Bisubstrate inhibitors of 6-hydroxymethyl-7,8-dihydroptein pyrophosphokinase: Toward cell permeability.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a key enzyme in the folate biosynthesis pathway. It catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). HPPK is essential for microorganisms but is absent in mammals. Yet, it is not the target of any existing antibiotics. Hence, this enzyme is an attractive target for developing novel antimicrobial agents. A wealth of structural and mechanistic information has provided solid basis for structure-based design of HPPK inhibitors. Our bisubstrate inhibitors were initially created by linking 6-hydroxymethylpterin to adenosine through 2, 3, or 4 phosphate groups (HPnA, n = 2, 3, or 4), among which HP4A exhibited the highest binding affinity (Kd = 0.47 ± 0.04 µM). Further development was carried out based on high-resolution structures of HPPK in complex with HP4A. Replacing the phosphate bridge with a piperidine linked thioether eliminated multiple negative charges of the bridge. Substituting the pterin moiety with 7,7-dimethyl-7,8-dihydropterin improved the binding affinity. Arming the piperidine ring with a carboxyl group and oxidizing the thioether further enhanced the potency, resulting in a druglike inhibitor of HPPK (Kd = 0.047 ± 0.007 µM). None of these inhibitors, however, exhibits bacterial cell permeability. It is most likely due to the lack of active folate transporters in bacteria. Replacing the pterin moiety with a 7-deazagaunine moiety, we have obtained a novel bisubstrate inhibitor (HP-101) showing observable cell permeability toward a Gram-positive bacterium. Here, we report the in vitro activity of HP-101 and its structure in complex with HPPK, providing a framework for structure-based further development.

Bisubstrate inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: Transition state analogs for high affinity binding.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a key enzyme in the folate biosynthesis pathway. It catalyzes pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). HPPK is essential for microorganisms but absent in mammals; therefore, it is an attractive target for developing novel antimicrobial agents. Previously, based on our studies of the structure and mechanism of HPPK, we created first-generation bisubstrate inhibitors by linking 6-hydroxymethylpterin to adenosine through phosphate groups, and developed second-generation inhibitors by replacing the phosphate bridge with a linkage that contains a piperidine moiety. Here, we report third-generation inhibitors designed based on the piperidine-containing inhibitor, mimicking the transition state. We synthesized two such inhibitors, characterized their protein-binding and enzyme inhibition properties, and determined their crystal structures in complex with HPPK, advancing the development of such bisubstrate analog inhibitors.

Overproduction of a Dominant Mutant of the Conserved Era GTPase Inhibits Cell Division in Escherichia coli.

Cell growth and division are coordinated, ensuring homeostasis under any given growth condition, with division occurring as cell mass doubles. The signals and controlling circuit(s) between growth and division are not well understood; however, it is known in Escherichia coli that the essential GTPase Era, which is growth rate regulated, coordinates the two functions and may be a checkpoint regulator of both. We have isolated a mutant of Era that separates its effect on growth and division. When overproduced, the mutant protein Era647 is dominant to wild-type Era and blocks division, causing cells to filament. Multicopy suppressors that prevent the filamentation phenotype of Era647 either increase the expression of FtsZ or decrease the expression of the Era647 protein. Excess Era647 induces complete delocalization of Z rings, providing an explanation for why Era647 induces filamentation, but this effect is probably not due to direct interaction between Era647 and FtsZ. The hypermorphic ftsZ* allele at the native locus can suppress the effects of Era647 overproduction, indicating that extra FtsZ is not required for the suppression, but another hypermorphic allele that accelerates cell division through periplasmic signaling, ftsL*, cannot. Together, these results suggest that Era647 blocks cell division by destabilizing the Z ring.IMPORTANCE All cells need to coordinate their growth and division, and small GTPases that are conserved throughout life play a key role in this regulation. One of these, Era, provides an essential function in the assembly of the 30S ribosomal subunit in Escherichia coli, but its role in regulating E. coli cell division is much less well understood. Here, we characterize a novel dominant negative mutant of Era (Era647) that uncouples these two activities when overproduced; it inhibits cell division by disrupting assembly of the Z ring, without significantly affecting ribosome production. The unique properties of this mutant should help to elucidate how Era regulates cell division and coordinates this process with ribosome biogenesis.

Cyclic depsipeptides as potential cancer therapeutics.

