Solution Conformation and Dynamics of the HIV-1 Integrase Core Domain

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) is a critical enzyme involved in infection. It catalyzes two reactions to integrate the viral cDNA into the host genome, 3′ processing and strand transfer, but the dynamic behavior of the active site during catalysis of these two processes remains poorly characterized. NMR spectroscopy can reveal important structural details about enzyme mechanisms, but to date the IN catalytic core domain has proven resistant to such an analysis. Here, we present the first NMR studies of a soluble variant of the catalytic core domain. The NMR chemical shifts are found to corroborate structures observed in crystals, and confirm prior studies suggesting that the α4 helix extends toward the active site. We also observe a dramatic improvement in NMR spectra with increasing MgCl₂ concentration. This improvement suggests a structural transition not only near the active site residues but also throughout the entire molecule as IN binds Mg²⁺. In particular, the stability of the core domain is linked to the conformation of its C-terminal helix, which has implications for relative domain orientation in the full-length enzyme. ¹⁵N relaxation experiments further show that, although conformationally flexible, the catalytic loop of IN is not fully disordered in the absence of DNA. Indeed, automated chemical shift-based modeling of the active site loop reveals several stable clusters that show striking similarity to a recent crystal structure of prototype foamy virus IN bound to DNA.     

A cooperative and specific DNA-binding mode of HIV-1 integrase depends on the nature of the metallic cofactor and involves the zinc-containing N-terminal domain

