Coordination Of Divalent Metal Ions Research Articles

Structural and functional insights into the enzymatic plasticity of the SARS-CoV-2 NiRAN domain

The enzymatic activity of the SARS-CoV-2 nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain is essential for viral propagation, with three distinct activities associated with modification of the nsp9N terminus, NMPylation, RNAylation, and deRNAylation/capping via a GDP-polyribonucleotidyltransferase reaction. The latter two activities comprise an unconventional mechanism for initiating viral RNA 5' cap formation, while the role of NMPylation is unclear. The structural mechanisms for these diverse enzymatic activities have not been properly delineated. Here, we determine high-resolution cryoelectron microscopy (cryo-EM) structures of catalytic intermediates for the NMPylation and deRNAylation/capping reactions, revealing diverse nucleotide binding poses and divalent metal ion coordination sites to promote its repertoire of activities. The deRNAylation/capping structure explains why GDP is a preferred substrate for the capping reaction over GTP. Altogether, these findings enhance our understanding of the promiscuous coronaviral NiRAN domain, a therapeutic target, and provide an accurate structural platform for drug development.

Palladium Complexes of N-Methylcorroles.

Alkylation of one of the inner-core nitrogen atoms is one possible approach to obtain dianionic corrole ligands, suitable for the coordination of divalent metal ions, such as PdII . Inner-core N-methylation can be obtained by treating the corrole with CH3 I, but the reaction conditions should be optimized to limit the formation of the dimethylated derivative. Two regioisomers, the N-21 and the N-22 methyl derivatives are obtained from the reaction, with the first product achieved in a higher amount. Structural characterization of the reaction products evidenced the distortion induced by the introduction of the methyl groups; the N-methylcorroles are chiral compounds, and the enantiomers were separated by chromatography, with their absolute configuration assigned by ECD computation. Palladium insertion was achieved in the case of monosubstituted corroles, but not with the dimethylated macrocycle; X-ray characterization of the complexes showed the distortion of the macrocycles. The Pd complexes do not show luminescence emission, but are able to produce singlet oxygen upon irradiation. The PdII complexes were also inserted in human serum albumin (HSA) and dispersed in water; in this case, the protein protects the corroles from photobleaching, and a switch from the type II to the type I mechanism in reactive oxygen species (ROS) production is observed.

Structural features stabilized by divalent cation coordination within hepatitis E virus ORF1 are critical for viral replication.

Hepatitis E virus (HEV) is an RNA virus responsible for over 20 million infections annually. HEV's open reading frame (ORF)1 polyprotein is essential for genome replication, though it is unknown how the different subdomains function within a structural context. Our data show that ORF1 operates as a multifunctional protein, which is not subject to proteolytic processing. Supporting this model, scanning mutagenesis performed on the putative papain-like cysteine protease (pPCP) domain revealed six cysteines essential for viral replication. Our data are consistent with their role in divalent metal ion coordination, which governs local and interdomain interactions that are critical for the overall structure of ORF1; furthermore, the 'pPCP' domain can only rescue viral genome replication in trans when expressed in the context of the full-length ORF1 protein but not as an individual subdomain. Taken together, our work provides a comprehensive model of the structure and function of HEV ORF1.

A biologically stable DNAzyme that efficiently silences gene expression in cells.

Efforts to use RNA-cleaving DNA enzymes (DNAzymes) as gene-silencing agents in therapeutic applications have stalled due to their low efficacy in clinical trials. Here we report a xeno-nucleic-acid-modified version of the classic DNAzyme 10-23 that achieves multiple-turnover activity under cellular conditions and resists nuclease digestion. The new reagent, X10-23, overcomes the problem of product inhibition, which limited previous 10-23 designs, using molecular chemotypes with DNA, 2'-fluoroarabino nucleic acid and α-L-threofuranosyl nucleic acid backbone architectures that balance the effects of enhanced biological stability with RNA hybridization and divalent metal ion coordination. In cultured mammalian cells, X10-23 facilitates persistent gene silencing by efficiently degrading exogenous and endogenous messenger RNA transcripts. Together, these results demonstrate that new molecular chemotypes can improve the activity and stability of DNAzymes, and may provide a new route for nucleic acid enzymes to reach the clinic.