Cyclic depsipeptides are polypeptides in which one or more amino acid is replaced by a hydroxy acid, resulting in the formation of at least one ester bond in the core ring structure. Many natural cyclic depsipeptides possessing intriguing structural and biological properties, including antitumor, antifungal, antiviral, antibacterial, anthelmintic, and anti-inflammatory activities, have been identified from fungi, plants, and marine organisms. In particular, the potent effects of cyclic depsipeptides on tumor cells have led to a number of clinical trials evaluating their potential as chemotherapeutic agents. Although many of the trials have not achieved the desired results, romidepsin (FK228), a bicyclic depsipeptide that inhibits histone deacetylase, has been shown to have clinical efficacy in patients with refractory cutaneous T-cell lymphoma and has received Food and Drug Administration approval for use in treatment. In this review, we discuss antitumor cyclic depsipeptides that have undergone clinical trials and focus on their structural features, mechanisms, potential applications in chemotherapy, and pharmacokinetic and toxicity data. The results of this study indicate that cyclic depsipeptides could be a rich source of new cancer therapeutics.

Inhibition of HIV-1 release by ADAM metalloproteinase inhibitors.

HIV-1 gp120 glycan binding to C-type lectin adhesion receptor L-selectin/CD62L on CD4 T cells facilitates viral attachment and entry. Paradoxically, the adhesion receptor impedes HIV-1 budding from infected T cells and the viral release requires the shedding of CD62L. To systematically investigate CD62L-shedding mediated viral release and its potential inhibition, we screened compounds specific for serine-, cysteine-, aspartyl-, and Zn-dependent proteases for CD62L shedding inhibition and found that a subclass of Zn-metalloproteinase inhibitors, including BB-94, TAPI, prinomastat, GM6001, and GI25423X, suppressed CD62L shedding. Their inhibition of HIV-1 infections correlated with enzymatic suppression of both ADAM10 and 17 activities and expressions of these ADAMs were transiently induced during the viral infection. These metalloproteinase inhibitors are distinct from the current antiretroviral drug compounds. Using immunogold labeling of CD62L, we observed association between budding HIV-1 virions and CD62L by transmission electron microscope, and the extent of CD62L-tethering of budding virions increased when the receptor shedding is inhibited. Finally, these CD62L shedding inhibitors suppressed the release of HIV-1 virions by CD4 T cells of infected individuals and their virion release inhibitions correlated with their CD62L shedding inhibitions. Our finding reveals a new therapeutic approach targeted at HIV-1 viral release.

Structure of Helicobacter pylori dihydroneopterin aldolase suggests a fragment-based strategy for isozyme-specific inhibitor design.

Dihydroneopterin aldolase (DHNA) is essential for folate biosynthesis in microorganisms. Without a counterpart in mammals, DHNA is an attractive target for antimicrobial agents. Helicobacter pylori infection occurs in human stomach of over 50% of the world population, but first-line therapies for the infection are facing rapidly increasing resistance. Novel antibiotics are urgently needed, toward which structural information on potential targets is critical. We have determined the crystal structure of H. pylori DHNA (HpDHNA) in complex with a pterin molecule (HpDHNA:Pterin) at 1.49-Å resolution. The HpDHNA:Pterin complex forms a tetramer in crystal. The tetramer is also observed in solution by dynamic light scattering and confirmed by small-angle X-ray scattering. To date, all but one reported DHNA structures are octameric complexes. As the only exception, ligand-free Mycobacterium tuberculosis DHNA (apo-MtDHNA) forms a tetramer in crystal, but its active sites are only partially formed. In contrast, the tetrameric HpDHNA:Pterin complex has well-formed active sites. Each active site accommodates one pterin molecule, but the exit of active site is blocked by two amino acid residues exhibiting a contact distance of 5.2 â€‹Å. In contrast, the corresponding contact distance in Staphylococcus aureus DHNA (SaDHNA) is twice the size, ranging from 9.8 to 10.5 â€‹Å, for ligand-free enzyme, the substrate complex, the product complex, and an inhibitor complex. This large contact distance indicates that the active site of SaDHNA is wide open. We propose that this isozyme-specific contact distance (ISCD) is a characteristic feature of DHNA active site. Comparative analysis of HpDHNA and SaDHNA structures suggests a fragment-based strategy for the development of isozyme-specific inhibitors.

