Yuxing Chen

Human and viral nucleoside/nucleotide kinases involved in antiviral drug activation: Structural and catalytic properties

Antiviral nucleoside and nucleotide analogs, essential for the treatment of viral infections in the absence of efficient vaccines, are prodrug forms of the active compounds that target the viral DNA polymerase or reverse transcriptase. The activation process requires several successive phosphorylation steps catalyzed by different kinases, which are present in the host cell or encoded by some of the viruses. These activation reactions often are rate-limiting steps and are thus open to improvement. We review here the structural and enzymatic properties of the enzymes that carry out the activation of analogs used in therapy against human immunodeficiency virus and against DNA viruses such as hepatitis B, herpes and poxviruses. Four major classes of drugs are considered: thymidine analogs, non-natural L-nucleosides, acyclic nucleoside analogs and acyclic nucleoside phosphonate analogs. Their efficiency as drugs depends both on the low specificity of the viral polymerase that allows their incorporation into DNA, but also on the ability of human/viral kinases to provide the activated triphosphate active forms at a high concentration at the right place. Two distinct modes of action are considered, depending on the origin of the kinase (human or viral). If the human kinases are house-keeping enzymes that belong to the metabolic salvage pathway, herpes and poxviruses encode for related enzymes. The structures, substrate specificities and catalytic properties of each of these kinases are discussed in relation to drug activation.

The ternary structure of the double-headed arrowhead protease inhibitor API-A complexed with two trypsins reveals a novel reactive site conformation.

The double-headed arrowhead protease inhibitors API-A and -B from the tubers of Sagittaria sagittifolia (Linn) feature two distinct reactive sites, unlike other members of their family. Although the two inhibitors have been extensively characterized, the identities of the two P1 residues in both API-A and -B remain controversial. The crystal structure of a ternary complex at 2.48 Å resolution revealed that the two trypsins bind on opposite sides of API-A and are 34 Å apart. The overall fold of API-A belongs to the β-trefoil fold and resembles that of the soybean Kunitz-type trypsin inhibitors. The two P1 residues were unambiguously assigned as Leu87 and Lys145, and their identities were further confirmed by site-directed mutagenesis. Reactive site 1, composed of residues P5 Met83 to P5′ Ala92, adopts a novel conformation with the Leu87 completely embedded in the S1 pocket even though it is an unfavorable P1 residue for trypsin. Reactive site 2, consisting of residues P5 Cys141 to P5′ Glu150, binds trypsin in the classic mode by employing a two-disulfide-bonded loop. Analysis of the two binding interfaces sheds light on atomic details of the inhibitor specificity and also promises potential improvements in enzyme activity by engineering of the reactive sites.

X-ray structure of Mycobacterium tuberculosis nucleoside diphosphate kinase

Introduction. Nucleoside Diphosphate (NDP) kinases transfer the -phosphate of a nucleoside or deoxynucleoside triphosphate, usually ATP, to a nucleoside or deoxynucleoside diphosphate, yielding the substrates of RNA and DNA synthesis. The genes, present in almost all organisms, code for polypeptide chains of about 150 residues with very similar sequences and folds. Nevertheless, the quaternary structure (reviewed in Lascu et al.) is not conserved: most NDP kinases are hexamers, but tetramers are found in some bacteria. The tetramer is illustrated by the X-ray structure of the Myxococcus xanthus enzyme, the hexamer, by those of Dictyostelium, Drosophila, and several human and bovine isoforms (reviewed in Janin et al.). The eukaryotic gene products are 10–12 residues longer at the C-terminus than in Myxococcus. Because major intersubunit contacts implicate these residues in the hexamer, short-chain bacterial NDP kinases were assumed to be tetramers like Myxococcus. We present here the 2.6 Å X-ray structure of the enzyme from Mycobacterium tuberculosis, which has an even shorter polypeptide chain, and show that it forms a very stable hexamer despite the missing interactions.

Crystal structure of Saccharomyces cerevisiae cytoplasmic thioredoxin reductase Trr1 reveals the structural basis for species-specific recognition of thioredoxin

Thioredoxin reductase (TrxR) is a member of the pyridine nucleotide-disulfide oxidoreductase family of the flavoenzymes. It can use a dithiol-disulfide active-site to transfer reducing equivalents from NADPH to thioredoxin (Trx), via the cofactor FAD. In Saccharomyces cerevisiae, the cytoplasmic thioredoxin reductase Trr1 plays an important role in multiple cellular events under the control of transcription factor Yap1 and/or Rho5. Here we present the crystal structure of Trr1 at the resolution of 2.8 A, the first fungal TrxR structure. Structural analysis shows it shares a very similar overall structure to Escherichia coli TrxR. However, fine comparisons indicate some distinct differences at the Trx recognition sites. These differences might be responsible to the species-specific recognition of Trx, which has been demonstrated by previous biochemical assays.