New insights into coordination chemistry of Monensin A towards divalent metal ions

Complexation reactions of polyether ionophorous antibiotic Monensin A with divalent metal ions were studied in methanolic solutions by means of synchrotron radiation circular dichroism (SRCD) spectroscopy. Data showed that in the presence of organic base (Et4NOH) Monensin A forms two types of metal complexes depending on metal-to-ligand molar ratio. Complexes of composition [M(Mon)2(H2O)2] (Mon– = Monensinate A anion; M = Co2+, Mn2+, Mg2+, Ca2+, Ni2+, Zn2+, Cd2+) exist mainly in the presence of ligand excess (M2+:Mon– ~ 1:10–1:2), while the increase of metal(II) ion concentration results in the formation of new [M(Mon)(H2O)]+ species (M2+:Mon– ~ 1:1–6:1), not detected up to now with these metal ions. Experimental data showed that coordination of divalent metal ions to Monensinate A anion significantly influences the SRCD spectral pattern in different manner. Based on sets of SRCD experiments, species distribution, individual spectra of [M(Mon)2(H2O)2] and [M(Mon)(H2O)]+ as well as their conditional stability constants were calculated. The relative affinity of Monensin A to bind divalent metal ions demonstrates the metal ion selectivity of the ligand.

Inner-Sphere Coordination of Divalent Metal Ion with Nucleobase in Catalytic RNA.

Identification of the function of metal ions and the RNA moieties, particularly nucleobases, that bind metal ions is important in RNA catalysis. Here we combine single-atom and abasic substitutions to probe functions of conserved nucleobases in ribonuclease P (RNase P). Structural and biophysical studies of bacterial RNase P propose direct coordination of metal ions by the nucleobases of conserved uridine and guanosine in helix P4 of the RNA subunit (P RNA). To biochemically probe the function of metal ion interactions, we substituted the universally conserved bulged uridine (U51) in the P4 helix of circularly permuted Bacillus subtilis P RNA with 4-thiouridine, 4-deoxyuridine, and abasic modifications and G378/379 with 2-aminopurine, N7-deazaguanosine, and 6-thioguanosine. The functional group modifications of U51 decrease RNase P-catalyzed phosphodiester bond cleavage 16- to 23-fold, as measured by the single-turnover cleavage rate constant. The activity of the 4-thiouridine RNase P is partially rescued by addition of Cd(II) or Mn(II) ions. This is the first time a metal-rescue experiment provides evidence for inner-sphere divalent metal ion coordination with a nucleobase. Modifications of G379 modestly decrease the cleavage activity of RNase P, suggesting outer-sphere coordination of O6 on G379 to a metal ion. These data provide biochemical evidence for catalytically important interactions of the P4 helix of P RNA with metal ions, demonstrating that the bulged uridine coordinates at least one catalytic metal ion through an inner-sphere interaction. The combination of single-atom and abasic nucleotide substitutions provides a powerful strategy to probe functions of conserved nucleobases in large RNAs.

The Thermodynamics of Divalent Metals Binding to Taurine/α-Ketoglutarate-Dependent Oxygenase (TAUD)