HIV-1 integrase catalyzes the insertion of the viral genome into chromosomal DNA. We characterized the structural determinants of the 3′-processing reaction specificity—the first reaction of the integration process—at the DNA-binding level. We found that the integrase N-terminal domain, containing a pseudo zinc-finger motif, plays a key role, at least indirectly, in the formation of specific integrase–DNA contacts. This motif mediates a cooperative DNA binding of integrase that occurs only with the cognate/viral DNA sequence and the physiologically relevant Mg²⁺ cofactor. The DNA-binding was essentially non-cooperative with Mn²⁺ or using non-specific/random sequences, regardless of the metallic cofactor. 2,2′-Dithiobisbenzamide-1 induced zinc ejection from integrase by covalently targeting the zinc-finger motif, and significantly decreased the Hill coefficient of the Mg²⁺-mediated integrase–DNA interaction, without affecting the overall affinity. Concomitantly, 2,2′-dithiobisbenzamide-1 severely impaired 3′-processing (IC₅₀ = 11–15 nM), suggesting that zinc ejection primarily perturbs the nature of the active integrase oligomer. A less specific and weaker catalytic effect of 2,2′-dithiobisbenzamide-1 is mediated by Cys 56 in the catalytic core and, notably, accounts for the weaker inhibition of the non-cooperative Mn²⁺-dependent 3′-processing. Our data show that the cooperative DNA-binding mode is strongly related to the sequence-specific DNA-binding, and depends on the simultaneous presence of the Mg²⁺ cofactor and the zinc effector.Integration of HIV-1 DNA into the host genome ensures stable maintenance of the viral genome in the host organism and, therefore, is a key process in the virus life cycle. Integrase (IN) is responsible for two distinct, consecutive catalytic steps in the integration process (1). The first of these two reactions is 3′-processing, which corresponds to the specific cleavage of two nucleotides from the 3′-ends of the linear viral DNA. The hydroxyl groups of newly recessed 3′-ends are then used in the second reaction— strand transfer—for the covalent joining of viral and cellular (or target) DNAs, resulting in full-site integration. For both reactions, IN functions as a multimer, most likely a dimer for 3′-processing and a tetramer (dimer of a dimer) for concerted integration (2–7). Two other reactions occur in vitro, a disintegration reaction that represents, in first approximation, the reversal of the half-site integration process (8) and a specific internal cleavage occurring on a symmetrical DNA site (9). All reactions require a metallic cofactor, Mg²⁺ or Mn²⁺, and, except for disintegration (10,11), all reactions require the full-length IN. There are several experimental evidences to suggest that Mg²⁺ is more physiologically relevant as a cofactor, particularly because Mg²⁺-dependent catalysis exhibits weaker non-specific endonucleolytic cleavage and the tolerance of sequence variation at the ends of the viral DNA is much greater in the presence of Mn²⁺ than in the presence of Mg²⁺ (12–15).The emergence of viral strains resistant against available drugs and the dynamic nature of the HIV-1 genome support a continued effort towards the discovery and characterization of novel targets and anti-viral drugs. Due to its central role in the HIV-1 life cycle, IN represents a promising therapeutic target. In the past, in vitro IN assays were extensively used to find IN inhibitors (16). Current inhibitors can be separated into two main classes, depending on their mechanisms of action: (i) Compounds that competitively prevent the DNA binding of IN to the viral DNA. These compounds are mainly directed against the 3′-processing reaction as they bind to the donor site within the catalytic site—i.e. the ‘specific’ DNA-binding site for the viral DNA -. This group is referred to as ‘integrase DNA-binding inhibitors’ (INBI) and includes styrylquinoline compounds (17,18). (ii) The second class includes compounds that cannot bind to the DNA-free IN. They bind to the pre-formed IN–viral DNA complex. These compounds preferentially inhibit strand transfer over the 3′-processing reaction [this family of compounds is referred to as ‘integrase strand transfer inhibitors’ (INSTI)], probably by displacing the viral DNA end from the active site (7,19–21). It is not clear whether this mechanism alone accounts for the inhibitory properties of INSTIs or whether these compounds also prevent the binding of the target DNA to the acceptor site—i.e. the ‘non-specific’ DNA binding site. INSTI compounds have generally good ex vivo activity against HIV replication, probably due to their ability to inhibit pre-assembled viral DNA/IN complexes. Raltegravir which is currently used in clinical treatment of HIV-1 belongs to this class. For both anti-IN classes, resistance mutations were identified (17,20,22,23). Difficulties in deeply understanding their mechanisms of action are closely related to the absence of structural data that clearly delineate the donor and the acceptor DNA binding sites in the active site. Although structural information is now available regarding the IN–viral DNA interaction, based on the recent crystal structure of the full-length primate foamy virus (PFV-1) IN in complex with its cognate processed viral DNA, the target DNA binding mode and the precise location of the acceptor site remains open to debate (7). Moreover, it is a difficult task to experimentally discriminate between the two DNA binding sites and no significant or only modest difference can be evidenced in vitro (depending on the method used for monitoring IN–DNA interactions) between the HIV-1 IN binding to the cognate viral DNA sequence and a non-specific random sequence in terms of overall affinity, suggesting that the specific and the non-specific DNA-binding modes display similar binding free energies (5,24). The basis of DNA binding specificity remains essentially unknown.HIV-1 IN (288 amino acids) contains three functional domains. The central domain or catalytic core domain (IN^50–213 or CC) contains the catalytic triad (DDE) that coordinates one or two metallic cofactors [probably a pair coordinated by three carboxylate groups of the triad, based on the X-ray structure of the PFV-1 IN (7)] and is essential for enzymatic activity; this domain alone can perform the disintegration reaction (10,11). This domain is flanked by the N-terminal (IN^1-49) and the C-terminal (IN^214–288) domains. The C-terminal domain is involved in IN–DNA contacts, together with the CC domain (25,26). The N-terminal domain contains a conserved non-conventional HHCC motif that binds zinc to ensure proper domain folding and promotes IN multimerization (27–29). It is worth noting that the integrity of the HHCC motif is crucial in the stringent Mg²⁺-context but appears dispensable under the less stringent Mn²⁺ condition (30), suggesting, at least, an indirect role of the zinc-binding domain in the establishment of specific and physiologically relevant IN–DNA complexes. In the structure of the PFV-1 IN–viral DNA complex, the N-terminal domain is also involved in the interaction with DNA (7).In this article, we found that IN binds cooperatively to the cognate viral DNA sequence only in the presence of Mg²⁺. The presence of Mn²⁺ or, most importantly, the use of non-specific random sequences, regardless of the metallic cofactor, dramatically reduced the Hill coefficient. This finding suggests that the cooperative DNA-binding mode of IN is strongly related to the formation of specific IN–DNA contacts. To gain deeper insight into the role of the zinc-binding domain in the cooperative/multimerization process, in relationship with the establishment of specific protein–DNA contacts, we studied the effect of DIBA-1 (2,2′-dithiobisbenzamide-1) (Figure 1A) on IN activity. This compound is a zinc ejector affecting many proteins containing zinc fingers, including HIV-1 nucleocapsid or estrogen receptor (31–34). Here, we found that DIBA-1 induced zinc ejection from the IN N-terminal domain by covalently targeting the HHCC motif. In the presence of Mg²⁺, DIBA-1 did not affect significantly the overall affinity of IN for the DNA substrate but dramatically reduced the Hill coefficient. Concomitantly, DIBA-1 strongly inhibited the catalytic step, with IC₅₀ values against the 3′-processing reaction of 11–15 nM. Interestingly, we found a secondary DIBA-1 binding site in the catalytic core (involving residue Cys 56), suggesting a second mechanism of action of DIBA-1, independent of zinc ejection. The prevalence of the two distinct mechanisms was dependent on the cofactor context, with the second one accounting for the weaker DIBA-1 inhibitory effect under Mn²⁺ conditions (IC₅₀ = 115–126 nM). DIBA-1 behaves as a non-competitive/catalytic inhibitor that did not disturb the fractional saturation of DNA sites, regardless of the mechanism considered. Open in a separate windowFigure 1.DIBA-1 induces the ejection of zinc from IN. (A) Structure of 2,2′-dithiobisbenzamide-1 (DIBA-1) (MW, 614 Da). (B) Ejection of zinc was measured by optical absorbance at 495 nm using a sample containing full-length IN (1 µM) and PAR (10⁻⁴ M) in a Tris buffer (20 mM pH 7.0) containing 15% DMSO (v/v) in the absence of reducing agent (black squares) or in the presence of 4 mM β-mercaptoethanol (white circles). The concentration of DIBA-1 for complete zinc release—[DIBA-1]_eq—was estimated graphically. This value was determined as a function of the initial concentration of IN (C). The slope of the straight line indicates that the zinc ejection coincides with the reaction of two DIBA-1 molecules per IN protomer.Altogether, our results show that, although it is a difficult task to discriminate between the specific viral sequence and a non-specific random sequence in terms of overall affinity, these sequences lead to distinct DNA-binding properties in terms of cooperativity. Moreover, our results highlight that the Mg²⁺-dependent catalytic activity of IN is strongly sensitive to the loss of cooperative DNA binding. Such a cooperative DNA-binding mode accounts for specific activity and requires: (i) the cognate viral DNA sequence, (ii) Mg²⁺ as a catalytic cofactor and (iii) zinc which can be considered as a positive allosteric effector. Development of non-competitive compounds acting on the N-terminal domain may be of interest for anti-IN pharmacology.     

