The P4 metal binding site in RNase P RNA affects active site metal affinity through substrate positioning

Although helix P4 in the catalytic domain of the RNase P ribozyme is known to coordinate magnesium ions important for activity, distinguishing between direct and indirect roles in catalysis has been difficult. Here, we provide evidence for an indirect role in catalysis by showing that while the universally conserved bulge of helix P4 is positioned 5 nt downstream of the cleavage site, changes in its structure can still purturb active site metal binding. Because changes in helix P4 also appear to alter its position relative to the pre-tRNA cleavage site, these data suggest that P4 contributes to catalytic metal ion binding through substrate positioning.     

The P15-loop of Escherichia coli RNase P RNA is an autonomous divalent metal ion binding domain.

We have studied the structure and divalent metal ion binding of a domain of the ribozyme RNase P RNA that is involved in base pairing with its substrate. Our data suggest that the folding of this internal loop, the P15-loop, is similar irrespective of whether it is part of the full-length ribozyme or part of a model RNA molecule. We also conclude that this element constitutes an autonomous divalent metal ion binding domain of RNase P RNA and our data suggest that certain specific chemical groups within the P15-loop participate in coordination of divalent metal ions. Substitutions of the Sp- and Rp-oxygens with sulfur at a specific position in this loop result in a 2.5-5-fold less active ribozyme, suggesting that Mg2+ binding at this position contributes to function. Our findings strengthen the concept that small RNA building blocks remain basically unchanged when removed from their structural context and thus can be used as models for studies of their potential function and structure within native RNA molecules.     

Dynamics and metal ion binding in the U6 RNA intramolecular stem-loop as analyzed by NMR

The U6 RNA intramolecular stem-loop (ISL) is a conserved component of the spliceosome, and contains an essential metal ion binding site centered between a protonated adenine, A79, and U80. Correlated with protonation of A79, U80 undergoes a base-flipping conformational change accompanied by significant helical movement. We have investigated the dynamics of the U6 ISL by analyzing the power dependence of 13C NMR relaxation rates in the rotating frame. The data provide evidence that the conformational transition is centered around an exchange lifetime of 84 micros. The U80 nucleotide displays low internal mobility on the picosecond time-scale at pH 7.0 but high internal mobility at pH 6.0, in agreement with the global transition resulting in the base of U80 adopting a looped-out conformation with increased dynamic disorder. A kinetic analysis suggests that the conformational change, rather than adenine protonation, is the rate-limiting step in the pathway of the conformational transition. Two nucleotides, U70 and U80, were found from chemical shift perturbation mapping to interact with the magnesium ion, with apparent K(d) values in the micromolar to millimolar range. These nucleotides also displayed metal ion-induced elevation of R1 rates, which can be explained by a model that assumes dynamic metal ion coordination concomitant with an induced higher shielding anisotropy for the base 13C nuclei. Addition of Mg2+ shifts the conformational equilibrium toward the high-pH (base-stacked) structure, accompanied by a significant drop in the apparent pK(a) of A79.     

Studying metal ion binding properties of a three-way junction RNA by heteronuclear NMR

Self-splicing group II introns are highly structured RNA molecules, containing a characteristic secondary and catalytically active tertiary structure, which is formed only in the presence of Mg(II). Mg(II) initiates the first folding step governed by the κζ element within domain 1 (D1κζ). We recently solved the NMR structure of D1κζ derived from the mitochondrial group II intron ribozyme Sc.ai5γ and demonstrated that Mg(II) is essential for its stabilization. Here, we performed a detailed multinuclear NMR study of metal ion interactions with D1κζ, using Cd(II) and cobalt(III)hexammine to probe inner- and outer-sphere coordination of Mg(II) and thus to better characterize its binding sites. Accordingly, we mapped ¹H, ¹⁵N, ¹³C, and ³¹P spectral changes upon addition of different amounts of the metal ions. Our NMR data reveal a Cd(II)-assisted macrochelate formation at the 5′-end triphosphate, a preferential Cd(II) binding to guanines in a helical context, an electrostatic interaction in the ζ tetraloop receptor and various metal ion interactions in the GAAA tetraloop and κ element. These results together with our recently published data on Mg(II) interaction provide a much better understanding of Mg(II) binding to D1κζ, and reveal how intricate and complex metal ion interactions can be.     

