Production of mutant dihydrofolate reductases of Lactobacillus casei for nuclear magnetic resonance spectroscopy

Gene, 100 (1991) 219-224 0 1991 Elsevier Science Publishers B.V. 0378-l 119/91/$03.50

219

GENE 04009

Production of mutant dihydrofolate reductases of Lactobacilfus cusei for nuclear magnetic resonance spectroscopy (Recombinant DNA; antibiotic; antineoplastic; site-directed mutagenesis; high-level expression; NMR peak assignment; isotopic labelling)

Julie Andrews*,

Steven J. Minter and R. Wayne Davies

Department of Biochemistry and Applied Molecular Biology, U.M.I.S.T.,

Manchester M60 1QD (U.K.) Tel. (44-61)2363311

Received by: J.R. Kinghom: 12 December 1990 Revised: 4 January 1991 Accepted: 5 January 1991

SUMMARY

Seven mutations (L4P, W21L, D26E, D26N, R57H, R57K and T63Q) affecting residues of dihydrofolate reductase of suspected of being important in substrate, inhibitor, or cofactor binding, were made by gapped-duplex site-directed mutagenesis. Expression of the L. casei dhfr gene required the removal of nucleotide sequences flanking the coding region. Temperature-inducible expression from the 2 pL promoter of plasmid pPLc28 allowed synthesis and subsequent aflinity purification of five mutant proteins in amounts and purity su%cient for nuclear magnetic resonance (NMR) spectroscopic analysis (100 mg or more) from lo-liter cultures. W21L required the growth of 40-liter batches, and L4P was not found. Using a two-plasmid system with ~~1857 providing 1 repressor and pMAC5-14 expressing the mutant gene, any auxotrophic strain of Escherichia coli can be used as a host, allowing isotopic labelling of each amino acid of any protein for rapid NMR peak assignment. Lactobacillus casei,

INTRODUCTION

Dihydrofolate reductase (DHFR) is the target of antibacterial drugs (e.g., trimethoprim) and antineoplastic drugs such as MTX (Blakley et al., 1985). The L. casei enzyme has been used in a large body of work in which Correspondenceto: Dr. R.W. Davies at his present address, Robertson Institute of Biotechnology, Department of Genetics, University of Glasgow, Church Street, Glasgow, Gil 5JS Scotland (U.K.) Tel. (44-41)3398855, ext. 5102; Fax (44-41)3305994. * Present address: Department of Pharmacy, University of Manchester, Manchester Ml3 9PL (U.K.) Tel. (44-61)2752400. Abbreviations: aa, amino acid(s); bp, base pair(s); 2D, two-dimensional; DHFR, diiydrofolate reductase; DTT, dithiothreitol; kb, kilobase or 1000 bp; L., Lactobacillus; LB, Luria-Bertani (broth); MTX, methotrexate; NMR, nuclear magnetic resonance; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; PCR, polymerase chain reaction; SD, Shine and Dalgarno ribosome-binding sequence; Tp, trimethoprim; wt, wild type. dhfr, gene encoding DHFR;

changes in the mode of binding that accompany structural modifications of substrate analogues have been monitored by NMR spectroscopy. The development of site-directed mutagenesis technology allowed the extension of these structure-activity studies to include studies of enzymes with specific replacements of aa side-chains suspected of involvement in ligand binding. A key component of this approach has been the development in this laboratory of a system for the reliable mutagenesis of the L. casei dhfr gene, and high-level expression and affinity purification of mutant forms of the protein. The L. casei dhfr gene was first cloned (Davies and Gronenborn, 1982) and sequenced (Andrews et al., 1985). Seven aa exchanges were chosen which would lead to further insight into substrate, inhibitor or cofactor binding (L4P, W21L, D26E, D26N, R57H, R57K and T63Q). We present here the procedures used to successfully generate six of these mutant proteins in amounts and purity sufficient for NMR spectroscopy (all but L4P). Important results

220 have already been obtained by spectroscopic (Birdsall et al., 1989; Jimenez et al., 1989) and kinetic (Andrews et al., 1989) study of some of these mutations, and further work is underway. The system can be routinely used to produce other mutant proteins. An adaptation, in which the components of the regulated expression system are delivered on two plasmids, is described which allows all 20 auxotrophic strains of E. coli to be used for biosynthetic isotopic labelling of proteins for NMR spectroscopy. This approach is particularly valuable for those contemplating NMR studies on new and large proteins.