The invariant glutamate of human PrimPol DxE motif is critical for its Mn2+-dependent distinctive activities

PrimPol is a human primase/polymerase specialized in downstream repriming of stalled forks during both nuclear and mitochondrial DNA replication. Like most primases and polymerases, PrimPol requires divalent metal cations, as Mg²⁺ or Mn²⁺, used as cofactors for catalysis. However, little is known about the consequences of using these two metal cofactors in combination, which would be the most physiological scenario during PrimPol-mediated reactions, and the individual contribution of the putative carboxylate residues (Asp¹¹⁴, Glu¹¹⁶ and Asp²⁸⁰) acting as metal ligands. By site-directed mutagenesis in human PrimPol, we confirmed the catalytic relevance of these three carboxylates, and identified Glu¹¹⁶ as a relevant enhancer of distinctive PrimPol reactions, which are highly dependent on Mn²⁺. Herein, we evidenced that PrimPol Glu¹¹⁶ contributes to error-prone tolerance of 8oxodG more markedly when both Mg²⁺ and Mn²⁺ ions are present. Moreover, Glu¹¹⁶ was important for TLS events mediated by primer/template realignments, and crucial to achieving an optimal primase activity, processes in which Mn²⁺ is largely preferred. EMSA analysis of PrimPol:ssDNA:dNTP pre-ternary complex indicated a critical role of each metal ligand, and a significant impairment when Glu¹¹⁶ was changed to a more conventional aspartate. These data suggest that PrimPol active site requires a specific motif A (DxE) to favor the use of Mn²⁺ ions in order to achieve optimal incoming nucleotide stabilization, especially required during primer synthesis.     

On the Divalent Metal Ion Dependence of DNA Cleavage by Restriction Endonucleases of the EcoRI Family

Restriction endonucleases of the PD…D/EXK family need Mg²⁺ for DNA cleavage. Whereas Mg²⁺ (or Mn²⁺) promotes catalysis, Ca²⁺ (without Mg²⁺) only supports DNA binding. The role of Mg²⁺ in DNA cleavage by restriction endonucleases has elicited many hypotheses, differing mainly in the number of Mg²⁺ involved in catalysis. To address this problem, we measured the Mg²⁺ and Mn²⁺ concentration dependence of DNA cleavage by BamHI, BglII, Cfr10I, EcoRI, EcoRII (catalytic domain), MboI, NgoMIV, PspGI, and SsoII, which were reported in co-crystal structure analyses to bind one (BglII and EcoRI) or two (BamHI and NgoMIV) Me²⁺ per active site. DNA cleavage experiments were carried out at various Mg²⁺ and Mn²⁺ concentrations at constant ionic strength. All enzymes show a qualitatively similar Mg²⁺ and Mn²⁺ concentration dependence. In general, the Mg²⁺ concentration optimum (between ∼ 1 and 10 mM) is higher than the Mn²⁺ concentration optimum (between ∼ 0.1 and 1 mM). At still higher Mg²⁺ or Mn²⁺ concentrations, the activities of all enzymes tested are reduced but can be reactivated by Ca²⁺. Based on these results, we propose that one Mg²⁺ or Mn²⁺ is critical for restriction enzyme activation, and binding of a second Me²⁺ plays a role in modulating the activity. Steady-state kinetics carried out with EcoRI and BamHI suggest that binding of a second Mg²⁺ or Mn²⁺ mainly leads to an increase in K_m, such that the inhibitory effect of excess Mg²⁺ or Mn²⁺ can be overcome by increasing the substrate concentration. Our conclusions are supported by molecular dynamics simulations and are consistent with the structural observations of both one and two Me²⁺ binding to these enzymes.     

