Anticancer Drugs of Lysine Specific Histone Demethylase-1 (LSD1) Display Variable Inhibition on Nucleosome Substrates.

Lysine specific demethylase-1 (LSD1) serves as a regulator of transcription and represents a promising epigenetic target for anticancer treatment. LSD1 inhibitors are in clinical trials for the treatment of Ewing's sarcoma (EWS), acute myeloid leukemia, and small cell lung cancer, and the development of robust inhibitors requires accurate methods for probing demethylation, potency, and selectivity. Here, the inhibition kinetics on the H3K4me2 peptide and nucleosome substrates was examined, comparing the rates of demethylation in the presence of reversible [CC-90011 (PD) and SP-2577 (SD)] and irreversible [ORY-1001 (ID) and tranylcypromine (TCP)] inhibitors. Inhibitors were also subject to viability studies in three human cell lines and Western blot assays to monitor H3K4me2 nucleosome levels in EWS (TC-32) cells, enabling a correlation of drug potency, inhibition in vitro, and cell-based studies. For example, SP-2577, a drug in clinical trials for EWS, inhibits activity on small peptide substrates (Ki = 60 ± 20 nM) using an indirect coupled assay but does not inhibit demethylation on H3K4me2 peptides or nucleosomes using direct Western blot approaches. In addition, the drug has no effect on H3K4me2 levels in TC-32 cells. These data show that SP-2577 is not an LSD1 enzyme inhibitor, although the drug may function independent of demethylation due to its cytotoxic selectivity in TC-32 cells. Taken together, this work highlights the pitfalls of using coupled assays to ascribe a drug's mode of action, emphasizes the use of physiologically relevant substrates in epigenetic drug targeting strategies, and provides insight into the development of substrate-selective inhibitors of LSD1.

An exon-biased biophysical approach and NMR spectroscopy define the secondary structure of a conserved helical element within the HOTAIR long non-coding RNA.

HOTAIR is a large, multi-exon spliced non-coding RNA proposed to function as a molecular scaffold and competes with chromatin to bind to histone modification enzymes. Previous sequence analysis and biochemical experiments identified potential conserved regions and characterized the full length HOTAIR secondary structure. Here, we examine the thermodynamic folding properties and structural propensity of the individual exonic regions of HOTAIR using an array of biophysical methods and NMR spectroscopy. We demonstrate that different exons of HOTAIR contain variable degrees of heterogeneity, and identify one exonic region, exon 4, that adopts a stable and compact fold under low magnesium concentrations. Close agreement of NMR spectroscopy and chemical probing unambiguously confirm conserved base pair interactions within the structural element, termed helix 10 of exon 4, located within domain I of human HOTAIR. This combined exon-biased and integrated biophysical approach introduces a new strategy to examine conformational heterogeneity in lncRNAs and emphasizes NMR as a key method to validate base pair interactions and corroborate large RNA secondary structures.

Solving the Conundrum: Widespread Proteins Annotated for Urea Metabolism in Bacteria Are Carboxyguanidine Deiminases Mediating Nitrogen Assimilation from Guanidine.

Free guanidine is increasingly recognized as a relevant molecule in biological systems. Recently, it was reported that urea carboxylase acts preferentially on guanidine, and consequently, it was considered to participate directly in guanidine biodegradation. Urea carboxylase combines with allophanate hydrolase to comprise the activity of urea amidolyase, an enzyme predominantly found in bacteria and fungi that catalyzes the carboxylation and subsequent hydrolysis of urea to ammonia and carbon dioxide. Here, we demonstrate that urea carboxylase and allophanate hydrolase from Pseudomonas syringae are insufficient to catalyze the decomposition of guanidine. Rather, guanidine is decomposed to ammonia through the combined activities of urea carboxylase, allophanate hydrolase, and two additional proteins of the DUF1989 protein family, expansively annotated as urea carboxylase-associated family proteins. These proteins comprise the subunits of a heterodimeric carboxyguanidine deiminase (CgdAB), which hydrolyzes carboxyguanidine to N-carboxyurea (allophanate). The genes encoding CgdAB colocalize with genes encoding urea carboxylase and allophanate hydrolase. However, 25% of urea carboxylase genes, including all fungal urea amidolyases, do not colocalize with cgdAB. This subset of urea carboxylases correlates with a notable Asp to Asn mutation in the carboxyltransferase active site. Consistent with this observation, we demonstrate that fungal urea amidolyase retains a strong substrate preference for urea. The combined activities of urea carboxylase, carboxyguanidine deiminase and allophanate hydrolase represent a newly recognized pathway for the biodegradation of guanidine. These findings reinforce the relevance of guanidine as a biological metabolite and reveal a broadly distributed group of enzymes that act on guanidine in bacteria.