EXPERIMENTAL

AND DISCUSSION

(a) Choice of aa exchanges The set of mutant L.. cusei DHFR described here was chosen with the goal of using NMR spectroscopy to test existing hypotheses concerning the role of particular

ARG 57

Y I

d’ //’,n f I

\

(D26N)

MT:

x

:

k

i

I

*y

1

(R57K)

ALA?7

I

i-i I I O-----HeOrH-----0~

LEIJ

114

THRI16 Y

Fig. 1. Hydrogen-bonding network for the DHFR substrate analogue MTX showing individual aa residues thought to be involved in substrate binding. Mutated residues are marked with an asterisk and the changes are given in parentheses. L4P: Leu4 is one of the hydrophobic residues in the substrate-binding site (Leu4, Ala9’ and Leuii4) that are thought to interact with substrate via the backbone carbonyl group. The aim was to remove one of these interactions to investigate its role in the specific binding of the 4-NH, group of inhibitors. Normally, the side group to be mutated is the R substituent of the aa, but in this case we wished to modify the carbonyl group, so the only option available was to convert this residue into a secondary amide, i.e., Pro. W21L: The indole ring of Trp*’ is in hy~ophobic contact with the nicotin~de ring of NADP( + ) and via a water molecule with the pteridine ring of MTX and the carboxyl of AspZ6. This residue might be important in the co-operativity of ligand binding which is usually observed for L. casei DHFR and which may contribute to the Tp selectivity of bacterial compared to vertebrate enzymes. W21L is a radical exchange which might be expected to abolish

aa residues interacting with substrate and inhibitors. Four of the aa proposed to form hydrogen bonds with the substrate have been mutated: Leu4, Trp”, Asp26 and Arg57. Fig. 1 shows the proposed hydrogen bonding network of aa with the inhibitor MTX. The degree of evolutionary conservation of residues was also used as a criterion. X-ray crystal structures available from several DHFR proteins show striking structural similarity, particularly in the active site region, despite considerable variability in aa sequence. The choice of replacement residues was dictated by consideration of steric hindrance and NMR visibility (e.g., Trp2’ + Leu, Args7 + His) as well as the specific question being posed in each case. All the proposed changes were initially modelled using an energy minimization program, to check whether the planned mutation was likely to produce any gross conformations distortions of the protein structure. The rationale for the choice of each of the seven aa exchanges is described in the legend to Fig. 1.

both the hydrophobic contact with the nicotinamide ring and also the H-bond to the pteridine ring. D26E: Asp26 is conserved throughout all bacterial and protozoal DHFRs while all the vertebrate enzymes have glutamate. D26 has been proposed as the proton donor in the catalytic reaction and is known from x-ray crystal structures to interact via two hydrogen bonds with the pteridine ring of the 2,4diaminopteridine group of i~ibitors. The D26E exchange is designed to make the active site of L. casei DHFR more like a vertebrate enzyme. It can be seen from molecular modelhng studies, however, that D26 is at the beginning of the B-helix and it was not obvious whether there would be suIIIcient space in the active site for the additional C-C bond ofthe Glu residue. Molecular modelling indicated that if the first part of the helix were to move outwards by about 1 A, to accommodate the increased length of the side chain, then the hydrogen bonding to the pteridine ring could remain intact and no further conformational change would be required. DMN: AspZ6+ Asn was designed to assess the role of the charge in inhibitor binding and during catalysis. The replacement of the carboxyl group by a carboxamide might be expected to prevent proton donation and hence to have a drastic effect on catalysis, while at the same time having very little effect on the ~onfo~ation of the protein. R57H The compIetely conserved residue Args’ binds the cr-carboxylate of the Glu moiety of MTX and was therefore considered to be important in the binding of certain groups of inhibitors. Changing Arg5’ + His should retain interaction with the y-carboxyl of any folate in the same way as His28 reacts with the y-carboxyl portion of any folate, with some conformational perturbation. The new Hiss’ would also be imme~ately NMR visible. R57K: Args7 + Lys is much more conservative in structurat terms but the loss of an NH, group would only allow the formation of a single H-bond as compared with the pair of H-bonds which are normally formed with Arg present. T63Q: Thr63 was mutated in order to assess its role in cofactor binding and Gln was selected as the replacement residue since it might allow a switch of cofactor specificity away from NADPH and towards NADH. Since the hydroxyl ofThr63 is doubt to form a H-bond to the phosphate group, Thr6’ + Gin should produce not only a change in charge, but also a considerable degree of steric hindrance. Molecular modelling studies have suggested that this type of change could prevent the 2’-phosphate part of the cofactor from binding to the enzyme and instead the Gln could make a H-bond to the oxygen of NADH.