Solution structure and metal-ion binding of the P4 element from bacterial RNase P RNA

We determined the solution structure of two 27-nt RNA hairpins and their complexes with cobalt(III)-hexammine (Co(NH3)3+(6)) by NMR spectroscopy. The RNA hairpins used in this study are the P4 region from Escherichia coli RNase P RNA and a C-to-U mutant that confers altered divalent metal-ion specificity (Ca2+ replaces Mg2+) for catalytic activity of this ribozyme. Co(NH3)3+(6) is a useful spectroscopic probe for Mg(H2O)2+(6)-binding sites because both complexes have octahedral symmetry and have similar radii. The thermodynamics of binding to both RNA hairpins was studied using chemical shift changes upon titration with Mg2+, Ca2+, and Co(NH3)3+(6). We found that the equilibrium binding constants for each of the metal ions was essentially unchanged when the P4 model RNA hairpin was mutated, although the NMR structures show that the RNA hairpins adopt different conformations. In the C-to-U mutant a C.G base pair is replaced by U.G, and the conserved bulged uridine in the P4 wild-type stem shifts in the 3' direction by 1 nt. Intermolecular NOE cross-peaks between Co(NH3)3+(6) and RNA protons were used to locate the site of Co(NH3)3+(6) binding to both RNA hairpins. The metal ion binds in the major groove near a bulge loop, but is shifted 5' by more than 1 bp in the mutant. The change of the metal-ion binding site provides a possible explanation for changes in catalytic activity of the mutant RNase P in the presence of Ca2+.     

Rp-phosphorothioate modifications in RNase P RNA that interfere with tRNA binding.

We have used Rp-phosphorothioate modifications and a binding interference assay to analyse the role of phosphate oxygens in tRNA recognition by Escherichia coli ribonuclease P (RNase P) RNA. Total (100%) Rp-phosphorothioate modification at A, C or G positions of RNase P RNA strongly impaired tRNA binding and pre-tRNA processing, while effects were less pronounced at U positions. Partially modified E. coli RNase P RNAs were separated into tRNA binding and non-binding fractions by gel retardation. Rp-phosphorothioate modifications that interfered with tRNA binding were found 5' of nucleotides A67, G68, U69, C70, C71, G72, A130, A132, A248, A249, G300, A317, A330, A352, C353 and C354. Manganese rescue at positions U69, C70, A130 and A132 identified, for the first time, sites of direct metal ion coordination in RNase P RNA. Most sites of interference are at strongly conserved nucleotides and nine reside within a long-range base-pairing interaction present in all known RNase P RNAs. In contrast to RNase P RNA, 100% Rp-phosphorothioate substitutions in tRNA showed only moderate effects on binding to RNase P RNAs from E. coli, Bacillus subtilis and Chromatium vinosum, suggesting that pro-Rp phosphate oxygens of mature tRNA contribute relatively little to the formation of the tRNA-RNase P RNA complex.     

The 1.3 A resolution structure of the RNA tridecamer r(GCGUUUGAAACGC): metal ion binding correlates with base unstacking and groove contraction

Metal ions play a key role in RNA folding and activity. Elucidating the rules that govern the binding of metal ions is therefore an essential step for better understanding the RNA functions. High-resolution data are a prerequisite for a detailed structural analysis of ion binding on RNA and, in particular, the observation of monovalent cations. Here, the high-resolution crystal structures of the tridecamer duplex r(GCGUUUGAAACGC) crystallized under different conditions provides new structural insights on ion binding on GAAA/UUU sequences that exhibit both unusual structural and functional properties in RNA. The present study extends the repertory of RNA ion binding sites in showing that the two first bases of UUU triplets constitute a specific site for sodium ions. A striking asymmetric pattern of metal ion binding in the two equivalent halves of the palindromic sequence demonstrates that sequence and its environment act together to bind metal ions. A highly ionophilic half that binds six metal ions allows, for the first time, the observation of a disodium cluster in RNA. The comparison of the equivalent halves of the duplex provides experimental evidences that ion binding correlates with structural alterations and groove contraction.     