G-quadruplex RNA binding and recognition by the lysine-specific histone demethylase-1 enzyme.

Lysine-specific histone demethylase 1 (LSD1) is an essential epigenetic regulator in metazoans and requires the co-repressor element-1 silencing transcription factor (CoREST) to efficiently catalyze the removal of mono- and dimethyl functional groups from histone 3 at lysine positions 4 and 9 (H3K4/9). LSD1 interacts with over 60 regulatory proteins and also associates with lncRNAs (TERRA, HOTAIR), suggesting a regulatory role for RNA in LSD1 function. We report that a stacked, intramolecular G-quadruplex (GQ) forming TERRA RNA (GG[UUAGGG]8UUA) binds tightly to the functional LSD1-CoREST complex (Kd ≈ 96 nM), in contrast to a single GQ RNA unit ([UUAGGG]4U), a GQ DNA ([TTAGGG]4T), or an unstructured single-stranded RNA. Stabilization of a parallel-stranded GQ RNA structure by monovalent potassium ions (K(+)) is required for high affinity binding to the LSD1-CoREST complex. These data indicate that LSD1 can distinguish between RNA and DNA as well as structured versus unstructured nucleotide motifs. Further, cross-linking mass spectrometry identified the primary location of GQ RNA binding within the SWIRM/amine oxidase domain (AOD) of LSD1. An ssRNA binding region adjacent to this GQ binding site was also identified via X-ray crystallography. This RNA binding interface is consistent with kinetic assays, demonstrating that a GQ-forming RNA can serve as a noncompetitive inhibitor of LSD1-catalyzed demethylation. The identification of a GQ RNA binding site coupled with kinetic data suggests that structured RNAs can function as regulatory molecules in LSD1-mediated mechanisms.

Structural Roles of Noncoding RNAs in the Heart of Enzymatic Complexes.

Over billions of years of evolution, nature has embraced proteins as the major workhorse molecules of the cell. However, nearly every aspect of metabolism is dependent upon how structured RNAs interact with proteins, ligands, and other nucleic acids. Key processes, including telomere maintenance, RNA processing, and protein synthesis, require large RNAs that assemble into elaborate three-dimensional shapes. These RNAs can (i) act as flexible scaffolds for protein subunits, (ii) participate directly in substrate recognition, and (iii) serve as catalytic components. Here, we juxtapose the near atomic level interactions of three ribonucleoprotein complexes: ribonuclease P (involved in 5' pre-tRNA processing), the spliceosome (responsible for pre-mRNA splicing), and telomerase (an RNA-directed DNA polymerase that extends the ends of chromosomes). The focus of this perspective is profiling the structural and dynamic roles of RNAs at the core of these enzymes, highlighting how large RNAs contribute to molecular recognition and catalysis.

Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA.

Ribonuclease (RNase) P is the universal ribozyme responsible for 5'-end tRNA processing. We report the crystal structure of the Thermotoga maritima RNase P holoenzyme in complex with tRNA(Phe). The 154 kDa complex consists of a large catalytic RNA (P RNA), a small protein cofactor and a mature tRNA. The structure shows that RNA-RNA recognition occurs through shape complementarity, specific intermolecular contacts and base-pairing interactions. Soaks with a pre-tRNA 5' leader sequence with and without metal help to identify the 5' substrate path and potential catalytic metal ions. The protein binds on top of a universally conserved structural module in P RNA and interacts with the leader, but not with the mature tRNA. The active site is composed of phosphate backbone moieties, a universally conserved uridine nucleobase, and at least two catalytically important metal ions. The active site structure and conserved RNase P-tRNA contacts suggest a universal mechanism of catalysis by RNase P.

Structural and functional insights into the N-terminus of Schizosaccharomyces pombe Cdc5.

