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Biomedical subjects

N Yao

Publications and source records attributed to N Yao.

At least 37 records · Page 2Linked to original sources
PubMed1998

Helicase, a target for novel inhibitors of hepatitis C virus.

Virus-encoded enzymes of hepatitis C virus (HCV) were identified from its genome sequence. This allows application of drug discovery strategies which rely on inhibition of enzymes unique to HCV. Discovery of high-affinity inhibitors is facilitated by knowledge of the target enzyme's three-dimensional structure. For development of inhibitors of the HCV helicase, which belongs to a class of enzymes for which little structural information is available, the impact of structural information on the drug discovery process is greater than for targets belonging to well-characterized classes of enzyme. Here the structure of the HCV helicase is described. Regions required for enzymatic activity, which are also the preferred sites of drug interaction, are highlighted.

Acid Anhydride Hydrolases↗
PubMed1997

An interaction between DNA ligase I and proliferating cell nuclear antigen: implications for Okazaki fragment synthesis and joining.

Although three human genes encoding DNA ligases have been isolated, the molecular mechanisms by which these gene products specifically participate in different DNA transactions are not well understood. In this study, fractionation of a HeLa nuclear extract by DNA ligase I affinity chromatography resulted in the specific retention of a replication protein, proliferating cell nuclear antigen (PCNA), by the affinity resin. Subsequent experiments demonstrated that DNA ligase I and PCNA interact directly via the amino-terminal 118 aa of DNA ligase I, the same region of DNA ligase I that is required for localization of this enzyme at replication foci during S phase. PCNA, which forms a sliding clamp around duplex DNA, interacts with DNA pol delta and enables this enzyme to synthesize DNA processively. An interaction between DNA ligase I and PCNA that is topologically linked to DNA was detected. However, DNA ligase I inhibited PCNA-dependent DNA synthesis by DNA pol delta. These observations suggest that a ternary complex of DNA ligase I, PCNA and DNA pol delta does not form on a gapped DNA template. Consistent with this idea, the cell cycle inhibitor p21, which also interacts with PCNA and inhibits processive DNA synthesis by DNA pol delta, disrupts the DNA ligase I-PCNA complex. Thus, we propose that after Okazaki fragment DNA synthesis is completed by a PCNA-DNA pol delta complex, DNA pol delta is released, allowing DNA ligase I to bind to PCNA at the nick between adjacent Okazaki fragments and catalyze phosphodiester bond formation.

Chromatography, Affinity↗
PubMed1997

A complex consisting of human replication factor C p40, p37, and p36 subunits is a DNA-dependent ATPase and an intermediate in the assembly of the holoenzyme.

Human replication factor C (hRFC) is a multi-subunit protein complex capable of supporting proliferating cell nuclear antigen (PCNA)-dependent DNA synthesis by DNA polymerases delta and epsilon. The hRFC complex consists of five different subunits with apparent molecular masses of 140, 40, 38, 37, and 36 kDa. We have previously reported the expression of a three-subunit core complex, consisting of the p40, p37, and p36 subunits following coupled in vitro transcription-translation of the cDNAs encoding these proteins (Uhlmann, F., Cai, J., Flores-Rozas, H., Dean, F. B., Finkelstein, J. , O'Donnell, M., and Hurwitz, J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6521-6526). Here we describe the isolation of a stable complex composed of the p40, p37, and p36 subunits of hRFC from baculovirus-infected insect cells. The purified p40.p37.p36 complex, like the five-subunit RFC, contained DNA-dependent ATPase activity that was stimulated by PCNA, preferentially bound to primed DNA templates, interacted with PCNA, and was capable of unloading PCNA from singly-nicked circular DNA. In contrast to the five-subunit RFC, the three-subunit core complex did not load PCNA onto DNA. The p40. p37.p36 complex inhibited the elongation of primed DNA templates catalyzed by the DNA polymerase delta holoenzyme. Incubation of the p40.p37.p36 complex with the hRFC p140 and p38 subunits formed the five-subunit hRFC complex that supported PCNA-dependent DNA synthesis by DNA polymerase delta.

Adenosine Triphosphatases↗
PubMed1997

Structure of the hepatitis C virus RNA helicase domain.

Helicases are nucleotide triphosphate (NTP)-dependent enzymes responsible for unwinding duplex DNA and RNA during genomic replication. The 2.1 A resolution structure of the HCV helicase from the positive-stranded RNA hepatitis C virus reveals a molecule with distinct NTPase and RNA binding domains. The structure supports a mechanism of helicase activity involving initial recognition of the requisite 3' single-stranded region on the nucleic acid substrate by a conserved arginine-rich sequence on the RNA binding domain. Comparison of crystallographically independent molecules shows that rotation of the RNA binding domain involves conformational changes within a conserved TATPP sequence and untwisting of an extended antiparallel beta-sheet. Location of the TATPP sequence at the end of an NTPase domain beta-strand structurally homologous to the 'switch region' of many NTP-dependent enzymes offers the possibility that domain rotation is coupled to NTP hydrolysis in the helicase catalytic cycle.