Interaction of a goose-type lysozyme with chitin oligosaccharides as determined by NMR spectroscopy

The interaction between a goose-type lysozyme from ostrich egg white (OEL) and chitin oligosaccharides [(GlcNAc)(n) (n = 2, 4 and 6)] was studied by nuclear magnetic resonance (NMR) spectroscopy. A stable isotope-labelled OEL was produced in Pichia pastoris, and backbone resonance assignments for the wild-type and an inactive mutant (E73A OEL) were achieved using modern multi-dimensional NMR techniques. NMR titration was performed with (GlcNAc)(n) for mapping the interaction sites of the individual oligosaccharides based on the shifts in the two-dimensional heteronuclear single quantum correlation (HSQC) resonances. In wild-type OEL, the interaction sites for (GlcNAc)(n) were basically similar to those determined by X-ray crystallography. In E73A OEL, however, the interaction sites were spread more widely over the substrate-binding cleft than expected, due to the multiple modes of binding. The association constant for E73A OEL and (GlcNAc)(6) calculated from the shifts in the Asp97 resonance (7.2 × 10(3) M(-1)) was comparable with that obtained by isothermal titration calorimetry (5.3 × 10(3) M(-1)). The interaction was enthalpy-driven as judged from the thermodynamic parameters (ΔH = -6.1 kcal/mol and TΔS = -1.0 kcal/mol). This study provided novel insights into the oligosaccharide binding mechanism and the catalytic residues of the enzymes belonging to family GH-23.     

Evidence for a polynuclear metal ion binding site in the catalytic domain of ribonuclease P RNA

Interactions with divalent metal ions are essential for the folding and function of the catalytic RNA component of the tRNA processing enzyme ribonuclease P (RNase P RNA). However, the number and location of specific metal ion interactions in this large, highly structured RNA are poorly understood. Using atomic mutagenesis and quantitative analysis of thiophilic metal ion rescue we provide evidence for metal ion interactions at the pro-R(P) and pro-S(P) non-bridging phosphate oxygens at nucleotide A67 in the universally conserved helix P4. Moreover, second-site modifications within helix P4 and the adjacent single stranded region (J3/4) provide the first evidence for metal ion interactions with nucleotide base functional groups in RNase P RNA and reveal the presence of an additional metal ion important for catalytic function. Together, these data are consistent with a cluster of metal ion interactions in the P1-P4 multi-helix junction that defines the catalytic core of the RNase P ribozyme.     

Solution structure of human Mts1 (S100A4) as determined by NMR spectroscopy

Mts1 is a member of the S100 family of Ca2+-binding proteins and is implicated in promoting tumor progression and metastasis. To better understand the structure-function relationships of this protein and to begin characterizing its Ca2+-dependent interaction with protein binding targets, the three-dimensional structure of mts1 was determined in the apo state by NMR spectroscopy. As with other S100 protein family members, mts1 is a symmetric homodimer held together by noncovalent interactions between two helices from each subunit (helices 1, 4, 1', and 4') to form an X-type four-helix bundle. Each subunit of mts1 has two EF-hand Ca2+-binding domains: a pseudo-EF-hand (or S100-hand) and a typical EF-hand that are brought into proximity by a small two-stranded antiparallel beta-sheet. The S100-hand is formed by helices 1 and 2, and is similar in conformation to other members of the S100 family. In the typical EF-hand, the position of helix 3 is similar to that of another member of the S100 protein family, calcyclin (S100A6), and less like that of other S100 family members for which three-dimensional structures are available in the calcium-free state (e.g., S100B and S100A1). The differences in the position of helix 3 in the apo state of these four S100 proteins are likely due to variations in the amino acid sequence in the C-terminus of helix 4 and in loop 2 (the hinge region) and could potentially be used to subclassify the S100 protein family.     