Taurine/α-ketoglutarate (αKG) dioxygenase (TauD) is a nonheme iron(II) and αKG dependent metalloenzyme, which catalyzes the hydroxylation of taurine leading to its decomposition into aminoacetaldehyde and sulfite, where sulfite is a key sulfur containing metabolite in E. coli. The nonheme iron(II) center in TauD is formed from two histidine side chain residues and a glutamic acid coordinating to one face of the octahedral coordination geometry. This common metal binding motif has been termed the 2-His-1-carboxylate facial triad and is found in a number of nonheme manganese, iron, and cobalt containing proteins. Here we have focused our efforts to measure the thermodynamic driving forces that lead to formation of these bioinorganic centers in biology, by studying divalent metal ion coordination to TauD using isothermal titration calorimetry. Titrations of metal complexes into the metal-free (apo) TauD and the corresponding chelation experiments were performed under anaerobic environment.The thermodynamic terms associated with cobalt(II), iron(II), and manganese(II) binding to apoTauD were deconvoluted from complex experiments, where the pH and buffer independent binding constant (K) were measured to be 2.9 × 109, 2.4 × 107, and 9.8 × 105, respectively. (The corresponding ΔG values were calculated to be −12.8 kcal/mol, −10.1 kcal/mol, and −8.2 kcal/mol, respectively.) Interestingly the measured enthalpy changes for these binding events (ΔH) are −17.8 kcal/mol, −12.8 kcal/mol, and −12.2 kcal/mol, respectively. These data are fully consistent with the Irving-Williams series, which suggest there is increasing affinity for transition metal ions from left to right across the periodic table. However, it seems this the increasing affinity is derived from increasing favorability of both the related ΔH and ΔS terms.

Processable and Robust MoS2 Paper Chemically Cross‐Linked with Polymeric Ligands by the Coordination of Divalent Metal Ions

Inorganic graphene analogues (IGAs) are a huge and fascinating family of compounds that have extraordinary electronic, mechanical, and thermal properties. However, one of the largest problems that face the industrial application of IGAs is their poor processability, which has led to a "bottlenecking" in the development of freestanding, large-area, IGA-based thin-film devices. Herein, we report a facile and cost-efficient method to chemically modify IGAs by using their abundant coordination atoms (S, O, and N). Taking MoS2 as an example, we have prepared homogeneous "solution" systems, in which MoS2 nanosheets are chemically cross-linked through a carboxylate-containing polymeric ligand, poly(methyl methacrylate) (PMMA), by copper-ion coordination. Bonding interactions between C=O bonds and sulfur atoms through copper ions were confirmed by various characterization techniques, such as UV/Vis, FTIR, and Raman spectroscopy and XPS. By using our method, freestanding MoS2 paper with significantly improved mechanical properties was obtained, thus laying the basis for the mass production of large-area MoS2-based thin-film devices. Furthermore, copper-ion coordination was also applied to MoS2/PMMA nanocomposites. Direct and strong nanofiller/matrix bonding interactions facilitate efficient load transfer and endow the polymeric nanocomposites with an excellent reinforcement effect. This method may pave a new way to high-strength polymeric nanocomposites with superior frictional properties, flame retardance, and oxidation resistance.

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.

Structural insights into complete metal ion coordination from ternary complexes of B family RB69 DNA polymerase.

We have captured a preinsertion ternary complex of RB69 DNA polymerase (RB69pol) containing the 3' hydroxyl group at the terminus of an extendable primer (ptO3') and a nonhydrolyzable 2'-deoxyuridine 5'-α,β-substituted triphosphate, dUpXpp, where X is either NH or CH(2), opposite a complementary templating dA nucleotide residue. Here we report four structures of these complexes formed by three different RB69pol variants with catalytically inert Ca(2+) and four other structures with catalytically competent Mn(2+) or Mg(2+). These structures provide new insights into why the complete divalent metal-ion coordination complexes at the A and B sites are required for nucleotidyl transfer. They show that the metal ion in the A site brings ptO3' close to the α-phosphorus atom (Pα) of the incoming dNTP to enable phosphodiester bond formation through simultaneous coordination of both ptO3' and the nonbridging Sp oxygen of the dNTP's α-phosphate. The coordination bond length of metal ion A as well as its ionic radius determines how close ptO3' can approach Pα. These variables are expected to affect the rate of bond formation. The metal ion in the B site brings the pyrophosphate product close enough to Pα to enable pyrophosphorolysis and assist in the departure of the pyrophosphate. In these dUpXpp-containing complexes, ptO3' occupies the vertex of a distorted metal ion A coordination octahedron. When ptO3' is placed at the vertex of an undistorted, idealized metal ion A octahedron, it is within bond formation distance to Pα. This geometric relationship appears to be conserved among DNA polymerases of known structure.

