster resonance energy transfer (FRET) spectroscopy. Initial non-equilibrium force ramp measurements revealed force-dependent docking distribution, suggesting a highly selective docked state at suboptimal Mn2+ ion concentration. Follow-up high-resolution equilibrium fixed-force measurements directly measured docking and undocking rate constants versus force to determine docking and undocking mechanics, including transition states and pathways, at varying Mn2+ vs Mg2+ concentrations. Simultaneous FRET measurements confirmed specific conformational transitions. Docking and undocking were symmetric processes with nearly equal force sensitivity and a transition state located nearly midway between the docked and undocked states. Mn2+ sensitivity appears to optimize at a concentration where further increasing Mn2+ competes with Mg2+ ions for the non-specific divalent metal ion binding site.

A structure-function analysis of chlorophyllase reveals a mechanism for activity regulation dependent on disulfide bonds

Chlorophyll pigments are used by photosynthetic organisms to facilitate light capture and mediate the conversion of sunlight into chemical energy. Due to the indispensable nature of this pigment and its propensity to form reactive oxygen species, organisms heavily invest in its biosynthesis, recycling, and degradation. One key enzyme implicated in these processes is chlorophyllase, an α/β hydrolase that hydrolyzes the phytol tail of chlorophyll pigments to produce chlorophyllide molecules. This enzyme was discovered a century ago, but despite its importance to diverse photosynthetic organisms, there are still many missing biochemical details regarding how chlorophyllase functions. Here, we present the 4.46-Å resolution crystal structure of chlorophyllase from Triticum aestivum. This structure reveals the dimeric architecture of chlorophyllase, the arrangement of catalytic residues, an unexpected divalent metal ion-binding site, and a substrate-binding site that can accommodate a diverse range of pigments. Further, this structure exhibits the existence of both intermolecular and intramolecular disulfide bonds. We investigated the importance of these architectural features using enzyme kinetics, mass spectrometry, and thermal shift assays. Through this work, we demonstrated that the oxidation state of the Cys residues is imperative to the activity and stability of chlorophyllase, illuminating a biochemical trigger for responding to environmental stress. Additional bioinformatics analysis of the chlorophyllase enzyme family reveals widespread conservation of key catalytic residues and the identified "redox switch" among other plant chlorophyllase homologs, thus revealing key details regarding the structure-function relationships in chlorophyllase.

Minocycline and tigecycline form higher-order Ca2+ complexes of stronger affinity than tetracycline

Tetracycline (TC), minocycline (MIN) and tigecycline (TIG) share a divalent metal ion binding site essential for mediating their antibiotic function. Isothermal titration calorimetry (ITC) and UV–Vis absorbance measurements combined with the Job’s Method were used to study their interaction with Ca2+. 1H NMR was also used to probe the interaction of TIG with Ca2+. Results from ITC showed that MIN and TIG bind Ca2+ more strongly than TC by 10–16 and 8–12 times, respectively, at pH 6.8, or by 23–56 and 20–42 times, respectively, at pH 7.5, in NEM and Tris buffers. The higher affinities can be attributed to the electron donating dimethylamino group at C7 of MIN and TIG which strengthened the Ca2+–antibiotic bonds. Both ITC and the Job’s Method showed that all three antibiotics formed higher order Ca2+ complexes at pH 6.8 in the form of 1Ca2+–3TC, 1Ca2+–3MIN and 2Ca2+–3TIG. As pH was increased to 7.5, TC preferred a lower order complex (1Ca2+–1TC) while MIN and TIG persisted in their respective higher order complex. The preference of MIN and TIG for higher order complexes is attributed to their stronger affinity for Ca2+ and more lipophilic nature. The different complexation mode in the 2Ca2+–3TIG versus 1Ca2+–3MIN complex is attributed to the N-t-butylglycylamido group, which is unique to TIG, participating in Ca2+ coordination along with the β-ketoenol moiety common to all three antibiotics. These results are consistent with TIG having substantially higher affinity for the bacterial ribosome, broader spectrum of antimicrobial activity and evasion of classic tetracycline-resistance mechanisms and with MIN being more effective than TC as an anti-inflammatory drug. These results do not correlate with their relative oral bioavailability. The current findings may aid in the rational design of novel tetracycline derivatives.