Crystal structure of the yeast cytoplasmic thioredoxin Trx2

Rui Bao, Yuxing Chen,* Ya-Jun Tang, Joel Janin, and Cong-Zhao Zhou* Protein Research Institute, Tongji University, Shanghai 200092, People’s Republic of China Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, Anhui 230026, People’s Republic of China School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, People’s Republic of China Yeast Structural Genomics, IBBMC UMR 8619, Universite Paris-Sud, 91405-Orsay, France

Structural and kinetic analysis of Saccharomyces cerevisiae thioredoxin Trx1: implications for the catalytic mechanism of GSSG reduced by the thioredoxin system

Thioredoxin (Trx) and glutathione/glutaredoxin (GSH/Grx) systems play the dominant role in cellular redox homeostasis. Recently the Trx system has been shown to be responsible to control the balance of GSH/GSSG once the glutathione reductase system is not available. To decipher the structural basis of electron transfer from the Trx system to GSSG, we solved the crystal structures of oxidized Trx1 and glutathionylated Trx1Cys33Ser mutant at 1.76 and 1.80 A, respectively. Comparative structural analysis revealed a key residue Met35 involved in the Trx-GSSG recognition. Subsequent mutagenesis and kinetic studies proved that Met35Arg mutation could alter the apparent K(m) and V(max) values of the reaction. These findings gave us the structural insights into GSSG reduction catalyzed by the Trx system.

Structural and mechanistic analyses of yeast mitochondrial thioredoxin Trx3 reveal putative function of its additional cysteine residues

The yeast Saccharomyces cerevisiae Trx3 is a key member of the thioredoxin system to control the cellular redox homeostasis in mitochondria. We solved the crystal structures of yeast Trx3 in oxidized and reduced forms at 1.80 and 2.10 A, respectively. Besides the active site, the additional cysteine residue Cys69 also undergoes a significant redox-correlated conformational change. Comparative structural analyses in combination with activity assays revealed that residue Cys69 could be S-nitrosylated in vitro. S-nitrosylation of Cys69 will decrease the activity of Trx3 by 20%, which is comparable to the effect of the Cys69Ser mutation. Taken together, these findings provided us some new insights into the putative function of the additional cysteine residues of Trx3.

Nucleoside Diphosphate Kinase and the Activation of Antiviral Phosphonate Analogs of Nucleotides: Binding Mode and Phosphorylation of Tenofovir Derivatives

Tenofovir is an acyclic phosphonate analog of deoxyadenylate used in AIDS and hepatitis B therapy. We find that tenofovir diphosphate, its active form, can be produced by human nucleoside diphosphate kinase (NDPK), but with low efficiency, and that creatine kinase is significantly more active. The 1.65 Å x-ray structure of NDPK in complex with tenofovir mono- and diphosphate shows that the analogs bind at the same site as natural nucleotides, but in a different conformation, and make only a subset of the Van der Waals and polar interactions made by natural substrates, consistent with their comparatively low affinity for the enzyme.

Adenosine phosphonoacetic acid is slowly metabolized by NDP kinase.

NDP kinase catalyzes the last step in the phosphorylation of nucleotides. It is also involved in the activation by cellular kinases of nucleoside analogs used in antiviral therapies. Adenosine phosphonoacetic acid, a close analog of ADP already proposed as an inhibitor of ribonucleotide reductase, was found to be a poor substrate for human NDP kinase, as well as a weak inhibitor with an equilibrium dissociation constant of 0.6 mM to be compared to 0.025 mM for ADP. The X-ray structure of a complex of adenosine phosphonoacetic acid and the NDP kinase from Dictyostelium was determined to 2.0 A resolution showing that the analog adopts a binding mode similar to ADP, but that no magnesium ion is present at the active site. As ACP may also interfere with other cellular kinases, its potential as a drug targeting NDP kinase or ribonucleotide reductase is likely to be limited due to strong side effects. The design of new molecules with a narrower specificity and a stronger affinity will benefit from the detailed knowledge of the complex ACP-NDP kinase.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of mitochondrial thioredoxin Trx3 from Saccharomyces cerevisiae

There are three thioredoxin isoforms in the yeast Saccharomyces cerevisiae: two cytosolic/nuclear thioredoxins, Trx1 and Trx2, and one mitochondrial thioredoxin, Trx3. In the present work, S. cerevisiae Trx3 overexpressed in Escherichia coli was purified and crystallized. The Trx3 crystals were obtained by the hanging-drop vapour-diffusion method. A data set diffracting to 2.0 A resolution was collected from a single crystal. The crystal belongs to space group P3(1), with unit-cell parameters a = b = 49.57, c = 94.55 A, alpha = beta = 90, gamma = 120 degrees. The asymmetric unit is assumed to contain two subunits of Trx3, with a V(M) value of 2.62 A(3) Da(-1) and a solvent content of 53%.