A Divalent Cation Stabilizes the Active Conformation of the B. subtilis RNase P·Pre-tRNA Complex: A Role for an Inner-Sphere Metal Ion in RNase P

Metal ions interact with RNA to enhance folding, stabilize structure, and, in some cases, facilitate catalysis. Assigning functional roles to specifically bound metal ions presents a major challenge in analyzing the catalytic mechanisms of ribozymes. Bacillus subtilis ribonuclease P (RNase P), composed of a catalytically active RNA subunit (PRNA) and a small protein subunit (P protein), catalyzes the 5′-end maturation of precursor tRNAs (pre-tRNAs). Inner-sphere coordination of divalent metal ions to PRNA is essential for catalytic activity but not for the formation of the RNase P·pre-tRNA (enzyme–substrate, ES) complex. Previous studies have demonstrated that this ES complex undergoes an essential conformational change (to the ES ⁎ conformer) before the cleavage step. Here, we show that the ES ⁎ conformer is stabilized by a high-affinity divalent cation capable of inner-sphere coordination, such as Ca(II) or Mg(II). Additionally, a second, lower-affinity Mg(II) activates cleavage catalyzed by RNase P. Structural changes that occur upon binding Ca(II) to the ES complex were determined by time-resolved Förster resonance energy transfer measurements of the distances between donor–acceptor fluorophores introduced at specific locations on the P protein and pre-tRNA 5′ leader. These data demonstrate that the 5′ leader of pre-tRNA moves 4 to 6 Å closer to the PRNA·P protein interface during the ES-to-ES ⁎ transition and suggest that the metal-dependent conformational change reorganizes the bound substrate in the active site to form a catalytically competent ES ⁎ complex.

Site‐specific inhibition of integrin αvβ3‐vitronectin association by a ser‐asp‐val sequence through an Arg‐Gly‐Asp‐binding site of the integrin

A functional proteomic technology using protein chip and molecular simulation was used to demonstrate a novel biomolecular interaction between P11, a peptide containing the Ser-Asp-Val (SDV) sequence and integrin alpha v beta 3. P11 (HSDVHK) is a novel antagonistic peptide of integrin alpha v beta 3 screened from hexapeptide library through protein chip system. An in silico docking study and competitive protein chip assay revealed that the SDV sequence of P11 is able to create a stable inhibitory complex onto the vitronectin-binding site of integrin alpha v beta 3. The Arg-Gly-Asp (RGD)-binding site recognition by P11 was site specific because the P11 was inactive for the complex formation of a denatured form of integrin-vitronectin. P11 showed a strong antagonism against alpha v beta 3-GRGDSP interaction with an IC(50) value of 25.72+/-3.34 nM, whereas the value of GRGDSP peptide was 1968.73+/-444.32 nM. The binding-free energies calculated from the docking simulations for each P11 and RGD peptide were -3.99 and -3.10 kcal/mol, respectively. The free energy difference between P11 and RGD corresponds to approximately a 4.5-fold lower K(i) value for the P11 than the RGD peptide. The binding orientation of the docked P11 was similar to the crystal structure of the RGD in alpha v beta 3. The analyzed docked poses suggest that a divalent metal-ion coordination was a common driving force for the formation of both SDV/alpha v beta 3 and RGD/alpha v beta 3 complexes. This is the first report on the specific recognition of the RGD-binding site of alpha v beta 3 by a non-RGD containing peptide using a computer-assisted proteomic approach.