Tissue Factor Residues That Modulate Magnesium-Dependent Rate Enhancements of the Tissue Factor/Factor VIIa Complex

The blood coagulation cascade is initiated when the cell-surface complex of factor VIIa (FVIIa, a trypsin-like serine protease) and tissue factor (TF, an integral membrane protein) proteolytically activates factor X (FX). Both FVIIa and FX bind to membranes via their γ-carboxyglutamate-rich domains (GLA domains). GLA domains contain seven to nine bound Ca(2+) ions that are critical for their folding and function, and most biochemical studies of blood clotting have employed supraphysiologic Ca(2+) concentrations to ensure saturation of these domains with bound Ca(2+). Recently, it has become clear that, at plasma concentrations of metal ions, Mg(2+) actually occupies two or three of the divalent metal ion-binding sites in GLA domains, and that these bound Mg(2+) ions are required for full function of these clotting proteins. In this study, we investigated how Mg(2+) influences FVIIa enzymatic activity. We found that the presence of TF was required for Mg(2+) to enhance the rate of FX activation by FVIIa, and we used alanine-scanning mutagenesis to identify TF residues important for mediating this response to Mg(2+). Several TF mutations, including those at residues G164, K166, and Y185, blunted the ability of Mg(2+) to enhance the activity of the TF/FVIIa complex. Our results suggest that these TF residues interact with the GLA domain of FX in a Mg(2+)-dependent manner (although effects of Mg(2+) on the FVIIa GLA domain cannot be ruled out). Notably, these TF residues are located within or immediately adjacent to the putative substrate-binding exosite of TF.

The Chaperone‐like Activity and Structure of Mutant H119G of Rat Lens αB‐crystallin: A Study of Divalent Metal Ion Binding Site

AbstractThe molecular chaperone αB‐crystallin, the major player in maintaining the transparency of the eye lens, preventing the aggregation of stress‐damaged and aging lens proteins from aggregation. In nonlenticular cells, it is involved in various neurological diseases, diabetes, and cancer. The role of some metal ions in the αB‐crystallin biology has been reported. Theoretical calculations have proposed that the coordination sites involving His101, His119, Lys121, His18 and Glu99 of human αB‐crystallin were the binding sites for divalent metal ions. Our previous mutagenesis study suggested that His18 rat lens αB‐crystallin is a crucial binding site for Cu(II) and Zn(II) in terms of chaperone‐like activity and structure. In this study mutant H119G of rat lens αB‐crystalin was cloned and expressed to investigate whether His119 is the coordination binding site. Copper and zinc at 1 mM concentration significantly increase the chaperone‐like activity in wild type αB‐crystalin, whereas zinc, copper and magnesium at 1 mM reduced the activity of H119G significantly. The results from chaperone‐like activity, ANS fluorescence measurement and Far‐and Near‐UV CD studies suggest that the replacement of His119 with Glycine resulted in a conformational and minor environmental changes that decrease chaperone‐like activity in the presence of divalent ions suggested that His119 was a crucial binding site for Cu(II) and Zn(II), which was similar to our previous study results of His18. Both results together suggest that His18 and His119 coordinates each other for the binding site of Cu(II) and Zn(II) in terms of improving the chaperone‐like activity and stability of crystallin/metal ion complex.