The spliceosome is a dynamic macromolecular machine composed of five small nuclear ribonucleoparticles (snRNPs), the NineTeen Complex (NTC), and other proteins that catalyze the removal of introns mature to form the mature message. The NTC, named after its founding member Saccharomyces cerevisiae Prp19, is a conserved spliceosome subcomplex composed of at least nine proteins. During spliceosome assembly, the transition to an active spliceosome correlates with stable binding of the NTC, although the mechanism of NTC function is not understood. Schizosaccharomyces pombe Cdc5, a core subunit of the NTC, is an essential protein required for pre-mRNA splicing. The highly conserved Cdc5 N-terminus contains two canonical Myb (myeloblastosis) repeats (R1 and R2) and a third domain (D3) that was previously classified as a Myb-like repeat. Although the N-terminus of Cdc5 is required for its function, how R1, R2, and D3 each contribute to functionality is unclear. Using a combination of yeast genetics, structural approaches, and RNA binding assays, we show that R1, R2, and D3 are all required for the function of Cdc5 in cells. We also show that the N-terminus of Cdc5 binds RNA in vitro. Structural and functional analyses of Cdc5-D3 show that, while this domain does not adopt a Myb fold, Cdc5-D3 preferentially binds double-stranded RNA. Our data suggest that the Cdc5 N-terminus interacts with RNA structures proposed to be near the catalytic core of the spliceosome.

The bacterial ribonuclease P holoenzyme requires specific, conserved residues for efficient catalysis and substrate positioning.

RNase P is an RNA-based enzyme primarily responsible for 5'-end pre-tRNA processing. A structure of the bacterial RNase P holoenzyme in complex with tRNAPhe revealed the structural basis for substrate recognition, identified the active site location, and showed how the protein component increases functionality. The active site includes at least two metal ions, a universal uridine (U52), and P RNA backbone moieties, but it is unclear whether an adjacent, bacterially conserved protein loop (residues 52-57) participates in catalysis. Here, mutagenesis combined with single-turnover reaction kinetics demonstrate that point mutations in this loop have either no or modest effects on catalytic efficiency. Similarly, amino acid changes in the 'RNR' region, which represent the most conserved region of bacterial RNase P proteins, exhibit negligible changes in catalytic efficiency. However, U52 and two bacterially conserved protein residues (F17 and R89) are essential for efficient Thermotoga maritima RNase P activity. The U52 nucleotide binds a metal ion at the active site, whereas F17 and R89 are positioned >20 Å from the cleavage site, probably making contacts with N(-4) and N(-5) nucleotides of the pre-tRNA 5'-leader. This suggests a synergistic coupling between transition state formation and substrate positioning via interactions with the leader.

TERRA-LSD1 phase separation promotes R-loop formation for telomere maintenance in ALT cancer cells.

The telomere repeat-containing RNA (TERRA) forms R-loops to promote homology-directed DNA synthesis in the alternative lengthening of telomere (ALT) pathway. Here we report that TERRA contributes to ALT via interacting with the lysine-specific demethylase 1A (LSD1 or KDM1A). We show that LSD1 localizes to ALT telomeres in a TERRA dependent manner and LSD1 function in ALT is largely independent of its demethylase activity. Instead, LSD1 promotes TERRA recruitment to ALT telomeres via RNA binding. In addition, LSD1 and TERRA undergo phase separation, driven by interactions between the RNA binding properties of LSD1 and the G-quadruplex structure of TERRA. Importantly, the formation of TERRA-LSD1 condensates enriches the R-loop stimulating protein Rad51AP1 and increases TERRA-containing R-loops at telomeres. Our findings suggest that LSD1-TERRA phase separation enhances the function of R-loop regulatory molecules for ALT telomere maintenance, providing a mechanism for how the biophysical properties of histone modification enzyme-RNA interactions impact chromatin function.

Structure and functional implications of a complex containing a segment of U6 RNA bound by a domain of Prp24.

U6 RNA plays a critical role in pre-mRNA splicing. Assembly of U6 into the spliceosome requires a significant structural rearrangement and base-pairing with U4 RNA. In the yeast Saccharomyces cerevisiae, this process requires the essential splicing factor Prp24. We present the characterization and structure of a complex containing one of Prp24's four RNA recognition motif (RRM) domains, RRM2, and a fragment of U6 RNA. NMR methods were used to identify the preferred U6 binding sequence of RRM2 (5'-GAGA-3'), measure the affinity of the interaction, and solve the structure of RRM2 bound to the hexaribonucleotide AGAGAU. Interdomain contacts observed between RRM2 and RRM3 in a crystal structure of the free protein are not detectable in solution. A structural model of RRM1 and RRM2 bound to a longer segment of U6 RNA is presented, and a partial mechanism for Prp24's annealing activity is proposed.

Effect of buffer general acid-base catalysis on the stereoselectivity of ester and thioester H/D exchange in D2O.