Development of a high-throughput assay for the HIV-1 integrase disintegration reaction

Both HIV-1 integrase (IN) and the central catalytic domain of IN (IN-CCD) catalyze the disintegration reaction in vitro. In this study, IN and IN-CCD proteins were expressed and purified, and a high-throughput format enzyme-linked immunosorbent assay (ELISA) was developed for the disintegration reaction. IN exhibited a marked preference for Mn²⁺ over Mg²⁺ as the divalent cation cofactor in disintegration. Baicalein, a known IN inhibitor, was found to be an IN-CCD inhibitor. The assay is sensitive and specific for the study of disintegration reaction as well as for the in vitro identification of antiviral drugs targeting IN, especially targeting IN-CCD.     

Identification by Mn2+ rescue of two residues essential for the proton transfer of tRNase Z catalysis

Thermotoga maritima tRNase Z cleaves pre-tRNAs containing the ₇₄CCA₇₆ sequence precisely after the A₇₆ residue to create the mature 3′ termini. Its crystal structure has revealed a four-layer αβ/βα sandwich fold that is typically found in the metallo-β-lactamase superfamily. The well-conserved six histidine and two aspartate residues together with metal ions are assumed to form the tRNase Z catalytic center. Here, we examined tRNase Z variants containing single amino acid substitutions in the catalytic center for pre-tRNA cleavage. Cleavage by each variant in the presence of Mg²⁺ was hardly detected, although it is bound to pre-tRNA. Surprisingly, however, Mn²⁺ ions restored the lost Mg²⁺-dependent activity with two exceptions of the Asp52Ala and His222Ala substitutions, which abolished the activity almost completely. These results provide a piece of evidence that Asp-52 and His-222 directly contribute the proton transfer for the catalysis.     

Conserved Structural Chemistry for Incision Activity in Structurally Non-homologous Apurinic/Apyrimidinic Endonuclease APE1 and Endonuclease IV DNA Repair Enzymes

Non-coding apurinic/apyrimidinic (AP) sites in DNA form spontaneously and as DNA base excision repair intermediates are the most common toxic and mutagenic in vivo DNA lesion. For repair, AP sites must be processed by 5′ AP endonucleases in initial stages of base repair. Human APE1 and bacterial Nfo represent the two conserved 5′ AP endonuclease families in the biosphere; they both recognize AP sites and incise the phosphodiester backbone 5′ to the lesion, yet they lack similar structures and metal ion requirements. Here, we determined and analyzed crystal structures of a 2.4 Å resolution APE1-DNA product complex with Mg²⁺ and a 0.92 Å Nfo with three metal ions. Structural and biochemical comparisons of these two evolutionarily distinct enzymes characterize key APE1 catalytic residues that are potentially functionally similar to Nfo active site components, as further tested and supported by computational analyses. We observe a magnesium-water cluster in the APE1 active site, with only Glu-96 forming the direct protein coordination to the Mg²⁺. Despite differences in structure and metal requirements of APE1 and Nfo, comparison of their active site structures surprisingly reveals strong geometric conservation of the catalytic reaction, with APE1 catalytic side chains positioned analogously to Nfo metal positions, suggesting surprising functional equivalence between Nfo metal ions and APE1 residues. The finding that APE1 residues are positioned to substitute for Nfo metal ions is supported by the impact of mutations on activity. Collectively, the results illuminate the activities of residues, metal ions, and active site features for abasic site endonucleases.     

Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis

Alkaline exonuclease and single-strand DNA (ssDNA) annealing proteins (SSAPs) are key components of DNA recombination and repair systems within many prokaryotes, bacteriophages and virus-like genetic elements. The recently sequenced β-proteobacterium Laribacter hongkongensis (strain HLHK9) encodes putative homologs of alkaline exonuclease (LHK-Exo) and SSAP (LHK-Bet) proteins on its 3.17 Mb genome. Here, we report the biophysical, biochemical and structural characterization of recombinant LHK-Exo protein. LHK-Exo digests linear double-stranded DNA molecules from their 5′-termini in a highly processive manner. Exonuclease activities are optimum at pH 8.2 and essentially require Mg²⁺ or Mn²⁺ ions. 5′-phosphorylated DNA substrates are preferred over dephosphorylated ones. The crystal structure of LHK-Exo was resolved to 1.9 Å, revealing a ‘doughnut-shaped’ toroidal trimeric arrangement with a central tapered channel, analogous to that of λ-exonuclease (Exo) from bacteriophage-λ. Active sites containing two bound Mg²⁺ ions on each of the three monomers were located in clefts exposed to this central channel. Crystal structures of LHK-Exo in complex with dAMP and ssDNA were determined to elucidate the structural basis for substrate recognition and binding. Through structure-guided mutational analysis, we discuss the roles played by various active site residues. A conserved two metal ion catalytic mechanism is proposed for this class of alkaline exonucleases.     