Kinetic mechanism of human DNA ligase I

DNA ligase I (Lig1) is essential for DNA replication and repair. It catalyzes the joining of single‐strand DNA breaks via three chemical reactions. In the first step the enzyme is activated by transferring the adenylyl group from ATP to an active site lysine; in the second step the adenylyl group is transferred to the 5′‐phosphate at the nick; in the third step the new phosphodiester bond is formed and AMP is released. We used transient kinetic approaches to determine the minimal kinetic mechanism for human Lig1 in the presence of Mg2+ and other divalent metal ions. Although all three reactions catalyzed by Lig1 require divalent metal ions, individual steps exhibit different affinity for metal and are susceptible to inhibition by different metal ions. These different patterns of inhibition suggest that there are multiple divalent metal ion binding sites and that the metal sites change throughout the reaction cycle. This work provides deeper insight into the mechanism of DNA ligation and illustrates how divalent metal ions can inhibit a single enzyme via very different mechanisms. The results have implications for abortive ligation, which generates blocked (adenylylated) 5′ends and for the inhibition of DNA ligation by transition metals.

The C-terminal domain of the 2b protein of Cucumber mosaic virus is stabilized by divalent metal ion coordination

The main function of the 2b protein of Cucumber mosaic virus (CMV) is binding permanently the double stranded siRNA molecules in the suppression process of post-transcriptional gene silencing (PTGS). The crystal structure of the homologue Tomato aspermy virus (TAV) 2b protein is known, but without the C-terminal domain. The biologically active form is a tetramer: four 2b protein molecules and two siRNA duplexes. Regarding the complete 2b protein structure, we performed a molecular dynamics (MD) simulation of the whole siRNA–2b ribonucleoprotein complex. Unfortunately, the C-terminal domain is proved to be partially unstructured. Multiple sequence alignment showed a well conserved motif between residues 94 and 105. The negatively charged residues of the C-terminal domain are supposed to take part in coordination of a divalent metal ion and stabilize the three-dimensional structure of the C-terminal domain. MD simulations were performed on the detached C-terminal domains (aa 65–110). 0.15M MgCl2, CaCl2, FeCl2 and ZnCl2 salt concentrations were used in the screening simulations. Among the tested divalent metal ions Mg2+ proved to be very successful because Asp95, Asp96 and Asp98 forms a quasi-permanent Mg2+ binding site. However the control computations have resulted in any (at least) divalent metal ion remains in the binding site after replacement of the bound Mg2+ ion. A quadruple mutation (Rs2DDTD/95–98/AAAA) was introduced into the position of the putative divalent metal ion binding site to analyze the biological relevance of molecular modeling derived hypothesis. The plant inoculation experiments proved that the movement of the mutant virus is slower and the symptoms are milder comparing to the wild type virus. These results demonstrate that the quadruple mutation weakens the stability of the 2b protein tetramer–siRNA ribonucleoprotein complex.

Crystal Structure Analysis Reveals Functional Flexibility in the Selenocysteine-Specific tRNA from Mouse

BackgroundSelenocysteine tRNAs (tRNASec) exhibit a number of unique identity elements that are recognized specifically by proteins of the selenocysteine biosynthetic pathways and decoding machineries. Presently, these identity elements and the mechanisms by which they are interpreted by tRNASec-interacting factors are incompletely understood.Methodology/Principal FindingsWe applied rational mutagenesis to obtain well diffracting crystals of murine tRNASec. tRNASec lacking the single-stranded 3′-acceptor end (ΔGCCARNASec) yielded a crystal structure at 2.0 Å resolution. The global structure of ΔGCCARNASec resembles the structure of human tRNASec determined at 3.1 Å resolution. Structural comparisons revealed flexible regions in tRNASec used for induced fit binding to selenophosphate synthetase. Water molecules located in the present structure were involved in the stabilization of two alternative conformations of the anticodon stem-loop. Modeling of a 2′-O-methylated ribose at position U34 of the anticodon loop as found in a sub-population of tRNASec in vivo showed how this modification favors an anticodon loop conformation that is functional during decoding on the ribosome. Soaking of crystals in Mn2+-containing buffer revealed eight potential divalent metal ion binding sites but the located metal ions did not significantly stabilize specific structural features of tRNASec.Conclusions/SignificanceWe provide the most highly resolved structure of a tRNASec molecule to date and assessed the influence of water molecules and metal ions on the molecule's conformation and dynamics. Our results suggest how conformational changes of tRNASec support its interaction with proteins.