Investigating the conservation pattern of a putative second terpene synthase divalent metal binding motif in plants

Terpene synthases (TPS) require divalent metal ion co-factors, typically magnesium, that are bound by a canonical DDXXD motif, as well as a putative second, seemingly less well conserved and understood (N/D)DXX(S/T)XXXE motif. Given the role of the Ser/Thr side chain hydroxyl group in ligating one of the three catalytically requisite divalent metal ions and the loss of catalytic activity upon substitution with Ala, it is surprising that Gly is frequently found in this ‘middle’ position of the putative second divalent metal binding motif in plant TPS. Herein we report mutational investigation of this discrepancy in a model plant diterpene cyclase, abietadiene synthase from Abies grandis (AgAS). Substitution of the corresponding Thr in AgAS with Ser or Gly decreased catalytic activity much less than substitution with Ala. We speculate that the ability of Gly to partially restore activity relative to Ala substitution for Ser/Thr stems from the associated reduction in steric volume enabling a water molecule to substitute for the hydroxyl group from Ser/Thr, potentially in a divalent metal ion coordination sphere. In any case, our results are consistent with the observed conservation pattern for this putative second divalent metal ion binding motif in plant TPS.

DNA Gyrase Requires DNA for Effective Two-Site Coordination of Divalent Metal Ions: Further Insight into the Mechanism of Enzyme Action

The catalytic properties of DNA gyrase, an A 2B 2 complex, are modulated by the presence of divalent metal ions. Using circular dichroism, protein melting experiments and enzyme activity assays, we investigated the correlation between the A 2B 2 conformation, the nature of the metal ion cofactor and the enzyme activity in the presence and absence of DNA substrate. At room temperature, DNA gyrase structure is not appreciably affected by Ca (2+) or Mg (2+) but is modified by Mn (2+). In addition, metal ions strongly affect the enzyme's thermal transitions, rendering the A 2B 2 structure more flexible. Using the B subunit, we were able to identify two distinct complexes with manganese ions. The first one exhibits a 1:1 stoichiometry and is not affected by the presence of DNA. The second complex is associated with a large protein structural modification that can be remarkably modulated by addition of the DNA substrate. This behavior is conserved in the reconstituted protein. Studies with two GyrB mutants indicate that Mn (2+) interference with the TOPRIM region modulates gyrase supercoiling activity. In particular, considering the need for two divalent metal ions for an efficient catalytic cleavage of the phosphodiester bond, our data suggest that residue D500 participates in the first complexation event (DNA-independent), whereas residue D498 is involved mainly in the second process. In conclusion, a combination of the ion features (ionic size, electronegativity, coordination sphere) operating at the level of the catalytic region and of the ion-driven modifications in overall enzyme structure and flexibility contribute to the mechanism of gyrase activity. An effectual role for DNA recruiting the second catalytic metal ion is envisaged.

Divalent Metal Ion Coordination by Residue T118 of Anthrax Toxin Receptor 2 Is Not Essential for Protective Antigen Binding

The protective antigen (PA) subunit of anthrax toxin interacts with the integrin-like I domains of either of two cellular receptors, ANTXR1 or ANTXR2. These I domains contain a metal ion-dependent adhesion site (MIDAS) made up of five non-consecutive amino acid residues that coordinate a divalent metal ion that is important for PA-binding. The MIDAS residues of integrin I domains shift depending upon whether the domain exists in a closed (ligand-unbound) or open (ligand-bound) conformation. Of relevance to this study, the MIDAS threonine residue coordinates the metal ion only in the open I domain conformation. Previously it was shown that the MIDAS threonine is essential for PA interaction with ANTXR1, a result consistent with the requirement that the I domain of that receptor adopts an open conformation for PA-binding [1]. Here we have tested the requirement for the MIDAS threonine of ANTXR2 for PA-binding. We show that the toxin can bind to a mutant receptor lacking the MIDAS threonine and that it can use that mutant receptor to intoxicate cultured cells. These findings suggest that an open-like configuration of the ANTXR2 MIDAS is not essential for the interaction with PA.