Ruthenium red as a carrier of electrons between external NADH and cytochrome c in rat liver mitochondria.

We have determined that Co²⁺, Ni²⁺ or Zn²⁺ may substitute for Mg²⁺ during DNA synthesis with $\text{E.}\text{coli}$ DNA polymerase I, sea urchin nuclear DNA polymerase and the DNA polymerase from avian myeloblastosis virus (AMV). In addition, the frequency of non-complementary nucleotide incorporation using AMV DNA polymerase was increased using Co²⁺ or Mn²⁺ as the metal activator. These results suggest that the fidelity of DNA synthesis may be influenced by the metal activator used during catalysis.     

Mutations in the Human Immunodeficiency Virus Type 1 Integrase D,D(35)E Motif Do Not Eliminate Provirus Formation

The core domain of human immunodeficiency virus type 1 (HIV-1) integrase (IN) contains a D,D(35)E motif, named for the phylogenetically conserved glutamic acid and aspartic acid residues and the invariant 35 amino acid spacing between the second and third acidic residues. Each acidic residue of the D,D(35)E motif is independently essential for the 3′-processing and strand transfer activities of purified HIV-1 IN protein. Using a replication-defective viral genome with a hygromycin selectable marker, we recently reported that a mutation at any of the three residues of the D,D(35)E motif produces a 10³- to 10⁴-fold reduction in infectious titer compared with virus encoding wild-type IN (A. D. Leavitt et al., J. Virol. 70:721–728. 1996). The infectious titer, as measured by the number of hygromycin-resistant colonies formed following infection of cells in culture, was less than a few hundred colonies per μg of p24. To understand the mechanism by which the mutant virions conferred hygromycin resistance, we characterized the integrated viral DNA in cells infected with virus encoding mutations at each of the three residues of the D,D(35)E motif. We found the integrated viral DNA to be colinear with the incoming viral genome. DNA sequencing of the junctions between integrated viral DNA and host DNA showed that (i) the characteristic 5-bp direct repeat of host DNA flanking the HIV-1 provirus was not maintained, (ii) integration often produced a deletion of host DNA, (iii) integration sometimes occurred without the viral DNA first undergoing 3′-processing, (iv) integration sites showed a strong bias for a G residue immediately adjacent to the conserved viral CA dinucleotide, and (v) mutations at each of the residues of the D,D(35)E motif produced essentially identical phenotypes. We conclude that mutations at any of the three acidic residues of the conserved D,D(35)E motif so severely impair IN activity that most, if not all, integration events by virus encoding such mutations are not IN mediated. IN-independent provirus formation may have implications for anti-IN therapeutic agents that target the IN active site.     

Metal Switch-controlled Myosin II from Dictyostelium discoideum Supports Closure of Nucleotide Pocket during ATP Binding Coupled to Detachment from Actin Filaments

G-proteins, kinesins, and myosins are hydrolases that utilize a common protein fold and divalent metal cofactor (typically Mg²⁺) to coordinate purine nucleotide hydrolysis. The nucleoside triphosphorylase activities of these enzymes are activated through allosteric communication between the nucleotide-binding site and the activator/effector/polymer interface to convert the free energy of nucleotide hydrolysis into molecular switching (G-proteins) or force generation (kinesins and myosin). We have investigated the ATPase mechanisms of wild-type and the S237C mutant of non-muscle myosin II motor from Dictyostelium discoideum. The S237C substitution occurs in the conserved metal-interacting switch-1, and we show that this substitution modulates the actomyosin interaction based on the divalent metal present in solution. Surprisingly, S237C shows rapid basal steady-state Mg²⁺- or Mn²⁺-ATPase kinetics, but upon binding actin, its MgATPase is inhibited. This actin inhibition is relieved by Mn²⁺, providing a direct and experimentally reversible linkage of switch-1 and the actin-binding cleft through the swapping of divalent metals in the reaction. Using pyrenyl-labeled F-actin, we demonstrate that acto·S237C undergoes slow and weak MgATP binding, which limits the rate of steady-state catalysis. Mn²⁺ rescues this effect to near wild-type activity. 2′(3′)-O-(N-Methylanthraniloyl)-ADP release experiments show the need for switch-1 interaction with the metal cofactor for tight ADP binding. Our results are consistent with strong reciprocal coupling of nucleoside triphosphate and F-actin binding and provide additional evidence for the allosteric communication pathway between the nucleotide-binding site and the filament-binding region.     