Effect of metal ion binding on the secondary structure of bovine alpha-lactalbumin as examined by infrared spectroscopy

We have examined the influence of monovalent and divalent cations on the secondary structure of bovine alpha-lactalbumin at neutral pH using Fourier-transform infrared spectroscopy. Our present studies are based on previously reported amide I' component band assignments for this protein [Prestrelski, S. J., Byler, D. M., & Thompson, M. P. (1991) Int. J. Pept. Protein Res. 37, 508-512]. The results indicate that upon dissolution, alpha-lactalbumin undergoes a small, but significant, time-dependent conformational change, regardless of the ions present. Additionally, these studies provide the first quantitative measure of the well-known secondary structural change which accompanies calcium binding. Results indicate that removal of Ca2+ from holo alpha-lactalbumin results in local unfolding of the Ca(2+)-binding loop; the spectra indicate that approximately 16% of the backbone chain changes from a rigid coordination complex to an unordered loop. We have also examined the effects of binding of several other metal ions. Our studies have revealed that binding of Mn2+ to apo alpha-lactalbumin (Ca(2+)-free), while inducing a small, but significant, conformational change, does not cause the alpha-lactalbumin backbone conformation to change to that of the holo (Ca(2+)-bound) form as characterized by infrared spectroscopy. Similar changes to those induced by Mn2+ are observed upon binding of Na+ to apo alpha-lactalbumin, and furthermore, even at very high concentrations (0.2 M), Na+ does not stabilize a structure similar to the holo form. Binding of Zn2+ to the apo form of alpha-lactalbumin does not result in significant backbone conformational changes, suggesting a rigid Zn(2+)-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of metal ion binding sites within the hairpin ribozyme domains by NMR

Cations play an important role in RNA folding and stabilization. The hairpin ribozyme is a small catalytic RNA consisting of two domains, A and B, which interact in the transition state in an ion-dependent fashion. Here we describe the interaction of mono-, di-, and trivalent cations with the domains of the ribozyme, as studied by homo- and heteronuclear NMR spectroscopy. Paramagnetic line broadening, chemical shift mapping, and intermolecular NOEs indicate that the B domain contains four to five metal binding sites, which bind Mn(2+), Mg(2+), and Co(NH(3))(6)(3+). There is no significant structural change in the B domain upon the addition of Co(NH(3))(6)(3+) or Mg(2+). No specific monovalent ion binding sites exist on the B domain, as determined by (15)NH(4)(+) binding studies. In contrast to the B domain, there are no observable metal ion interactions within the internal loop of the A domain. Model structure calculations of Mn(2+) interactions at two sites within the B domain indicate that the binding sites comprise major groove pockets lined with functional groups oriented so that multiple hydrogen bonds can be formed between the RNA and Mn(H(2)O)(6)(2+) or Co(NH(3))(6)(3+). Site 1 is very similar in geometry to a site within the P4-P6 domain of the Tetrahymena group I intron, while site 2 is unique among known ion binding sites. The site 2 ion interacts with a catalytically essential nucleotide and bridges two phosphates. Due to its location and geometry, this ion may play an important role in the docking of the A and B domains.     

Metal ion binding properties of hen ovalbumin and S-ovalbumin: characterization of the metal ion binding site by 31P NMR and water proton relaxation rate enhancements

In this study, water proton relaxation rate (PRR) enhancements have been used to characterize the binding of metal ions to native ovalbumin, ovalbumin in which phosphate has been enzymatically cleaved from one or both of the two protein phosphoserines, and a heat-stabilized form of the protein (S-ovalbumin). With Scatchard plots constructed from water PRR enhancements, it was found that native ovalbumin and S-ovalbumin had one strong binding site for Mn2+ ion (KD approximately equal to 6.0 X 10(-4) M). Alkaline phosphatase treated ovalbumin, a protein having a single phosphoserine, had one Mn2+ binding site of slightly weaker affinity (KD approximately equal to 8.3 X 10(-4) M), while acid phosphatase treated ovalbumin, a dephosphorylated protein, had two much weaker Mn2+ ion binding sites (KD approximately equal to 1.3 X 10(-3) M). Competitive binding studies on the native protein suggested that Zn2+ ion competes with Mn2+ for the single strong-affinity site (KD approximately equal to 6.1 X 10(-3) M) while Mg2+ and Ca2+ do not. In a second set of experiments, the paramagnetic contribution to the 31P spin-lattice (T1P) and spin-spin (T2P) relaxation times at three separate magnetic field strengths was measured. Correlation times tau c characterizing Mn2+-31P dipolar relaxation were estimated from the ratios of T1P/T2P at a single field and from the ratios of spin-lattice relaxation rates at three different field strengths. The correlation times so obtained, ranging from about 0.7 to 7.7 ns at the three field strengths, were used in calculating distances from the bound Mn2+ ion to the phosphoserines of native ovalbumin, S-ovalbumin, and alkaline phosphatase treated ovalbumins. It was determined that the phosphate of phosphoserine-68 was 5.95 +/- 0.26 and 6.29 +/- 0.18 A from the Mn2+ in the native and alkaline phosphatase treated protein, respectively, and 6.99 +/- 0.30 A away from the Mn2+ in S-ovalbumin. The phosphate of phosphoserine-344 was determined to be 5.31 +/- 0.20 and 5.75 +/- 0.10 A from the Mn2+ ion in native ovalbumin and S-ovalbumin, respectively. The 13C nucleus of [1-13C]galactose enzymatically transferred to the nonreducing end of the ovalbumin oligosaccharide chain was not found to be significantly relaxed by Mn2+ bound to the protein, even at 1:1 stoichiometric ratio of metal:protein. Using this, we estimate the nonreducing terminal of the ovalbumin oligosaccharide to be at least 39 A from the metal ion binding site on the protein.     

