A Putative Glutathione-binding Site in T4 Glutaredoxin Investigated by ...

1 downloads 0 Views 4MB Size Report
Mar 11, 1991 - TOM features (Cambillau et al., 1984) of FRODO (Jones, 1978) was used for the .... rabbit glutaredoxin (Hopper et al., 1989) with pig liver thiol-.
Vol . 266, No. 24, Issue of August 25, pp . 16105-16112, 1991 Printed in U.S.A.

OF BIOLOGICAL CHEMISTRY THEJOURNAL 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

A Putative Glutathione-binding Site in T4 Glutaredoxin Investigated by Site-directed Mutagenesis* (Received for publication, March 11, 1991)

Matti NikkolaS, Florence K. GleasonQ,Markku SaarinenS, Thorleif JoelsonS, Olof Bjornbergll, and Hans EklundS 11 From the $Department of Molecular Biology, Swedish Uniuersity of Agricultural Sciences, BiomedicalCenter, Box 590, S-75124 Uppsala, Sweden and the §Department of Plant Biology, University of Minnesota, St. Paul, Minnesota 55108

A glutathione monomer has been docked into the On infection of E. coli, phage T4 produces a protein, which active site cleft of T4 glutaredoxin (previously called was named T4 thioredoxin on the basis of its activity with T4 thioredoxin) using molecular graphics. The central thioredoxin reductase (Berglund, 1969). This protein can also part of the cleft is formed by the side chainof Tyr-16 be reduced efficiently by glutathione (Holmgren, 1978). To on one side and the residues Thr-64,Met-65, and Pro- carry out DNA synthesis effectively upon infection of the E. 66 on the other. The entire glutathione molecule fits coli host cell, the phage T4 has its own ribonucleotide reducwell into the cleft. A cis-peptide bond between the tase, which can be reduced by both the glutathioneand residues Met-65 and Pro-66 allows glutathione to bind thioredoxin systems via T4 thioredoxin (Holmgren, 1978). As in an anti-parallelfashion to residues 64-66. Hydro- we will show below this protein belongs to the glutaredoxin gen bonds can be formed between Met-65 and the family, and we therefore propose that it is called T4 glutareglutathione cysteine. This bindingpositions the glutadoxin. thione sulfur atom ideally for reaction with the glutaThe:rystal structure of T4 glutaredoxin has been refined redoxin disulfide. In themodel, glutathione can forma at A.’ Recently hydrogen bond tothehydroxylgroup of Tyr-16. at 2.0-A resolution and a mutant structure1.45 there has been great progress in the structural studies of the Charged interactions at opposite ends of the the binding cleft are provided by His-12 and Asp-80. The neg- enzymes with which T4 glutaredoxin interacts. Crystalloatively charged a-carboxyl group of glutathione may graphic structure determinations of the B1 subunit of the ribonucleotide reductase* and thethioredoxin reductase of E. interact with a positive helix dipole of the protein. Fifteenmutant T4 glutaredoxinshave been pro- coli are underway (Kuriyan et al., 1989).However, glutathione duced and assayed for glutathione binding by deter- still is structurally the best known of the molecules which interact with T4 glutaredoxin. mining thioltransferase activity. Mutant proteins with substitutions in the sides of the cleft (Tyr-16,Pro-66) Crystallographic studies of glutathione binding to T4 gluexhibited the most marked decreases in thioltransfer- taredoxin have not been possible. Soaking the crystals in a ase activity. Mutation of His-12 to a serine decreases solution containing glutathione breaks the crystals. In the the catalytic efficiency whereas substitutionof Asp-80 native crystals, the active site of one of the two molecules in by serine increases the catalytic efficiency. A double the asymmetric unit has extensive molecular contacts with mutant, D80S;H12S, has much less affinity for gluta- another molecule. The binding of the ligand could introduce thione than either single mutant. Substitutionof Cys- changes which disrupt the crystal interactions. Co-crystalli1 4 produces an inactive protein, whereas C17S retains zation experiments with glutathione or glutathione analogues some thioltransferase activity. have not been successful. Thus, the interaction between T4 glutaredoxin and glutathione has been studied indirectly. Molecular modeling with Escherichia coli has at least two different small disulfide- computer graphics was used to identify the most plausible containing proteinswhich can function as reducing agents for binding site for glutathione. These hypothetical interactions ribonucleotide reductase (see Holmgren, 1989 and Fuchs, were tested with mutant glutaredoxins constructed with site1989; for reviews). These redox proteins, thioredoxin and directed mutagenesis. The thioltransferase activity of the glutaredoxin, differ in the way they are reduced. Thioredoxin mutant proteins was used as a measure of their ability to is reduced by NADPH, catalyzed by the flavoprotein thiore- interact with glutathione. The analysis of the mutantproteins doxin reductase. Glutaredoxin is reduced by the abundant confirms most of the predictions of the glutathione-binding tripeptideglutathione, which, inturn, is also reduced by model. NADPH, in areaction catalyzed by the flavoprotein glutathione reductase (see Fig. 1). MATERIALSANDMETHODS * This work was supported by grants from the Swedish Ministry of Agriculture, the Swedish Natural Science Research Council, the Swedish National Board for Technical Developments, the Finnish Academy, and the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 7 Present address: Dept. of Biochemistry, Kemicentrum, S-22100 Lund, Sweden. 11 TOwhom correspondence should he addressed.