Unusually wide co-factor tolerance in a metalloenzyme; divalent metal ions modulate endo–exonuclease activity in T5 exonuclease

T5 5′–3′ exonuclease is a member of a homologous group of 5′ nucleases which require divalent metal co-factors. Structural and biochemical studies suggest that single-stranded DNA substrates thread through a helical arch or hole in the protein, thus bringing the phosphodiester backbone into close proximity with the active site metal co-factors. In addition to the expected use of Mg²⁺, Mn²⁺ and Co²⁺ as co-factors, we found that divalent zinc, iron, nickel and copper ions also supported catalysis. Such a range of co-factor utilisation is unusual in a single enzyme. Some co-factors such as Mn²⁺ stimulated the cleavage of double-stranded closed-circular plasmid DNA. Such endonucleolytic cleavage of circular double-stranded DNA cannot be readily explained by the threading model proposed for the cleavage of substrates with free 5′-ends as the hole observed in the crystal structure of T5 exonuclease is too small to permit the passage of double-stranded DNA. We suggest that such a substrate may gain access to the active site of the enzyme by a process which does not involve threading.     

Structure-based functional identification of Helicobacter pylori HP0268 as a nuclease with both DNA nicking and RNase activities

HP0268 is a conserved, uncharacterized protein from Helicobacter pylori. Here, we determined the solution structure of HP0268 using three-dimensional nuclear magnetic resonance (NMR) spectroscopy, revealing that this protein is structurally most similar to a small MutS-related (SMR) domain that exhibits nicking endonuclease activity. We also demonstrated for the first time that HP0268 is a nicking endonuclease and a purine-specific ribonuclease through gel electrophoresis and fluorescence spectroscopy. The nuclease activities for DNA and RNA were maximally increased by Mn²⁺ and Mg²⁺ ions, respectively, and decreased by Cu²⁺ ions. Using NMR chemical shift perturbations, the metal and nucleotide binding sites of HP0268 were determined to be spatially divided but close to each other. The lysine residues (Lys7, Lys11 and Lys43) are clustered and form the nucleotide binding site. Moreover, site-directed mutagenesis was used to define the catalytic active site of HP0268, revealing that this site contains two acidic residues, Asp50 and Glu54, in the metal binding site. The nucleotide binding and active sites are not conserved in the structural homologues of HP0268. This study will contribute to improving our understanding of the structure and functionality of a wide spectrum of nucleases.     

Mechanism for Inhibition of Deoxyribonuclease Activity by Antisera

The activity of bovine DNase, but not that of porcine DNase, is inhibited by antisera against bovine DNase, and vice versa. Inhibition of DNase is found in the immunoglobulin G-containing fractions, as shown by ion exchange chromatography. Inactive DNase, carboxymethylated specifically at the active site His¹³⁴, competes with active DNase and reverses the antisera inhibition of DNase, suggesting that the epitode responsible for inhibition does not contain the active site His¹³⁴. Alignment of the sequences of DNase of these two species shows that the greatest variation occurs between residues 153 and 163, within which are three consecutive peptide bonds, Lys-Trp-His-Leu, that are readily cleaved by trypsin, chymotrypsin, or thermolysin. The 8-hr digest of DNase by each of these three proteases has lost the ability to reverse antisera inhibition. The degree of antisera inhibition varies with the metal ion used as the activator for DNase-catalyzed reactions. When Mn²⁺, Co²⁺, or Mg²⁺ plus Ca²⁺ are used as activators, inhibition is approximately 50%. When pBR322 plasmid is used as substrate, gel electrophoresis shows that the DNase-catalyzed DNA hydrolysis produces a significant amount of double-strand cuts with Mn²⁺, Co²⁺, or Mg²⁺ plus Ca²⁺ as activators and antisera inhibit DNase action only on double-strand cuts. With only Mg²⁺ as the activator no double-strand cuts are observed, either in the presence or absence of antisera, and the DNase activity is not significantly inhibited. We conclude that antisera inhibition is due to antibody binding of the DNase polypeptide chain within residues 153 and 163. These residues are not crucial for catalysis, but are required for DNA binding, which results in double-strand cuts.     

Two distinct modes of metal ion binding in the nuclease active site of a viral DNA-packaging terminase: insight into the two-metal-ion catalytic mechanism

Many dsDNA viruses encode DNA-packaging terminases, each containing a nuclease domain that resolves concatemeric DNA into genome-length units. Terminase nucleases resemble the RNase H-superfamily nucleotidyltransferases in folds, and share a two-metal-ion catalytic mechanism. Here we show that residue K428 of a bacteriophage terminase gp2 nuclease domain mediates binding of the metal cofactor Mg²⁺. A K428A mutation allows visualization, at high resolution, of a metal ion binding mode with a coupled-octahedral configuration at the active site, exhibiting an unusually short metal-metal distance of 2.42 Å. Such proximity of the two metal ions may play an essential role in catalysis by generating a highly positive electrostatic niche to enable formation of the negatively charged pentacovalent phosphate transition state, and provides the structural basis for distinguishing Mg²⁺ from Ca²⁺. Using a metal ion chelator β-thujaplicinol as a molecular probe, we observed a second mode of metal ion binding at the active site, mimicking the DNA binding state. Arrangement of the active site residues differs drastically from those in RNase H-like nucleases, suggesting a drifting of the active site configuration during evolution. The two distinct metal ion binding modes unveiled mechanistic details of the two-metal-ion catalysis at atomic resolution.     