Coordination of Divalent Metal Ions in the Active Site of Poly(A)-specific Ribonuclease

Poly(A)-specific ribonuclease (PARN) is a highly poly(A)-specific 3'-exoribonuclease that efficiently degrades mRNA poly(A) tails. PARN belongs to the DEDD family of nucleases, and four conserved residues are essential for PARN activity, i.e. Asp-28, Glu-30, Asp-292, and Asp-382. Here we have investigated how catalytically important divalent metal ions are coordinated in the active site of PARN. Each of the conserved amino acid residues was substituted with cysteines, and it was found that all four mutants were inactive in the presence of Mg2+. However, in the presence of Mn2+, Zn2+, Co2+, or Cd2+, PARN activity was rescued from the PARN(D28C), PARN(D292C), and PARN(D382C) variants, suggesting that these three amino acids interact with catalytically essential metal ions. It was found that the shortest sufficient substrate for PARN activity was adenosine trinucleotide (A3) in the presence of Mg2+ or Cd2+. Interestingly, adenosine dinucleotide (A) was efficiently hydrolyzed in the presence of Mn2+, Zn2+, or Co2+, suggesting that the substrate length requirement for PARN can be modulated by the identity of the divalent metal ion. Finally, introduction of phosphorothioate modifications into the A substrate demonstrated that the scissile bond non-bridging phosphate oxygen in the pro-R position plays an important role during cleavage, most likely by coordinating a catalytically important divalent metal ion. Based on our data we discuss binding and coordination of divalent metal ions in the active site of PARN.

Conformational Analysis of Chirally Deuterated Tunicamycin as an Active Site Probe of UDP-N-Acetylhexosamine:Polyprenol-P N-Acetylhexosamine-1-P Translocases

Tunicamycins are potent inhibitors of UDP-N-acetyl-D-hexosamine:polyprenol-phosphate N-acetylhexosamine-1-phosphate translocases (D-HexNAc-1-P translocases), a family of enzymes involved in bacterial cell wall synthesis and eukaryotic protein N-glycosylation. Structurally, tunicamycins consist of an 11-carbon dialdose core sugar called tunicamine that is N-linked at C-1' to uracil and O-linked at C-11' to N-acetylglucosamine (GlcNAc). The C-11' O-glycosidic linkage is highly unusual because it forms an alpha/beta anomeric-to-anomeric linkage to the 1-position of the GlcNAc residue. We have assigned the (1)H and (13)C NMR spectra of tunicamycin and have undertaken a conformational analysis from rotating angle nuclear Overhauser effect (ROESY) data. In addition, chirally deuterated tunicamycins produced by fermentation of Streptomyces chartreusis on chemically synthesized, monodeuterated (S-6)-[(2)H(1)]glucose have been used to assign the geminal H-6'a, H-6'b methylene bridge of the 11-carbon dialdose sugar, tunicamine. The tunicamine residue is shown to assume pseudo-D-ribofuranose and (4)C(1) pseudo-D-galactopyranosaminyl ring conformers. Conformation about the C-6' methylene bridge determines the relative orientation of these rings. The model predicts that tunicamycin forms a right-handed cupped structure, with the potential for divalent metal ion coordination at 5'-OH, 8'-OH, and the pseudogalactopyranosyl 7'-O ring oxygen. The formation of tunicamycin complexes with various divalent metal ions was confirmed experimentally by MALDI-TOF mass spectrometry. Our data support the hypothesis that tunicamycin is a structural analogue of the UDP-D-HexNAc substrate and is reversibly coordinated to the divalent metal cofactor in the D-HexNAc-1-P translocase active site.

Evaluation by mutagenesis of the roles of His309, His315, and His319 in the coenzyme site of pig heart NADP-dependent isocitrate dehydrogenase.