As part of a comprehensive investigation on the stereochemistry of base-catalyzed 1,2-elimination and H/D exchange reactions of carbonyl compounds, we have found that the stereoselectivity of H/D exchange of 3-hydroxybutyryl N-acetylcysteamine (3) in D(2)O is strongly influenced by the presence of buffers. This buffer effect is also operative with a simple acyclic ester, ethyl 3-methoxybutanoate (7). Buffers whose general-acid components are cyclic tertiary ammonium ions are particularly effective in changing the stereoselectivity. (2)H NMR analysis showed that without buffer, H/D exchange of 3 produces 81-82% of the 2R*, 3R* diastereomer of 2-deuterio 3 (the anti product). In the presence of 0.33 M 3-quinuclidinone buffer, only 44% of the 2R*, 3R* diastereomer was formed. With ester 7, the stereoselectivity went from 93-94% in DO(-)/D(2)O to 60% in the presence of buffer. Phosphate buffer, as well as others, also showed substantial effects. The results are put into the context of what is known about the mechanism of H/D exchange of esters and thioesters, and the relevance of the buffer effect on the mechanism of the enoyl-CoA hydratase reaction is discussed. It is likely that hydrogen bonding in the enolate-buffer acid encounter complex is an important stereochemical determinant in producing a greater amount of the 2R*, 3S* diastereomer (the syn product). Studies that involve the protonation of enolate anions in D(2)O need to include the buffer general acid in any understanding of the stereoselectivity.

Dissecting Monomer-Dimer Equilibrium of an RNase P Protein Provides Insight Into the Synergistic Flexibility of 5' Leader Pre-tRNA Recognition.

Ribonuclease P (RNase P) is a universal RNA-protein endonuclease that catalyzes 5' precursor-tRNA (ptRNA) processing. The RNase P RNA plays the catalytic role in ptRNA processing; however, the RNase P protein is required for catalysis in vivo and interacts with the 5' leader sequence. A single P RNA and a P protein form the functional RNase P holoenzyme yet dimeric forms of bacterial RNase P can interact with non-tRNA substrates and influence bacterial cell growth. Oligomeric forms of the P protein can also occur in vitro and occlude the 5' leader ptRNA binding interface, presenting a challenge in accurately defining the substrate recognition properties. To overcome this, concentration and temperature dependent NMR studies were performed on a thermostable RNase P protein from Thermatoga maritima. NMR relaxation (R1, R2), heteronuclear NOE, and diffusion ordered spectroscopy (DOSY) experiments were analyzed, identifying a monomeric species through the determination of the diffusion coefficients (D) and rotational correlation times (τc). Experimental diffusion coefficients and τc values for the predominant monomer (2.17 ± 0.36 * 10-10 m2/s, τ c = 5.3 ns) or dimer (1.87 ± 0.40* 10-10 m2/s, τ c = 9.7 ns) protein assemblies at 45°C correlate well with calculated diffusion coefficients derived from the crystallographic P protein structure (PDB 1NZ0). The identification of a monomeric P protein conformer from relaxation data and chemical shift information enabled us to gain novel insight into the structure of the P protein, highlighting a lack of structural convergence of the N-terminus (residues 1-14) in solution. We propose that the N-terminus of the bacterial P protein is partially disordered and adopts a stable conformation in the presence of RNA. In addition, we have determined the location of the 5' leader RNA in solution and measured the affinity of the 5' leader RNA-P protein interaction. We show that the monomer P protein interacts with RNA at the 5' leader binding cleft that was previously identified using X-ray crystallography. Data support a model where N-terminal protein flexibility is stabilized by holoenzyme formation and helps to accommodate the 5' leader region of ptRNA. Taken together, local structural changes of the P protein and the 5' leader RNA provide a means to obtain optimal substrate alignment and activation of the RNase P holoenzyme.

DNA mimicry by a high-affinity anti-NF-kappaB RNA aptamer.

The binding of RNA molecules to proteins or other ligands can require extensive RNA folding to create an induced fit. Understanding the generality of this principle involves comparing structures of RNA before and after complex formation. Here we report the NMR solution structure of a 29-nt RNA aptamer whose crystal structure had previously been determined in complex with its transcription factor target, the p50(2) form of NF-kappaB. The RNA aptamer internal loop structure has pre-organized features that are also found in the complex, including non-canonical base pairing and cross-strand base stacking. Remarkably, the free RNA aptamer structure possesses a major groove that more closely resembles B-form DNA than RNA. Upon protein binding, changes in RNA structure include the kinking of the internal loop and distortion of the terminal tetraloop. Thus, complex formation involves both pre-formed and induced fit binding interactions. The high affinity of the NF-kappaB transcription factor for this RNA aptamer may largely be due to the structural pre-organization of the RNA that results in its ability to mimic DNA.