Crystal structure of Escherichia coli thiamine pyrophosphate-sensing riboswitch in the apo state.

The thiamine pyrophosphate (TPP)-sensing riboswitch is one of the earliest discovered and most widespread riboswitches. Numerous structural studies have been reported for this riboswitch bound with various ligands. However, the ligand-free (apo) structure remains unknown. Here, we report a 3.1 Å resolution crystal structure of Escherichia coli TPP riboswitch in the apo state, which exhibits an extended, Y-shaped conformation further supported by small-angle X-ray scattering data and driven molecular dynamics simulations. The loss of ligand interactions results in helical uncoiling of P5 and disruption of the key tertiary interaction between the sensory domains. Opening of the aptamer propagates to the gene-regulatory P1 helix and generates the key conformational flexibility needed for the switching behavior. Much of the ligand-binding site at the three-way junction is unaltered, thereby maintaining a partially preformed pocket. Together, these results paint a dynamic picture of the ligand-induced conformational changes in TPP riboswitches that confer conditional gene regulation.

Bisubstrate analog inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: new lead exhibits a distinct binding mode.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), a key enzyme in the folate biosynthesis pathway catalyzing the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin, is an attractive target for developing novel antimicrobial agents. Previously, we studied the mechanism of HPPK action, synthesized bisubstrate analog inhibitors by linking 6-hydroxymethylpterin to adenosine through phosphate groups, and developed a new generation of bisubstrate inhibitors by replacing the phosphate bridge with a piperidine-containing linkage. To further improve linker properties, we have synthesized a new compound, characterized its protein binding/inhibiting properties, and determined its structure in complex with HPPK. Surprisingly, this inhibitor exhibits a new binding mode in that the adenine base is flipped when compared to previously reported structures. Furthermore, the side chain of amino acid residue E77 is involved in protein-inhibitor interaction, forming hydrogen bonds with both 2' and 3' hydroxyl groups of the ribose moiety. Residue E77 is conserved among HPPK sequences, but interacts only indirectly with the bound MgATP via water molecules. Never observed before, the E77-ribose interaction is compatible only with the new inhibitor-binding mode. Therefore, this compound represents a new direction for further development.

Bisubstrate analogue inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: New design with improved properties.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), a key enzyme in the folate biosynthetic pathway, catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin. The enzyme is essential for microorganisms, is absent from humans, and is not the target for any existing antibiotics. Therefore, HPPK is an attractive target for developing novel antimicrobial agents. Previously, we characterized the reaction trajectory of HPPK-catalyzed pyrophosphoryl transfer and synthesized a series of bisubstrate analog inhibitors of the enzyme by linking 6-hydroxymethylpterin to adenosine through 2, 3, or 4 phosphate groups. Here, we report a new generation of bisubstrate analog inhibitors. To improve protein binding and linker properties of such inhibitors, we have replaced the pterin moiety with 7,7-dimethyl-7,8-dihydropterin and the phosphate bridge with a piperidine linked thioether. We have synthesized the new inhibitors, measured their K(d) and IC(50) values, determined their crystal structures in complex with HPPK, and established their structure-activity relationship. 6-Carboxylic acid ethyl ester-7,7-dimethyl-7,8-dihydropterin, a novel intermediate that we developed recently for easy derivatization at position 6 of 7,7-dimethyl-7,8-dihydropterin, offers a much high yield for the synthesis of bisubstrate analogs than that of previously established procedure.

New ways to derivatize at position 6 of 7,7-dimethyl-7,8-dihydropterin.

Reported are the synthesis of two intermediates for derivatization at position 6 of 7,7-dimethyl-7,8-dihydropterin: 6-carboxylic acid ethyl ester-7,7-dimethyl-7,8-dihydropterin, which is a novel compound, and 6-aldehyde-7,7-dimethyl-7,8-dihydropterin, which is synthesized by a new method with a yield of 90%.

Dual-Target Inhibitors of the Folate Pathway Inhibit Intrinsically Trimethoprim-Resistant DfrB Dihydrofolate Reductases.