Amino Acid Sequence↗
PubMed1997

A low energy short hydrogen bond in very high resolution structures of protein receptor--phosphate complexes.

A very short hydrogen bond between an Asp and a phosphate is established in two high resolution structures (0.98 and 1.05 A). A mutant complex that changes the Asp to an Asn, which forms a normal hydrogen bond, has a similar free energy of binding to the wild type complex, suggesting that the contribution of the short hydrogen bond is not extraordinarily strong.

Carrier Proteins↗
PubMed1996

Reconstitution of human replication factor C from its five subunits in baculovirus-infected insect cells.

Human replication factor C (RFC, also called activator 1) is a five-subunit protein complex (p140, p40, p38, p37, and p36) required for proliferating cell nuclear antigen (PCNA)-dependent processive DNA synthesis catalyzed by DNA polymerase delta or epsilon. Here we report the reconstitution of the RFC complex from its five subunits simultaneously overexpressed in baculovirus-infected insect cells. The purified baculovirus-produced RFC appears to contain equimolar levels of each subunit and was shown to be functionally identical to its native counterpart in (i) supporting DNA polymerase delta-catalyzed PCNA-dependent DNA chain elongation; (ii) catalyzing DNA-dependent ATP hydrolysis that was stimulated by PCNA and human single-stranded DNA binding protein; (iii) binding preferentially to DNA primer ends; and (iv) catalytically loading PCNA onto singly nicked circular DNA and catalytically removing PCNA from these DNA molecules.

Adenosine Triphosphatases↗
PubMed1996

Biomimetic Pathways for Assembling Inorganic Thin Films

Living organisms construct various forms of laminated nanocomposites through directed nucleation and growth of inorganics at self-assembled organic templates at temperatures below 100°C and in aqueous solutions. Recent research has focused on the use of functionalized organic surfaces to form continuous thin films of single-phase ceramics. Continuous thin films of mesostructured silicates have also been formed on hydrophobic and hydrophilic surfaces through a two-step mechanism. First, under acidic conditions, surfactant micellar structures are self-assembled at the solid/liquid interface, and second, inorganic precursors condense to form an inorganic-organic nanocomposite. Epitaxial coordination of adsorbed surfactant tubules is observed on mica and graphite substrates, whereas a random arrangement is observed on amorphous silica. The ability to process ceramic-organic nanocomposite films by these methods provides new technological opportunities.

Journal Article↗
PubMed1996

Maltose-binding protein containing an interdomain disulfide bridge confers a dominant-negative phenotype for transport and chemotaxis.

Bacterial substrate-binding proteins exist in an equilibrium among four forms: open/substrate-free, open/substrate-bound, closed/substrate-free, and closed/substrate-bound. Ligands stabilize the closed conformation, whereas the open conformation predominates in the substrate-free species. In its closed form, the NH2-terminal and COOH-terminal domains of maltose-binding protein (MBP) are proposed to be aligned to allow residues in both domains to interact simultaneously with complementary sites on the MalF and MalG proteins of the maltodextrin uptake system or with the Tar chemotactic signal transducer. However, the initial interaction might occur with an open/substrate-bound form of the binding protein, which would then close in contact with MalFG or Tar. Ligand would help stabilize this complex. We introduced cysteines (G69C and S337C) by site-directed mutagenesis into each domain of MBP and found that they formed an interdomain disulfide cross-link that should hold the protein in a closed conformation. This mutant MBP confers a dominant-negative phenotype for growth on maltose, for maltose transport, and for maltose chemotaxis. The growth and transport defects are partially reversed when the cells are exposed to the reducing agent dithiothreitol. We conclude that the cross-linked form of MBP competes with wild-type MBP in vivo for interaction with MalFG and Tar.

ATP-Binding Cassette Transporters↗
PubMed1996

Negative electrostatic surface potential of protein sites specific for anionic ligands.

Determination of the crystal structure of an "open" unliganded active mutant (T141D) form of the Escherichia coli phosphate receptor for active transport has allowed calculation of the electrostatic surface potential for it and two other comparably modeled receptor structures (wild type and D137N). A discovery of considerable implication is the intensely negative potential of the phosphate-binding cleft. We report similar findings for a sulfate transport receptor, a DNA-binding protein, and, even more dramatically, redox proteins. Evidently, for proteins such as these, which rely almost exclusively on hydrogen bonding for anion interactions and electrostatic balance, a noncomplementary surface potential is not a barrier to binding. Moreover, experimental results show that the exquisite specificity and high affinity of the phosphate and sulfate receptors for unions are insensitive to modulations of charge potential, but extremely sensitive to conditions that leave a hydrogen bond donor or acceptor unpaired.

Anions↗
PubMed1996

Modulation of a salt link does not affect binding of phosphate to its specific active transport receptor.