Structural characterization of tartrate dehydrogenase: a versatile enzyme catalyzing multiple reactions.

The first structure of an NAD-dependent tartrate dehydrogenase (TDH) has been solved to 2 A resolution by single anomalous diffraction (SAD) phasing as a complex with the intermediate analog oxalate, Mg(2+) and NADH. This TDH structure from Pseudomonas putida has a similar overall fold and domain organization to other structurally characterized members of the hydroxy-acid dehydrogenase family. However, there are considerable differences between TDH and these functionally related enzymes in the regions connecting the core secondary structure and in the relative positioning of important loops and helices. The active site in these complexes is highly ordered, allowing the identification of the substrate-binding and cofactor-binding groups and the ligands to the metal ions. Residues from the adjacent subunit are involved in both the substrate and divalent metal ion binding sites, establishing a dimer as the functional unit and providing structural support for an alternating-site reaction mechanism. The divalent metal ion plays a prominent role in substrate binding and orientation, together with several active-site arginines. Functional groups from both subunits form the cofactor-binding site and the ammonium ion aids in the orientation of the nicotinamide ring of the cofactor. A lysyl amino group (Lys192) is the base responsible for the water-mediated proton abstraction from the C2 hydroxyl group of the substrate that begins the catalytic reaction, followed by hydride transfer to NAD. A tyrosyl hydroxyl group (Tyr141) functions as a general acid to protonate the enolate intermediate. Each substrate undergoes the initial hydride transfer, but differences in substrate orientation are proposed to account for the different reactions catalyzed by TDH.

Structural Basis for DNase Activity of a Conserved Protein Implicated in CRISPR-Mediated Genome Defense

Acquired immunity in prokaryotes is achieved by integrating short fragments of foreign nucleic acids into clustered regularly interspaced short palindromic repeats (CRISPRs). This nucleic acid-based immune system is mediated by a variable cassette of up to 45 protein families that represent distinct immune system subtypes. CRISPR-associated gene 1 (cas1) encodes the only universally conserved protein component of CRISPR immune systems, yet its function is unknown. Here we show that the Cas1 protein is a metal-dependent DNA-specific endonuclease that produces double-stranded DNA fragments of approximately 80 base pairs in length. The 2.2 A crystal structure of the Cas1 protein reveals a distinct fold and a conserved divalent metal ion-binding site. Mutation of metal ion-binding residues, chelation of metal ions, or metal-ion substitution inhibits Cas1-catalyzed DNA degradation. These results provide a foundation for understanding how Cas1 contributes to CRISPR function, perhaps as part of the machinery for processing foreign nucleic acids.

Identification of neomycin B-binding site in T box antiterminator model RNA

The T box transcription antitermination mechanism regulates the expression of unique genes in many Gram-positive bacteria by responding, in a magnesium-dependent manner, to uncharged cognate tRNA base pairing with an antiterminator RNA element and other regions of the 5′-untranslated region. Model T box antiterminator RNA is known to bind aminoglycosides, ligands that typically bind RNA in divalent metal ion-binding sites. In this study, enzymatic footprinting and spectroscopic assays were used to identify and characterize the binding site of neomycin B to an antiterminator model RNA. Neomycin B binds the antiterminator bulge nucleotides in an electrostatic-dependent manner and displaces 3–4 monovalent cations, indicating that the antiterminator likely contains a divalent metal ion-binding site. Neomycin B facilitates rather than inhibits tRNA binding indicating that bulge-targeted inhibitors that bind the antiterminator via non-electrostatic interactions may be the more optimal candidates for antiterminator-targeted ligand design.