Trimethoprim (TMP) is widely used to treat infections in humans and in livestock, accelerating the incidence of TMP resistance. The emergent and largely untracked type II dihydrofolate reductases (DfrBs) are intrinsically TMP-resistant plasmid-borne Dfrs that are structurally and evolutionarily unrelated to chromosomal Dfrs. We report kinetic characterization of the known DfrB family members. Their kinetic constants are conserved and all are poorly inhibited by TMP, consistent with TMP resistance. We investigate their inhibition with known and novel bisubstrate inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK). Importantly, all are inhibited by the HPPK inhibitors, making these molecules dual-target inhibitors of two folate pathway enzymes that are strictly microbial.

Design, synthesis, and anticancer activity evaluation of irreversible allosteric inhibitors of the ubiquitin-conjugating enzyme Ube2g2.

The RING finger-dependent ubiquitin ligase (E3) gp78, known as the tumor autocrine motility factor receptor, contributes to tumor progression. The protein interacts with its cognate ubiquitin-conjugating enzyme (E2), Ube2g2, via its RING domain and a unique region called G2BR that strongly binds to E2. The binding of G2BR to Ube2g2 allosterically enhances the binding of RING to E2, and the binding of RING triggers the departure of G2BR from E2 also in an allosteric fashion. Targeting these allosteric events, we developed a family of inhibitors that irreversibly block E2-E3 interactions and thereby eliminate the tumorigenic effect of gp78. One among 19 compounds screened with the NCI 60 tumor cell lines exhibited outstanding anticancer activities. At 10 µM, it caused >50% growth inhibition to 40% of the cell lines; at 100 µM, it showed lethiferous activity against most cell lines.

Reaction trajectory of pyrophosphoryl transfer catalyzed by 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase.

6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the Mg(2+)-dependent pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). The reaction follows a bi-bi mechanism with ATP as the first substrate and AMP and HP pyrophosphate (HPPP) as the two products. HPPK is a key enzyme in the folate biosynthetic pathway and is essential for microorganisms but absent from mammals. For the HPPK-catalyzed pyrophosphoryl transfer, a reaction coordinate is constructed on the basis of the thermodynamic and transient kinetic data we reported previously, and the reaction trajectory is mapped out with five three-dimensional structures of the enzyme at various liganded states. The five structures are apo-HPPK (ligand-free enzyme), HPPK.MgATP(analog) (binary complex of HPPK with its first substrate) and HPPK.MgATP(analog).HP (ternary complex of HPPK with both substrates), which we reported previously, and HPPK.AMP.HPPP (ternary complex of HPPK with both product molecules) and HPPK.HPPP (binary complex of HPPK with one product), which we present in this study.

Assignment of Proton Resonances and Conformational Characterization of Oligodeoxyribonucleic Acid d(CCGTACGG) in Solution.

Lycobetaine prepared from lycorine is a new anticancer agent. The experimental and quantum pharmacological studies revealed that lycobetaine can interact with DNA by intercalation, preferentially into GO base pairs. In order to provide detailed interaction model of lycobetaine-DNA, a self-complementary octanucleotide d(CCGTACGG) was designed and synthesized by using new HELP (high efficiency Liquid phase) According to its nature, the sample was prepared to the desired final concentration by adding salt and buffer solutions. Two-dimensional (1)H-(1)H COSY and NOESY spectra in 99.8% D(2)O and 95% H(2)O were recorded for the duplex, and the NMR techniques of presaturation and WATERGATE were applied to water suppression. Protons of every spin system were identified by their scalar couplings, then through their special couplings all protons in the molecule were assigned except the poorly resolved H5' and H5' ' resonances. The chemical shifts of exchangeable protons and NOE intensities of nonexchangeable protons indicate qualitatively that the d(CCGTACGG) helix is right-handed B-DNA in aqueous solution.

Structural enzymology and inhibition of the bi-functional folate pathway enzyme HPPK-DHPS from the biowarfare agent Francisella tularensis.