Modeling of the ComplexaThe structure of oxidized wild-type T4 glutaredoxin, refined at 2.0-A resolution to a conventional crystallographic residual of 20%’ was used in the docking studies and for the calculation of surface accessibility (Lee and Richards, 1971). Recently, one of themutant glutaredoxins (V15G;Y16P)has been crystallized and the structure solved by molecular replacement. The

H. Eklund, M. Ingelman, B.-0. Soderberg, T. Uhlin, P.Nordlund, M. Nikkola, T. Joelson, U. Sonnerstam, and K. Petrakos, submitted for publication. U. Uhlin, unpublished data.

16105

Glutathione Binding toT4 Glutaredoxin

16106

Thloredoxln-(SH)2 Thloredoxln-S2

QSSQ

FIG.2. Schematic drawing of the T4 glutaredoxinmolecule (based on a computer drawing using the program MolScript The secondary by Per Kraulis, University of Uppsala (1991)). structure elements are numbered in ascending order. The short fi-

2QSH

\

NADPWW

-

strands which are not in the main pleated sheet are called PA and PB. The nomenclature is revised as compared to Soderberg et al. (1978). aT refers to one turn of a-helix (see legend to Fig. 8). The active-site disulfide bridge is located in the amino end of helix al. Qlutathlone reductsee

NADP’

FIG. 1. Two different pathways are known for reduction of ribonucleotide reductase in E. coli. In the first one, thioredoxin is reduced by thioredoxin reductase and ribonucleotide reductase by thioredoxin. In the second pathway, glutaredoxin is reduced by glutathione which is reduced by glutathione reductase. Ribonucleotide reductase is reduced by glutaredoxin. Upon infection, the phage T4encoded glutaredoxin can use both reduction pathways. T4 glutaredoxin reduces the phage encoded ribonucleotide reductase. refined structure a t 1.45-A resolution shows no changes in theactive site except for the substitutedside chains.’ The accessible active-site surfaceof T4 glutaredoxin was computed with the program MS (Connolly, 1983). Detailed modeling with the TOM features (Cambillau et al., 1984) of FRODO (Jones, 1978) was used for the docking of the glutathione molecule to the active site of T 4 glutaredoxin. The program optimizes the bonding distances and the stereochemistry of interactions between small molecules and proteins by energy minimization. We have aligned the amino acid sequences for E. coli, calf thymus, rabbit, T4 glutaredoxins, and pig liver thioltransferase basedon theknown structure of T4 glutaredoxin and built models of them as described by Eklund etal. (1984). Mutagenesis and Sequencing-The construction andexpression of the mutant glutaredoxins V15G, V15P, Y16P, and V15G;Y16P have been described (Joelson et QL, 1990). The restof the mutant proteins described in this studywere constructed similarly with the “coupled priming” method of Carter et al. (1985) or using the Amersham sitedirected mutagenesis kit RPN.1523 (Nakamaye and Eckstein, 1986). The primers used in the mutagenesis were synthesized by G. Englund, Department of Immunology, University of Uppsala, or by Symbicom AB, Umei, Sweden. The mutations were confirmed by dideoxy sequencing (Sanger et d., 1977) the gene in the M13 vector and the final expression construct. The sequencing reactions were performed using T7 DNA polymerase (Tabor and Richardson, 1987). The enzymes, plasmids, phages, and bacterial strains used in the DNA work were as described by Joelson et al. (1990). Expression and Purification of Proteins-Wild-type and mutant T 4 glutaredoxins were expressed in E. coli N4830 as described by Joelson et ~ l (1990). . In the purification, DEAE-Sepharose CL-GB was substituted for DEAE-cellulose and fast protein liquid chromatography on a Mono-S column, for the CM-cellulose step. Glutaredoxins were eluted from the Mono-S column with a0-0.2 M gradient of NaCl in 50 mM MES, 2 mM EDTA, pH 6.5. Resins were products from Pharmacia LKB Biotechnology Inc. The proteins were concentrated using DIAFLO cells with YM5 membranes and Centricon 3 microconcentrators from Amicon. The purity of the proteins was determined by native (IO-15%, Phast System, Pharmacia) and sodium dodecyl sulfate-(HighDensity, Phast System, Pharmacia) polyacrylamide gel electrophoresis. The concentrations of the wildtype and mutant glutaredoxins T4 were estimated using the extinction