Identification of a Novel Nonstructural Protein, VP9, from White Spot Syndrome Virus: Its Structure Reveals a Ferredoxin Fold with Specific Metal Binding Sites

White spot syndrome virus (WSSV) is a major pathogen in shrimp aquaculture. VP9, a full-length protein of WSSV, encoded by open reading frame wsv230, was identified for the first time in the infected Penaeus monodon shrimp tissues, gill, and stomach as a novel, nonstructural protein by Western blotting, mass spectrometry, and immunoelectron microscopy. Real-time reverse transcription-PCR demonstrated that the transcription of VP9 started from the early to the late stage of WSSV infection as a major mRNA species. The structure of full-length VP9 was determined by both X-ray and nuclear magnetic resonance (NMR) techniques. It is the first structure to be reported for WSSV proteins. The crystal structure of VP9 revealed a ferredoxin fold with divalent metal ion binding sites. Cadmium sulfate was found to be essential for crystallization. The Cd2+ ions were bound between the monomer interfaces of the homodimer. Various divalent metal ions have been titrated against VP9, and their interactions were analyzed using NMR spectroscopy. The titration data indicated that VP9 binds with both Zn2+ and Cd2+. VP9 adopts a similar fold as the DNA binding domain of the papillomavirus E2 protein. Based on our present investigations, we hypothesize that VP9 might be involved in the transcriptional regulation of WSSV, a function similar to that of the E2 protein during papillomavirus infection of the host cells.

Principles of RNA Compaction: Insights from the Equilibrium Folding Pathway of the P4-P6 RNA Domain in Monovalent Cations

Counterions are required for RNA folding, and divalent metal ions such as Mg(2+) are often critical. To dissect the role of counterions, we have compared global and local folding of wild-type and mutant variants of P4-P6 RNA derived from the Tetrahymena group I ribozyme in monovalent and in divalent metal ions. A remarkably simple picture of the folding thermodynamics emerges. The equilibrium folding pathway in monovalent ions displays two phases. In the first phase, RNA molecules that are initially in an extended conformation enforced by charge-charge repulsion are relaxed by electrostatic screening to a state with increased flexibility but without formation of long-range tertiary contacts. At higher concentrations of monovalent ions, a state that is nearly identical to the native folded state in the presence of Mg(2+) is formed, with tertiary contacts that involve base and backbone interactions but without the subset of interactions that involve specific divalent metal ion-binding sites. The folding model derived from these and previous results provides a robust framework for understanding the equilibrium and kinetic folding of RNA.

Recruitment of divalent metal ions by incorporation of 4-thio-2'-deoxythymidine or 4-thio-2'-deoxyuridine into DNA.

The modified nucleosides 4-thio-2'-deoxyuridine (s4dU) and 4-thio-2'-deoxythymidine (s4dT) are incorporated into dinucleosides, and s4dT is incorporated into a DNA hairpin loop to provide divalent metal ion binding sites. Binding of two different metal ions to these sites is studied, including Cd(II) as an NMR spectroscopy probe and Cu(II) as a reactive metal ion for DNA cleavage. Binding of Cd(II) to 4-thiouridine (s4U) and s4dT nucleosides, s4dU- and s4dT-containing dinucleosides, and a hairpin loop oligonucleotide containing s4dT is monitored by following the change in UV-vis absorbance of the thionucleosides at 340 nm and 21 degrees C in solutions containing 20.0-40 mM buffer, 1.00 M NaCl, and 15.0 mM BaCl2. Cd(II) binds to the N3 deprotonated form of s4dT with a binding constant (K = 1.1 x 10(4) M(-1)) that is similar to that for Cd(II) binding to d(Tps4T) (K = 9.2 x 10(3) M(-1)). Apparent binding constants (Kapp) at pH 7.7 of Cd(II) to dinucleosides d(Gps4T), d(s4TpG), and d(Gps4U) are similar to those of their respective nucleosides s4U and s4dT, suggesting that neither the phosphate diester nor the second nucleoside has a major effect on Cd(II) binding. Binding of Cd(II) to s4U and d(Gps4U) is studied by use of 113Cd NMR and 1H NMR spectroscopy, respectively. Binding strength and stoichiometry of the Cd(II) complex with d(Gps4U) as studied by 1H NMR spectroscopy are similar to that obtained by UV-vis spectroscopy. Cd(II) binds strongly to s4dT in the loop portion of a DNA hairpin loop (Kapp = 2.7 x 10(3) M(-1) at pH 7.7). However, the hairpin loop is moderately destabilized by Cd(II) binding, with a decrease in T(m) of 14 degrees C in the presence of 10.0 mM Cd(II) as determined by optical melting experiments. Cu(II) oxidizes s4dT to form the disulfide of s4dT, limiting the usefulness of further studies with Cu(II).