UNLABELLED: Two valid targets for antibiotic development, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS), catalyze consecutive reactions in folate biosynthesis. In Francisella tularensis (Ft), these two activities are contained in a single protein, FtHPPK-DHPS. Although Pemble et al. (PLoS One 5, e14165) determined the structure of FtHPPK-DHPS, they were unable to measure the kinetic parameters of the enzyme. In this study, we elucidated the binding and inhibitory activities of two HPPK inhibitors (HP-18 and HP-26) against FtHPPK-DHPS, determined the structure of FtHPPK-DHPS in complex with HP-26, and measured the kinetic parameters for the dual enzymatic activities of FtHPPK-DHPS. The biochemical analyses showed that HP-18 and HP-26 have significant isozyme selectivity, and that FtHPPK-DHPS is unique in that the catalytic efficiency of its DHPS activity is only 1/260,000 of that of Escherichia coli DHPS. Sequence and structural analyses suggest that HP-26 is an excellent lead for developing therapeutic agents for tularemia, and that the very low DHPS activity is due, at least in part, to the lack of a key residue that interacts with the substrate p-aminobenzoic acid (pABA). A BLAST search of the genomes of ten F. tularensis strains indicated that the bacterium contains a single FtHPPK-DHPS. The marginal DHPS activity and the single copy existence of FtHPPK-DHPS in F. tularensis make this bacterium more vulnerable to DHPS inhibitors. Current sulfa drugs are ineffective against tularemia; new inhibitors targeting the unique pABA-binding pocket may be effective and less subject to resistance because any mutations introducing resistance may make the marginal DHPS activity unable to support the growth of F. tularensis. DATABASE: The coordinates and structure factors have been deposited in the Protein Data Bank under accession code 4PZV.

Wilms' tumorigenesis is altered by misexpression of the transcriptional co-activator, CITED1.

PURPOSE: Wilms' tumors arise from arrested differentiation of renal progenitor cells. CITED1 is a transcriptional regulator that blocks the metanephric mesenchymal-to-epithelial conversion and is expressed in the blastema of both the developing kidney and Wilms' tumors. We hypothesized that alterations of CITED1-dependent signaling promote persistence of blastema and thereby subject these pluripotent cells to future oncogenic events. METHODS: We used a retroviral delivery system to overexpress the full-length CITED1 (F/L) protein and 2 deletion mutants lacking either of its known functional domains, deltaSID (Smad-4 Interacting Domain) and deltaCR2 (Conserved Region 2; the CITED1 transactivation domain), in a human Wilms' tumor cell line that endogenously expresses CITED1. In vitro effects on cellular proliferation and apoptosis were assayed. In vivo effects on tumorigenesis, growth, proliferation, and apoptosis were determined after heterotransplantation into immunodeficient mice (n = 15 per cell line). RESULTS: In vitro, overexpression of CITED1-F/L significantly increased, whereas overexpression of the functionally inactivating mutant, CITED1-deltaCR2, significantly reduced cellular proliferation relative to the other lines (P < .0001). In vivo, Wilms' tumor incidence was significantly reduced in animals injected with cells overexpressing the mutant CITED1-deltaCR2 (7%) compared with CITED1-F/L (40%, P = .03) and CITED1-deltaSID (60%, P < .002). Similarly, mean tumor volume was least in the CITED1-deltaCR2 animals when compared with CITED1-F/L (P = .03) and CITED1-deltaSID animals (P < .005). Furthermore, the CITED1-deltaCR2 tumor showed the least cellular proliferation. Misexpression of CITED1 did not affect apoptosis either in vitro or in vivo. CONCLUSIONS: Overexpression of CITED1 in a human Wilms' tumor cell line significantly increases proliferation in vitro, whereas mutation of its functionally critical transactivation domain (deltaCR2) significantly reduces proliferation. This mutation further perturbs tumorigenesis and tumor growth after heterotransplantation into immunodeficient mice. We speculate that overexpression of CITED1 promotes expansion of a rapidly proliferating population of blastema and thereby induces an unstable environment highly susceptible to future oncogenic events.

CITED1 expression in Wilms' tumor and embryonic kidney.

Wilms' tumors, or nephroblastomas, are thought to arise from abnormal postnatal retention and dysregulated differentiation of nephrogenic progenitor cells that originate as a condensed metanephric mesenchyme within embryonic kidneys. We have previously shown that the transcriptional regulator CITED1 (CBP/p300-interacting transactivators with glutamic acid [E]/aspartic acid [D]-rich C-terminal domain) is expressed exclusively in these nephrogenic progenitor cells and is downregulated as they differentiate to form nephronic epithelia. In the current study, we show that CITED1 expression persists in blastemal cell populations of both experimental rat nephroblastomas and human Wilms' tumors, and that primary human Wilms' tumors presenting with disseminated disease show the highest level of CITED1 expression. Unlike the predominantly cytoplasmic subcellular localization of CITED1 in the normal developing kidney, CITED1 is clearly detectable in the nuclear compartment of Wilms' tumor blastema. These findings indicate that CITED1 is a marker of primitive blastema in Wilms' tumors and suggest that persistent expression and/or altered subcellular localization of CITED1 in the condensed metanephric mesenchyme could play a role in Wilms' tumor initiation and pathogenesis.