221 (b) Expression of wt Lactobacillus casei dlfiiin Escherichia coli The original clone of the L. casei dhj? gene (pWDLcB 1; Davies and Gronenborn, 1982) consisted of two BamHI fragments of L. cusei DNA in tandem. This clone was selected for dhfr expression by Tp resistance. One of the BumHI fragments contained the entire coding sequence (with the other fragment on the 3’ side relative to the gene), but gave no dhfr expression when subcloned alone. All subclones that gave Tp resistance always proved to contain both BumHI fragments in the same orientation. A 1145bp EcaRI-PstI fragment containing the coding region was cloned into M13mp8, giving M13mp8Lcl (Fig. 2) which was sequenced (Andrews et al., 1985). Processive deletion of this fragment by BAL 3 1 exonuclease was undertaken to generate a minimal coding region fragment that would definitely be expressible in E. coli. BAL 31-digested fragments were cloned into the E. coli expression vector pPLc28 (Remaut et al., 1981) with selection for Tp resistance. One product of BAL 31 digestion (pMT701; Fig. 2) retained the key elements of the L. cusei dhfr gene promoter. This clone produced L. cusei DHFR at high levels even at 28°C when the 1 p,_ promoter of the vector is inactive. The level of DHFR synthesis obtained was the same at 40°C when both promoters are potentially active and is equivalent to the level produced by pMT702 (see following paragraph) with the I p,_ promoter alone. This form of the L. cusei dhfr promoter is clearly a very strong constitutive promoter in E. coli. Deletion of 3’-flanking sequence, possibly combined with the loss of 87 bp at the 5’ end of the 1145-bp fragment, has released the promoter from suppression and from dependence on the downstream BamHI fragment. These interesting regulatory phenomena have not been further investigated. Another product of BAL 3 1 digestion retained only the SD sequence of the L. cusei dhfr gene, and had lost nt sequences between 145 and 333 bp 3’ to the coding sequence. This clone, pMT702, gave high-level temperature-inducible expression of L. cusei dhfr under the control L.casei --

of the 1 pL promoter of pPLc28. Both pMT701 and pMT702 give levels of 12-15% of total E. coli protein as L. cusei DHFR. The 649-bp insert of pMT702 was also expressed in pINIIIA1 (Masui et al., 1984) and in pMAC5-14 (another temperature-inducible A pL vector; Stanssens et al., 1989), yielding DHFR levels of 2-5 % and 14-18x of total cell protein, respectively. The 649-bp fragment was used in all subsequent experiments, and was cloned into M13mp8 to give M13mp802 which provided the material for mutagenesis experiments. (c) Site-directed mutagenesis Site-directed mutagenesis was initiated with L4P and T63Q as the target mutants, using both the double primer (Zoller and Smith, 1984) and gapped-duplex (Kramer et al., 1984) methods. In five separate experiments in which 1000 clones were tested for each mutant with both methods, it was shown that the gapped-duplex method was more efficient, and that the frequency of mutant clones obtained by both methods was considerably enhanced (at least lOO-fold) by using mismatch repair-deficient hosts (Table I), MutL and MutS being equally satisfactory. The gapped-duplex method was used in all subsequent mutagenesis experiments. For reasons that are not apparent, the frequencies of L. cusei dhfr gene mutation (Tables I and II) were well below those reported for other genes (Kramer et al., 1984) although amber-revertant control experiments gave the expected high frequency (Table I). The mutagenic primers used and other details are given in Table II. The recent development of PCR-based mutagenesis (Mullis and Faloona, 1987; Ho et al., 1989) makes the generation of specific mutations at a high frequency even simpler. (d) High-level DHFRs

expression

and

purification

of

mutant

All mutant proteins were expressed in E. coli using the 649-bp insert of pMT702 recloned into pPLc28 after mutagenesis in M13mp8. The host strain used was K12dHldtrp

promoter -35 -10 \I-7

&QRI mpBLc1

1

;

II 134

S D ATG II/ coding I t&zzz%C~~/7V 321

dhfr

TAA sequence gene

812

j i

i :

;

: :

pMT 701

pMT 702

t 87

mI

I y a72

i:S764Wp

’ 785bp

i

insert

insert

Fig. 2. Clones ofL. cusei DNA fragments containing dhfrgene sequences. Construct mp8Lcl is a clone of a 1145-bp EcoRI-PstI fragment from pWDLcB 1 (Davies and Gronenborn, 1982), in Ml3mp8. Plasmids pMT701 and 702 are TpR clones of a BAL 31-digested L. curei fragment from Ml3mp8Lcl in pPLc28 (Remaut et al., 1981). Numbers refer to nt counted from the EcoRI site in the L. cusei DNA fragment containing the dhfr gene. The dhp gene coding region is hatched; filled-in regions correspond to putative SD sites and consensus promoter elements in the L. casei sequence (Andrews et al., 1985).