Sequence alignment predicts that His(309) of pig heart NADP-dependent isocitrate dehydrogenase is equivalent to His(339) of the Escherichia coli enzyme, which interacts with the coenzyme in the crystal structure [Hurley et al. (1991) Biochemistry 30, 8671-8688], and porcine His(315) and His(319) are close to that site. The mutant porcine enzymes H309Q, H309F, H315Q, and H319Q were prepared by site-directed mutagenesis, expressed in E. coli, and purified. The H319Q mutant has K(m) values for NADP, isocitrate, and Mn(2+) similar to those of wild-type enzyme, and V(max) = 20.1, as compared to 37.8 micromol of NADPH min(-1) (mg of protein)(-1) for wild type. Thus, His(319) is not involved in coenzyme binding and has a minimal effect on catalysis. In contrast, H315Q exhibits a K(m) for NADP 40 times that of wild type and V(max) = 16.2 units/mg of protein, with K(m) values for isocitrate and Mn(2+) similar to those of wild type. These results implicate His(315) in the region of the NADP site. Replacement of His(309) by Q or F yields enzyme with no detectable activity. The His(309) mutants bind NADPH poorly, under conditions in which wild type and H319Q bind 1.0 mol of NADPH/mol of subunit, indicating that His(309) is important for the binding of coenzyme. The His(309) mutants bind isocitrate stoichiometrically, as do wild-type and the other mutant enzymes. However, as distinguished from the wild-type enzyme, the His(309) mutants are not oxidatively cleaved by metal isocitrate, implying that the metal ion is not bound normally. Since circular dichroism spectra are similar for wild type, H315Q, and H319Q, these amino acid substitutions do not cause major conformational changes. In contrast, replacement of His(309) results in detectable change in the enzyme's CD spectrum and therefore in its secondary structure. We propose that His(309) plays a significant role in the binding of coenzyme, contributes to the proper coordination of divalent metal ion in the presence of isocitrate, and maintains the normal conformation of the enzyme.

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.

Inhibition of the human immunodeficiency virus type 1 integrase by guanosine quartet structures.

An oligonucleotide (T30177) composed entirely of deoxyguanosine and thymidine has previously been shown to fold upon itself in the presence of potassium into a highly stable four-stranded DNA structure containing two stacked deoxyguanosine quartets (G4s). T30177 also protects host cells from the cytopathic effects of human immunodeficiency virus type 1 (HIV-1). We report that this G4 oligonucleotide is the most potent inhibitor of HIV-1 integrase identified to date, with IC50 values in the nanomolar range. Both the number of quartets formed and the sequence of the loops between the quartets are important for optimal activity. T30177 binds to HIV-1 integrase without being processed and blocks the binding of the normal viral DNA substrate to the enzyme. The normal DNA substrate was not able to compete off T30177 binding to HIV-1 integrase, indicating a tight binding of G4s to the enzyme. Experiments with truncated HIV-1 integrases indicate that the N-terminal region containing a putative zinc finger is required for inhibition by T30177 and that T30177 binds better to full-length or deletion mutant integrases containing the zinc finger region than to a deletion mutant consisting of only the central catalytic domain. The N-terminal region of integrase alone is able to bind efficiently to T30177, but not the linear viral DNA substrate, in the presence of zinc. Hence, G4s represent the first class of compounds that inhibit HIV-1 integrase by interacting with the enzyme N-terminal domain. The greater inhibitory potency of T30177 in buffer containing magnesium versus manganese suggests that divalent metal ion coordination along the phosphodiester backbone may play a role in the inhibitory activity. T30177 inhibited HIV-2 integrase with similar potency as HIV-1 but inhibited feline and simian immunodeficiency virus integrases at higher concentrations, suggesting selectivity can be achieved. We propose that novel AIDS therapies could be based upon guanosine quarters as inhibitors of HIV-1 integrase.