Crystal structures of Bacillus stearothermophilus adenylate kinase with bound Ap5A,Mg2+ Ap5A,and Mn2+ Ap5A reveal an intermediate lid position and six coordinate octahedral geometry for bound Mg2+ and Mn2+

Crystal structures of Bacillus stearothermophilus adenylate kinase with bound Ap₅A, Mn²⁺ Ap₅A, and Mg²⁺ Ap₅A have been determined by X-ray crystallography to resolutions of 1.6 Å, 1.85 Å, and 1.96 Å, respectively. The protein's lid domain is partially open, being both rotated and translated away from bound Ap₅A. The flexibility of the lid domain in the ternary state and its ability to transfer force directly to the the active site is discussed in light of our proposed entropic mechanism for catalytic turnover. The bound Zn²⁺ atom is demonstrably structural in nature, with no contacts other than its ligating cysteine residues within 5 Å. The B. stearothermophilus adenylate kinase lid appears to be a truncated zinc finger domain, lacking the DNA binding finger, which we have termed a zinc knuckle domain. In the Mg²⁺ Ap₅A and Mn²⁺ Ap₅A structures, Mg²⁺ and Mn²⁺ demonstrate six coordinate octahedral geometry. The interactions of the Mg²⁺-coordinated water molecules with the protein and Ap₅A phosphate chain demonstrate their involvement in catalyzing phosphate transfer. The protein selects for β-γ (preferred by Mg²⁺) rather than α-γ (preferred by Mn²⁺) metal ion coordination by forcing the ATP phosphate chain to have an extended conformation. Proteins 32:276–288, 1998. © 1998 Wiley-Liss, Inc.     

Metal binding by the D,DX35E motif of human immunodeficiency virus type 1 integrase: selective rescue of Cys substitutions by Mn2+ in vitro

The D,DX(35)E motif characteristic of retroviral integrase enzymes (INs) is expected to bind the required metal cofactors (Mg(2+) or Mn(2+)), but direct evidence for a catalytic role has been lacking. Here we used a metal rescue strategy to investigate metal binding. We established conditions for analysis of an activity of IN, disintegration, in both Mg(2+) and Mn(2+), and tested IN mutants with cysteine substitutions in each acidic residue of the D,DX(35)E motif. Mn(2+) but not Mg(2+) can bind tightly to Cys, so if metal binding at the acidic residues is mechanistically important, it is expected that the Cys-substituted enzymes would be active in the presence of Mn(2+) only. Of the three acidic residues, a strong metal rescue effect was obtained for D116C, a weaker rescue was seen for D64C, and no rescue was seen with E152C. Modest rescue could also be detected for D116C in normal integration in vitro. Comparison to Ser and Ala substitutions at D116 established that the rescue was selective for Cys. Further studies of the response to pH suggest that the metal cofactor may stabilize the deprotonated nucleophile active in catalysis, and studies of the response to NaCl titrations disclose an additional role for the metal cofactor in stabilizing the IN-DNA complex.     

Conformation and reactivity of DNA. IV. Base binding ability of transition metal ions to native DNA and effect on helix conformation with special reference to DNA-Zn(II) complex