Electrostatic interactions are among the key forces determining the structure and function of proteins. These are exemplified in the liganded form of the receptor, a phosphate binding protein from Escherichia coli. The phosphate, completely dehydrated and buried in the receptor, is bound by 12 hydrogen bonds as well as a salt link with Arg 135. We have modulated the ionic attraction while preserving the hydrogen bonds by mutating Asp 137, also salt linked to Arg 135, to Asn, Gly or Thr. High-resolution crystallographic analysis revealed that Gly and Thr (but not Asn) mutant proteins have incorporated a more electronegative Cl- in place of the Asp carboxylate. That no dramatic effect on phosphate affinity was produced by these ionic perturbations indicates a major role for hydrogen bonds and other local dipoles in the binding and charge stabilization of ionic ligands.

Asparagine↗
PubMed1996

Clamp loading, unloading and intrinsic stability of the PCNA, beta and gp45 sliding clamps of human, E. coli and T4 replicases.

BACKGROUND: The high speed and processivity of replicative DNA polymerases reside in a processivity factor which has been shown to be a ring-shaped protein. This protein ("sliding clamp') encircles DNA and tethers the catalytic unit to the template. Although in eukaryotic, prokaryotic and bacteriophage-T4 systems, the processivity factors are ring-shaped, they assume different oligomeric states. The Escherichia coli clamp (the beta subunit) is active as a dimer while the eukaryotic and T4 phage clamps (PCNA and gp45, respectively) are active as trimers. The clamp can not assemble itself on DNA. Instead, a protein complex known as a clamp loader utilizes ATP to assemble the ring around the primer-template. This study compares properties of the human PCNA clamp with those of E. coli and T4 phage. RESULTS: The PCNA ring is a stable trimer down to a concentration below 100 nM (Kd approximately 21 nM). On DNA, the PCNA clamp slides freely and dissociates from DNA slowly (t1/2 approximately 24 min). beta is more stable in solution (Kd < 60 PM) and on DNA (t1/2 approximately 1 h) than PCNA which may be explained by its simpler oligomeric state. The T4 gp45 clamp is a much less stable trimer than PCNA (Kd approximately 250 nM) and requires association with the polymerase to stabilize it on DNA as observed previously. The consequence of this cooperation between clamp and polymerase is that upon finishing a template and dissociation of the polymerase from DNA, the gp45 clamp spontaneously dissociates from DNA without assistance. However, the greater stability of the PCNA and beta clamps on DNA necessitates an active process for their removal. The clamp loaders (RFC and gamma complex) were also capable of unloading their respective clamps from DNA in the presence of ATP. CONCLUSIONS: The stability of the different clamps in solution correlates with their stability on DNA. Thus, the low stability of the T4 clamp explains the inability to isolate gp45 on DNA. The stability of the PCNA and beta clamps predicts they will require an unloading factor to recycle them on and off DNA during replication. The clamp loaders of PCNA and beta double as clamp unloaders presumably for the purpose of clamp recycling.

Bacteriophage T4↗
PubMed1995

Escherichia coli expression vectors containing a protein kinase recognition motif, His6-tag and hemagglutinin epitope.

Escherichia coli expression vectors, based on the pET system, were constructed to allow fusion of a protein kinase (PK) recognition motif, a hemagglutinin (HG) epitope-tag and a His6-tag at the N-terminal portion of a protein of interest. The fusion proteins, that result from expression using these vectors, can be phosphorylated in vitro using cAMP-dependent PK, immunoprecipitated using monoclonal antibody against the HG-epitope, and can be rapidly purified using a Ni2+ column.

Amino Acid Sequence↗
PubMed1994

Refined 1.89-A structure of the histidine-binding protein complexed with histidine and its relationship with many other active transport/chemosensory proteins.

The structure of the histidine-binding protein (HBP, M(r) = 26,100), involved solely in active transport, has been determined by the molecular replacement technique and refined to 1.89-A resolution and to an R-factor of 0.199. The structure is that of two protein molecules, each with a bound L-histidine, in the asymmetric unit. Replacement solution was achieved by using a model of the crystal structure of the ligand-free, open-cleft form of the lysine/arginine/ornithine-binding protein which was modified so that the two domains are close to each other by bending the hinge connecting the two domains. The bound histidine is held in place by 10 hydrogen bonds, 2 salt links, and about 60 van der Waals contacts. Elucidation of the HBP structure brings a total of eight different binding proteins structures determined in our laboratory, including those with specificities for monosaccharides, maltodextrins (linear and cyclic), aliphatic amino acids, and inorganic oxyanions. These structures comprise about a third of the entire family of periplasmic binding proteins which act as initial primary high-affinity receptors of active transport in Gram-negative bacteria. Two of the binding proteins with specificities for glucose/galactose and maltodextrins also serve in a similar capacity in chemotaxis. Though these proteins have different molecular weights (ranging from 26,000 to 40,000), amino acid sequences, and ligand specificities, their three-dimensional structures are similar overall. They are elongated (axial ratios of 2:1) and composed of two similar globular domains separated by a deep cleft wherein the ligand-binding site is located. These structures provide understanding of molecular recognition of a variety of ligands at the atomic level and functional roles of the binding proteins.

Bacterial Proteins↗