coefficients a t An~nwhich are found in Joelson et d . (1990). Enzyme Assays-The hydroxyethyl disulfide reductase assayswere performed as described in Holmgren (1978, 1985) and the sulfocysteine reductase, as reported by Gan and Wells (1986). Both these procedures measure the ability of T4 glutaredoxin to function as a glutathione disulfide oxidoreductase. Activity is measured in a coupled reaction where the resulting oxidized glutathione is reduced by excess glutathione reductase and NADPH (where GR, glutathione reductase; T4-Glx-S2, oxidized glutaredoxin; T4-GIx-(SH)*, reduced glutaredoxin, RSSR, the oxidized form of a disulfide substrate). NADPH + H ~ ( G ~ ~ ~ ~ Z G S H > ~ T ~ . G ~ XZRSH - S *

)c

GRRED NADP+

GSSG

T4-GLX-(SH)*

RSSR

The oxidation of NADPH was monitored a t 340 nm in a HewlettPackard model 8450A “split-beam’’ spectrophotometer or with a Shimadzu UV-260 spectrophotometer. Oxidized glutathione has been reported to be an inhibitor of E. coli glutaredoxin (Holmgren, 1979). Thus, excess amounts of glutathione reductase were used in the assay to keep glutathione in reduced state for the duration of the assay (approximately 2 min). Hydroxyethyl disulfide (HED):’was purchased from Aldrich. Sulfocysteine was a generous gift of Prof. B. Mannervik, Department of Biochemistry, Uppsala University. NADPH, reduced glutathione, and glutathione reductase were obtained from Sigma. RESULTS

Docking of Glutathionetothe T4 Glutaredoxin-In the proposedreactionmechanismforreduction of T4 glutaredoxin, a mixed disulfide is formed with glutathione (Holmgren, 1978). In the crystal structure of oxidized T4 glutaredoxin, only one of the cysteines is accessible to solvent. This exposed cysteine (Cys-14) is most likely the targetof the thiolate anion of glutathione in the primary reduction step. The glutathione molecule must enter the active site cleft with its sulfur atom approaching the Cys-14 sulfur atom of the disulfide (Fig. 2). The sides of the cleft areformedby residues His-12, Cys-14, and Tyr-16 andresidues Thr-64, Met-65, and Pro-66. The side chain of Tyr-16 forms one face of the central part of the cleft, while both main chain and side chain atoms of residues 64-66 contributeto the opposite face. The ends of the cleft are defined by Tyr-7 and by the amino end of the carboxy-terminal helix (Fig. 3). The cleft



The abbreviations used are: HED, hydroxyethyl disulfide; MES, 2-(N-morpholino)ethanesulfonic acid.

Glutathione Binding to T4 Glutaredoxin Gln 67 Met 65

-Pro66-