Structure and interactions of the first three RNA recognition motifs of splicing factor prp24.

The essential Saccharomyces cerevisiae pre-messenger RNA splicing protein 24 (Prp24) has four RNA recognition motifs (RRMs) and facilitates U6 RNA base-pairing with U4 RNA during spliceosome assembly. Prp24 is a component of the free U6 small nuclear ribonucleoprotein particle (snRNP) but not the U4/U6 bi-snRNP, and so is thought to be displaced from U6 by U4/U6 base-pairing. The interaction partners of each of the four RRMs of Prp24 and how these interactions direct U4/U6 pairing are not known. Here we report the crystal structure of the first three RRMs and the solution structure of the first two RRMs of Prp24. Strikingly, RRM 2 forms extensive inter-domain contacts with RRMs 1 and 3. These contacts occupy much of the canonical RNA-binding faces (beta-sheets) of RRMs 1 and 2, but leave the beta-sheet of RRM 3 exposed. Previously identified substitutions in Prp24 that suppress mutations in U4 and U6 spliceosomal RNAs cluster primarily in the beta-sheet of RRM 3, but also in a conserved loop of RRM 2. RNA binding assays and chemical shift mapping indicate that a large basic patch evident on the surface of RRMs 1 and 2 is part of a high affinity U6 RNA binding site. Our results suggest that Prp24 binds free U6 RNA primarily with RRMs 1 and 2, which may remodel the U6 secondary structure. The beta-sheet of RRM 3 then influences U4/U6 pairing through interaction with an unidentified ligand.

NMR resonance assignments of RNase P protein from Thermotoga maritima.

Ribonuclase P (RNase P) is an essential metallo-endonuclease that catalyzes 5' precursor-tRNA (ptRNA) processing and exists as an RNA-based enzyme in bacteria, archaea, and eukaryotes. In bacteria, a large catalytic RNA and a small protein component assemble to recognize and accurately cleave ptRNA and tRNA-like molecular scaffolds. Substrate recognition of ptRNA by bacterial RNase P requires RNA-RNA shape complementarity, intermolecular base pairing, and a dynamic protein-ptRNA binding interface. To gain insight into the binding specificity and dynamics of the bacterial protein-ptRNA interface, we report the backbone and side chain 1H, 13C, and 15N resonance assignments of the hyperthermophilic Thermatoga maritima RNase P protein in solution at 318 K. Our data confirm the formation of a stable RNA recognition motif (RRM) with intrinsic heterogeneity at both the N- and C-terminus of the protein, consistent with available structural information. Comprehensive resonance assignments of the bacterial RNase P protein serve as an important first step in understanding how coupled RNA binding and protein-RNA conformational changes give rise to ribonucleoprotein function.

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.

Emerging structural themes in large RNA molecules.

Extensive networks of tertiary interactions give rise to unique, highly organized domain architectures that characterize the three-dimensional structure of large RNA molecules. Formed by stacked layers of a near-planar arrangement of contiguous coaxial helices, large RNA molecules are relatively flat in overall shape. The functional core of these molecules is stabilized by a diverse set of tertiary interaction motifs that often bring together distant regions of conserved nucleotides. Although homologous RNAs from different organisms can be structurally diverse, they adopt a structurally conserved functional core that includes preassembled active and/or substrate binding sites. These findings broaden our understanding of RNA folding and tertiary structure stabilization, illustrating how large, complex RNAs assemble into unique structures to perform recognition and catalysis.

Identification of the SSB binding site on E. coli RecQ reveals a conserved surface for binding SSB's C terminus.