Mechanism of the conformational transitions in 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase as revealed by NMR spectroscopy.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), leading to the biosynthesis of folate cofactors. HPPK undergoes dramatic conformational changes during its catalytic cycle, and the conformational changes are essential for enzymatic catalysis. Thus, the enzyme is not only an attractive target for developing antimicrobial agents but also an excellent model system for studying the catalytic mechanism of enzymatic pyrophosphoryl transfer as well as the role of protein dynamics in enzymatic catalysis. In the present study, we report the NMR solution structures of the binary complex HPPK*MgAMPCPP and the ternary complex HPPK*MgAMPCPP*DMHP, where alpha,beta-methyleneadenosine triphosphate (AMPCPP) and 7, 7-dimethyl-6-hydroxypterin (DMHP) are the analogues of the substrates ATP and HP, respectively. The results suggest that the three catalytic loops of the binary complex of HPPK can assume multiple conformations in slow exchanges as evidenced by multiple sets of NMR signals for several residues in loops 2 and 3 and the very weak or missing NH cross-peaks for several residues in loops 1 and 3. However, the ternary complex shows only one set of NMR signals, and the cross-peak intensities are rather uniform, suggesting that the binding of the second substrate shifts the multiple conformations of the binary complex to an apparently single conformation of the ternary complex. The NMR behaviors and conformations of the binary complex HPPK*MgAMPCPP are significantly different from those of HPPK in complex with Mgbeta,gamma-methyleneadenosine triphosphate (MgAMPPCP). It is suggested that the conformational properties of the binary substrate complex HPPK*MgATP be represented by those of HPPK*MgAMPCPP, because MgAMPCPP is a better MgATP analogue for HPPK with respect to both binding affinity and bound conformation.

The transcriptional activity of CITED1 is regulated by phosphorylation in a cell cycle-dependent manner.

CITED1 is the founding member of the CITED family of cofactors that are involved in regulating a wide variety of CBP/p300-dependent transcriptional responses. In the present study, we show that the phosphorylation status of CITED1 changes during the cell cycle and affects its transcriptional cofactor activity. Tryptic mapping and mutagenesis studies identified five phosphorylated serine residues in CITED1. Phosphorylation of these residues did not affect CRM1-dependent nuclear export, but did decrease CITED1 binding to p300 and inhibited CITED1-dependent transactivation of Smad4 and p300. These results suggest that CITED1 functions as a cell cycle-dependent transcriptional cofactor whose activity is regulated by phosphorylation.

Is the critical role of loop 3 of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase in catalysis due to loop-3 residues arginine-84 and tryptophan-89? Site-directed mutagenesis, biochemical, and crystallographic studies.

Deletion mutagenesis, biochemical, and X-ray crystallographic studies have shown that loop 3 of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is required for the assembly of the active center, plays an important role in the stabilization of the ternary complex of HPPK with MgATP and 6-hydroxymethyl-7,8-dihydropterin (HP), and is essential for catalysis. Whether the critical functional importance of loop 3 is due to the interactions between residues R84 and W89 and the two substrates has been addressed by site-directed mutagenesis, biochemical, and X-ray crystallographic studies. Substitution of R84 with alanine causes little changes in the dissociation constants and kinetic constants of the HPPK-catalyzed reaction, indicating that R84 is not important for either substrate binding or catalysis. Substitution of W89 with alanine increases the K(d) for the binding of MgATP by a factor of 3, whereas the K(d) for HP increases by a factor of 6, which is due to the increase in the dissociation rate constant. The W89A mutation decreases the rate constant for the chemical step of the forward reaction by a factor of 15 and the rate constant for the chemical step of the reverse reaction by a factor of 25. The biochemical results of the W89A mutation indicate that W89 contributes somewhat to the binding of HP and more significantly to the chemical step. The crystal structures of W89A show that W89A has different conformations in loops 2 and 3, but the critical catalytic residues are positioned for catalysis. When these results are taken together, they suggest that the critical functional importance of loop 3 is not due to the interactions of the R84 guanidinium group or the W89 indole ring with the substrates.