Mapping divalent metal ion binding sites in a group II intron by Mn(2+)- and Zn(2+)-induced site-specific RNA cleavage.

The function of group II introns depends on positively charged divalent metal ions that stabilize the ribozyme structure and may be directly involved in catalysis. We investigated Mn2+- and Zn2+-induced site-specific RNA cleavage to identify metal ions that fit into binding pockets within the structurally conserved bI1 group II intron domains (DI-DVI), which might fulfill essential roles in intron function. Ten cleavage sites were identified in DI, two sites in DIII and two in DVI. All cleavage sites are located in the center or close to single-stranded and flexible RNA structures. Strand scissions mediated by Mn2+/Zn2+ are competed for by Mg2+, indicating the existence of Mg2+ binding pockets in physical proximity to the observed Mn2+-/Zn2+-induced cleavage positions. To distinguish between metal ions with a role in structure stabilization and those that play a more specific and critical role in the catalytic process of intron splicing, we combined structural and functional assays, comparing wild-type precursor and multiple splicing-deficient mutants. We identified six regions with binding pockets for Mg2+ ions presumably playing an important role in bI1 structure stabilization. Remarkably, assays with DI deletions and branch point mutants revealed the existence of one Mg2+ binding pocket near the branching A, which is involved in first-step catalysis. This pocket formation depends on precise interaction between the branching nucleotide and the 5' splice site, but does not require exon-binding site 1/intron binding site 1 interaction. This Mg2+ ion might support the correct placing of the branching A into the 'first-step active site'.

Aminoglycoside binding displaces a divalent metal ion in a tRNA-neomycin B complex.

Aminoglycosides bind to RNA and interfere with its function, and it has been suggested that aminoglycoside binding to RNA displaces essential divalent metal ions. Here we demonstrate that addition of various aminoglycosides inhibited Pb2+-induced cleavage of yeast tRNA(Phe). Cocrystallization of yeast tRNA(Phe) and an aminoglycoside, neomycin B, resulted in crystals that diffracted to 2.6 A and the structure of the complex was solved by molecular replacement. The structure shows that the neomycin B binding site overlaps with known divalent metal ion binding sites in yeast tRNA(Phe), providing direct evidence for the hypothesis that aminoglycosides displace metal ions. Additionally, the neomycin B binding site overlaps with major determinants for Escherichia coli phenylalanyl-tRNA-synthetase. Here we present data demonstrating that addition of neomycin B inhibited aminoacylation of E. coli tRNA(Phe) in the mid microM range. Given that aminoglycoside and metal ion binding sites overlap, we discuss that aminoglycosides can be considered as 'metal mimics'.

Monitoring the structure of Escherichia coli RNase P RNA in the presence of various divalent metal ions.

Lead(II)-induced cleavage can be used as a tool to probe conformational changes in RNA. In this report, we have investigated the conformation of M1 RNA, the catalytic subunit of Escherichia coli RNase P, by studying the lead(II)-induced cleavage pattern in the presence of various divalent metal ions. Our data suggest that the overall conformation of M1 RNA is very similar in the presence of Mg(2+), Mn(2+), Ca(2+), Sr(2+) and Ba(2+), while it is changed compared to the Mg(2+)-induced conformation in the presence of other divalent metal ions, Cd(2+) for example. We also observed that correct folding of some M1 RNA domains is promoted by Pb(2+), while folding of other domain(s) requires the additional presence of other divalent metal ions, cobalt(III) hexamine or spermidine. Based on the suppression of Pb(2+) cleavage at increasing concentrations of various divalent metal ions, our findings suggest that different divalent metal ions bind with different affinities to M1 RNA as well as to an RNase P hairpin-loop substrate and yeast tRNA(Phe). We suggest that this approach can be used to obtain information about the relative binding strength for different divalent metal ions to RNA in general, as well as to specific RNA divalent metal ion binding sites. Of those studied in this report, Mn(2+) is generally among the strongest RNA binders.