The composition of 2-keto aldoses in organic solvents as determined by NMR spectroscopy

The composition of the 2-keto aldoses D-glucosone (1), 6-deoxy-D-glucosone (2), D-allosone (3), and D-galactosone (4) in organic solvents has been determined using NMR spectroscopy. Whereas these keto aldoses form mixtures with up to 15 different isomers in water, the number of forms is significantly decreased in organic solvents. Equilibrium mixtures of 1, 2, and 4 in Me(2)SO, DMF, and pyridine consist to 70-90% of the prevailing alpha-1,5-pyranose form. Two bicyclic forms with a proportion of 80% are the main isomers of 3 in pyridine. Generally, forms with non-hydrated keto functions prevail in non-aqueous solutions.     

Surface potential effects on metal ion binding to phosphatidylcholine membranes 31P NMR study of lanthanide and calcium ion binding to egg-yolk lecithin vesicles

31P NMR of phosphatidylcholine (lecithin) from egg-yolk in sonicated vesicles has been measured in the presence of various ions. Addition of Ln3+ or Ca2+ shifted the 31P resonance of the phosphate groups of the outer surface of the vesicles. These shifts were measured at varied lanthanide or Ca2+ concentration at different ionic strengths obtained by addition of NaCl. The shifts induced by Tb3+ and Ca2+ have been analyzed using the theory of the diffuse double layer. Corrections were introduced for the effect of the ionic strength on the activities of the ions. The binding efficiency is shown to be controlled by the electrostatic potential produced by the bound cations at the membrane surface. This potential is slightly modified due to weak chloride binding. Binding constants have been derived.     

Importance of the +73/294 interaction in Escherichia coli RNase P RNA substrate complexes for cleavage and metal ion coordination

We have studied an interaction, the "73/294-interaction", between residues 294 in M1 RNA (the catalytic subunit of Escherichia coli RNase P) and +73 in the tRNA precursor substrate. The 73/294-interaction is part of the "RCCA-RNase P RNA interaction", which anchors the 3' R(+73)CCA-motif of the substrate to M1 RNA (interacting residues underlined). Considering that in a large fraction of tRNA precursors residue +73 is base-paired to nucleotide -1 immediately 5' of the cleavage site, formation of the 73/294-interaction results in exposure of the cleavage site. We show that the nature/orientation of the 73/294-interaction is important for cleavage site recognition and cleavage efficiency. Our data further suggest that this interaction is part of a metal ion-binding site and that specific chemical groups are likely to act as ligands in binding of Mg(2+) or other divalent cations important for function. We argue that this Mg(2+) is involved in metal ion cooperativity in M1 RNA-mediated cleavage. Moreover, we suggest that the 73/294-interaction operates in concert with displacement of residue -1 in the substrate to ensure efficient and correct cleavage. The possibility that the residue at -1 binds to a specific binding surface/pocket in M1 RNA is discussed. Our data finally rationalize why the preferred residue at position 294 in M1 RNA is U.