RecQ DNA helicases act in conjunction with heterologous partner proteins to catalyze DNA metabolic activities, including recombination initiation and stalled replication fork processing. For the prototypical Escherichia coli RecQ protein, direct interaction with single-stranded DNA-binding protein (SSB) stimulates its DNA unwinding activity. Complex formation between RecQ and SSB is mediated by the RecQ winged-helix domain, which binds the nine C-terminal-most residues of SSB, a highly conserved sequence known as the SSB-Ct element. Using nuclear magnetic resonance and mutational analyses, we identify the SSB-Ct binding pocket on E. coli RecQ. The binding site shares a striking electrostatic similarity with the previously identified SSB-Ct binding site on E. coli exonuclease I, although the SSB binding domains in the two proteins are not otherwise related structurally. Substitutions that alter RecQ residues implicated in SSB-Ct binding impair RecQ binding to SSB and SSB/DNA nucleoprotein complexes. These substitutions also diminish SSB-stimulated DNA helicase activity in the variants, although additional biochemical changes in the RecQ variants indicate a role for the winged-helix domain in helicase activity beyond SSB protein binding. Sequence changes in the SSB-Ct element are sufficient to abolish interaction with RecQ in the absence of DNA and to diminish RecQ binding and helicase activity on SSB/DNA substrates. These results support a model in which RecQ has evolved an SSB-Ct binding site on its winged-helix domain as an adaptation that aids its cellular functions on SSB/DNA nucleoprotein substrates.

Inhibition of bacteriophage lambda protein phosphatase by organic and oxoanion inhibitors.

Bacteriophage lambda protein phosphatase (lambdaPP) with Mn(2+) as the activating metal cofactor was studied using phosphatase inhibition kinetics and electron paramagnetic resonance (EPR) spectroscopy. Orthophosphate and the oxoanion analogues orthovanadate, tungstate, molybdate, arsenate, and sulfate were shown to inhibit the phosphomonoesterase activity of lambdaPP, albeit with inhibition constants (K(i)) that range over 5 orders of magnitude. In addition, small organic anions were tested as inhibitors. Phosphonoacetohydroxamic acid (PhAH) was found to be a strong competitive inhibitor (K(i) = 5.1 +/- 1.6 microM) whereas phosphonoacetic acid (K(i) = 380 +/- 45 microM) and acetohydroxamic acid (K(i) > 75 mM) modestly inhibited lambdaPP. Low-temperature EPR spectra of Mn(2+)-reconstituted lambdaPP in the presence of oxoanions and PhAH demonstrate that inhibitor binding decreases the spin-coupling constant, J, compared to the native enzyme. This suggests a change in the bridging interaction between Mn(2+) ions of the dimer due to protonation or replacement of a bridging ligand. Inhibitor binding also induces several spectral shifts. Hyperfine splitting characteristic of a spin-coupled (Mn(2+))(2) dimer is most prominent upon the addition of orthovanadate (K(i) = 0.70 +/- 0.20 microM) and PhAH, indicating that these inhibitors tightly interact with the (Mn(2+))(2) form of lambdaPP. These EPR and inhibition kinetic results are discussed in the context of establishing a common mechanism for the hydrolysis of phosphate esters by lambdaPP and other serine/threonine protein phosphatases.

Mn2+ is a native metal ion activator for bacteriophage lambda protein phosphatase.

Bacteriophage lambda protein phosphatase (lambdaPP) is a member of a large family of metal-containing phosphoesterases, including purple acid phosphatase, protein serine/threonine phosphatases, 5'-nucleotidase, and DNA repair enzymes such as Mre11. lambdaPP can be activated several-fold by various divalent metal ions, with Mn(2+) and Ni(2+) providing the most significant activation. Despite the extensive characterization of purified lambdaPP in vitro, little is known about the identity and stoichiometry of metal ions used by lambdaPP in vivo. In this report, we describe the use of metal analysis, activity measurements, and whole cell EPR spectroscopy to investigate in vivo metal binding and activation of lambdaPP. Escherichia coli cells overexpressing lambdaPP show a 22.5-fold increase in intracellular Mn concentration and less dramatic changes in the intracellular concentration of other biologically relevant metal ions compared to control cells that do not express lambdaPP. Phosphatase activity assessed using para-nitrophenylphosphate as substrate is increased 850-fold in cells overexpressing lambdaPP, indicating the presence of metal-activated enzyme in cell lysate. EPR spectra of intact cells overexpressing lambdaPP exhibit resonances previously attributed to mononuclear Mn(2+) and dinuclear [(Mn(2+))(2)] species bound to lambdaPP. Spin quantitation of EPR spectra of intact E. coli cells overexpressing lambdaPP indicates the presence of approximately 40 microM mononuclear Mn(2+)-lambdaPP and 60 microM [(Mn(2+))(2)]-lambdaPP. The data suggest that overexpression of lambdaPP results in a mixture of apo-, mononuclear-Mn(2+), and dinuclear-[(Mn(2+))(2)] metalloisoforms and that Mn(2+) is a physiologically relevant activating metal ion in E. coli.