Mechanism of action of Escherichia coli ribonuclease III. Stringent chemical requirement for the glutamic acid 117 side chain and Mn2+ rescue of the Glu117Asp mutant.

Escherichia coli ribonuclease III (EC 3.1.24) is a double-strand- (ds-) specific endoribonuclease involved in the maturation and decay of cellular, phage, and plasmid RNAs. RNase III is a homodimer and requires Mg(2+) to hydrolyze phosphodiesters. The RNase III polypeptide contains an N-terminal catalytic (nuclease) domain which exhibits eight highly conserved acidic residues, at least one of which (Glu117) is important for phosphodiester hydrolysis but not for substrate binding [Li and Nicholson (1996) EMBO J. 15, 1421-1433]. To determine the side chain requirements for activity, Glu117 was changed to glutamine or aspartic acid. The mutant proteins were purified as (His)(6)-tagged species, and both exhibited normal homodimeric behavior as shown by chemical cross-linking. The Glu117Gln mutant is unable to cleave substrate in vitro under all tested conditions but can bind substrate. The Glu117Asp mutant also is defective in cleavage while able to bind substrate. However, low level activity is observed at extended reaction times and high enzyme concentrations, with an estimated catalytic efficiency approximately 15 000-fold lower than that of RNase III. The activity of the Glu117Asp mutant but not that of the Glu117Gln mutant can be greatly enhanced by substituting Mn(2+) for Mg(2+), with the catalytic efficiency of the Glu117Asp-Mn(2+) holoenzyme approximately 400-fold lower than that of the RNase III-Mn(2+) holoenzyme. For RNase III, a Mn(2+) concentration of 1 mM provides optimal activity, while concentrations >5 mM are inhibitory. In contrast, the Glu117Asp mutant is not inhibited by high concentrations of Mn(2+). Finally, high concentrations of Mg(2+) do not inhibit RNase III nor relieve the Mn(2+)-dependent inhibition. In summary, these experiments establish the stringent functional requirement for a precisely positioned carboxylic acid group at position 117 and reveal two classes of divalent metal ion binding sites on RNase III. One site binds either Mg(2+) or Mn(2+) and supports catalysis, while the other site is specific for Mn(2+) and confers inhibition. Glu117 is important for the function of both sites. The implications of these findings on the RNase III catalytic mechanism are discussed.

Helix P4 is a divalent metal ion binding site in the conserved core of the ribonuclease P ribozyme.

The ribonuclease P ribozyme (RNase P RNA), like other large ribozymes, requires magnesium ions for folding and catalytic function; however, specific sites of metal ion coordination in RNase P RNA are not well defined. To identify and characterize individual nucleotide functional groups in the RNase P ribozyme that participate in catalytic function, we employed self-cleaving ribozyme-substrate conjugates that facilitate measurement of the effects of individual functional group modifications. The self-cleavage rates and pH dependence of two different ribozyme-substrate conjugates were determined and found to be similar to the single turnover kinetics of the native ribozyme. Using site-specific phosphorothioate substitutions, we provide evidence for metal ion coordination at the pro-Rp phosphate oxygen of A67, in the highly conserved helix P4, that was previously suggested by modification-interference experiments. In addition, we detect a new metal ion coordination site at the pro-Sp phosphate oxygen of A67. These findings, in combination with the proximity of A67 to the pre-tRNA cleavage site, support the conclusion that an important role of helix P4 in the RNase P ribozyme is to position divalent metal ions that are required for catalysis.