The effects of metal ions of the first-row transition and of alkaline earth metals on the DNA helix conformation have been studied by uv difference spectra, circular dichroism, and sedimentation measurements. At low ionic strength (10^?3 M NaClO₄) DNA shows a maximum in the difference absorption spectra in the presence of Zn²⁺, Mn²⁺, Co²⁺, Cd²⁺, and Ni²⁺ but not with Mg²⁺ or Ca²⁺. The amplitude of this maximum is dependent on GC content as revealed by detailed studies of the DNA-Zn²⁺ complex of eight different DNA's. Pronounced changes also occur in the CD spectra of DNA transition metal complexes. A transition appears up to a total ratio of approximately 1 Zn²⁺ per DNA phosphate at 10^?3 M NaClO₄; then no further change was observed up to high concentrations. The characteristic CD changes are strongly dependent on the double-helical structure of DNA and on the GC content of DNA. Differences were also observed in hydrodynamic properties of DNA metal complexes as revealed by the greater increase of the sedimentation coefficient of native DNA in the presence of transition metal ions. Spectrophotometric acid titration experiments and CD measurements at acidic pH clearly indicate the suppression of protonation of GC base-pair regions on the addition of transition metal ions to DNA. Similar effects were not observed with DNA complexes with alkaline earth metal ions such as Mg²⁺ or Ca²⁺. The data are interpreted in terms of a preferential interaction of Zn²⁺ and of other transition metal ions with GC sites by chelation to the N-7 of guanine and to the phosphate residue. The binding of Zn²⁺ to DNA disappears between 0.5 M and 1 M NaClO₄, but complex formation with DNA is observable again in the presence of highly concentrated solutions of NaClO₄ (3?7.2 M NaClO₄) or at 0.5 to 2 M Mn²⁺. At relatively high cation concentration Mg²⁺ is also effective in changing the DNA comformation. These structural alterations probably result from both the shielding of negatively charged phosphate groups and the breakdown of the water structure along the DNA helix. Differential effects in CD are also observed between Mn²⁺, Zn²⁺ on one hand and Mg²⁺ on the other hand under these conditions. The greater sensitivity of the double-helical conformation of DNA to the action of transition metal ions is due to the affinity of the latter to electron donating sites of the bases resulting from the d electronic configuration of the metal ions. An order of the relative phosphate binding ability to base-site binding ability in native DNA is obtained as follows: Mg²⁺, Ba²⁺, < Ca²⁺ < Fe²⁺, Ni²⁺, Co²⁺ < Mn²⁺, Zn²⁺ < Cd²⁺ < Cu²⁺. The metal-ion induced conformational changes of the DNA are explained by alternation of the winding angle between base pairs as occurs in the transition from B to C conformation. These findings are used for a tentative molecular interpretation of some effects of Zn²⁺ and Mn²⁺ in DNA synthesis reported in the literature.     

Characterization of the 3' exonuclease subunit DP1 of Methanococcus jannaschii replicative DNA polymerase D

The B-subunits associated with the replicative DNA polymerases are conserved from Archaea to humans, whereas the corresponding catalytic subunits are not related. The latter belong to the B and D DNA polymerase families in eukaryotes and archaea, respectively. Sequence analysis places the B-subunits within the calcineurin-like phosphoesterase superfamily. Since residues implicated in metal binding and catalysis are well conserved in archaeal family D DNA polymerases, it has been hypothesized that the B-subunit could be responsible for the 3′-5′ proofreading exonuclease activity of these enzymes. To test this hypothesis we expressed Methanococcus jannaschii DP1 (MjaDP1), the B-subunit of DNA polymerase D, in Escherichia coli, and demonstrate that MjaDP1 functions alone as a moderately active, thermostable, Mn²⁺-dependent 3′-5′ exonuclease. The putative polymerase subunit DP2 is not required. The nuclease activity is strongly reduced by single amino acid mutations in the phosphoesterase domain indicating the requirement of this domain for the activity. MjaDP1 acts as a unidirectional, non-processive exonuclease preferring mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreading exonuclease of archaeal family D DNA polymerase.     

Microscopic insight to specificity of metal ion cofactor in DNA cleavage by restriction endonuclease EcoRV

Restriction endonucleases protect bacterial cells against bacteriophage infection by cleaving the incoming foreign DNA into fragments. In presence of Mg²⁺ ions, EcoRV is able to cleave the DNA but not in presence of Ca²⁺, although the protein binds to DNA in presence of both metal ions. We make an attempt to understand this difference using conformational thermodynamics. We calculate the changes in conformational free energy and entropy of conformational degrees of freedom, like DNA base pair steps and dihedral angles of protein residues in Mg²⁺(A)-EcoRV-DNA complex compared to Ca²⁺(S)-EcoRV-DNA complex using all-atom molecular dynamics (MD) trajectories of the complexes. We find that despite conformational stability and order in both complexes, the individual degrees of freedom behave differently in the presence of two different metal ions. The base pairs in cleavage region are highly disordered in Ca²⁺(S)-EcoRV-DNA compared to Mg²⁺(A)-EcoRV-DNA. One of the acidic residues ASP90, coordinating to the metal ion in the vicinity of the cleavage site, is conformationally destabilized and disordered, while basic residue LYS92 gets conformational stability and order in Ca²⁺(S) bound complex than in Mg²⁺(A) bound complex. The enhanced fluctuations hinder placement of the metal ion in the vicinity of the scissile phosphate of DNA. Similar loss of conformational stability and order in the cleavage region is observed by the replacement of the metal ion. Considering the placement of the metal ion near scissile phosphate as requirement for cleavage action, our results suggest that the changes in conformational stability and order of the base pair steps and the protein residues lead to cofactor sensitivity of the enzyme. Our method based on fluctuations of microscopic conformational variables can be applied to understand enzyme activities in other protein-DNA systems.