222 TABLE 1 Comparison of double primer and gapped duplex mutagenesis of the Lactobacillus casei dhfi gene Mutagenesis protocol a

Double primer

Gapped duplex

Host strain repair proficiency b

Repair Repair Repair Repair Repair Repair

+ ve -ve (mutL) -ve (mutS) + ve -ve (mutL) -ve (mutS)

Mutant yield L4P’

T63Qd

AR’

0 6+2 8+3 0.2 20 f 4 18 + 5

0 4t2 5_+2 0.1 7&3 8&2

3&5 63 + 8 72 k 4

’ Double primer mutagenesis was carried out according to Zoller and Smith (1984). Gapped-duplex DNA for 18 mutagenesis reactions was made by mixing 0.2 pmol (1 pg) of the large EcoRI-ZfindIII fragment of M 13mp83 (double-stranded) and 1.5 pmol(3.9 pg) of M 13mp802 single strands and heating at 95°C for 3 min in 80 ~1 total volume of 200 mM KC1/15 mm Tris.HCl pH 7.5. For each mutagenesis, 4~1 of gapped duplex (50 ng) were mixed with 0.4 ~1 (2 pmol) of kinased oligo, boiled for 3 min and cooled to room temperature. The solution was adjusted to 100 mM KC]/25 mM Tris/lS mM MgCl,/l mM DTTjl mM ATP/25 PM each of the four dNTPs and equilibrated to 15°C. The reaction was stopped with 2 ~1 of 0.25 M EDTA and heating at 65’ C for 10 min. Ten ~1 of the mix was added to 200 ~1 of cold-competent BMH 71-18 mutL orBMH71-18mutSARer 1 honice,3minat37”C,andgrowthat37”C for 2 h, the phage supernatant was collected. Phage were plated at 200 per plate on W71-18 or MK30-3. Mutants were identified by selective plaque hybridization carried out according to Zoller and Smith (1984). b E. coli strains defective in mismatch repair (Repair -ve) used were BMH 71-18 mutL or mutS (A lac-proA, thi, supE; F’, Ia0 ZAMl5, proA +B + , mutL or mutS215 : :TnlO), described in Kramer et al. (1984). ’ Leu4 -+ Pro. d Thr6j -+ Gln. ’ AR, amber reversion control experiment carried out according to Kramer et al. (1984).

4% of total cell protein at 6 h of growth at 40°C (Fig. 3). Subsequent studies with W21L mutant protein have shown that it binds the NADPH cofactor very poorly (Birdsall et al., 1989). The fact that the wt DHFR is not normally found as an apoenzyme but always as a binary or tertiary complex with ligands (Fierke et al., 1987) is consistent with NADPH binding being a factor in protein stability. This may provide an explanation for the reduced and transient appearance of W21L after induction. This example supports the utility of an inducible system for protein expression. The specific activities relative to wt of D26E, T63Q, R57K, W21L, D26N, R57H and L4P were 100,50, 10, 5, 2, 1 and 0, respectively. D26E, D26N, R57H and T63Q were all purified by standard MTX affinity chromatography as for the wt protein. The low specific activity of D26N and R57H necessitated the use of SDS-PAGE after the puriIication. W21L bound well to MTX columns, but was rapidly inactivated under elution conditions. It was purified successfully in stable form using NADPH afIinity columns, despite its 400-fold reduced affinity for NADPH. A reasonable amount of pure protein required for a series of NMR experiments is 100 mg. One-dimensional NMR experiments used 7 mg of DHFR per tube at 1 mM. 2D NMR experiments require a minimum concentration of 2 mM protein or 14 mg per tube for COSY experiments and preferably 3-4 mM protein (21-28 mg) for NOESY studies. For wt and D26E, D26N, R57H and R57K mutant DHFRs, yields of 150 mg of purified protein are obtained from only lo-liter cultures. W21L required the growth of 40 liters to obtain 100 mg of pure protein. (e) A two-plasmid system with auxotrophic host strains for isotopic labelling

(Remaut et al., 1981) which produces the temperaturesensitive phage I repressor encoded by gene ~I857 inserted into the chromosome. The D26E, D26N, R57H and R57K mutant L. casei DHFRs were all produced at the same rate and to the same level as wt L. casei DHFR in this system, yielding a plateau level of 12% of total cell protein. L4P was the only mutant protein that was not detectable. The proline may disrupt the b-sheet structure, adversely affecting folding and/or stability. The same observation has been made for the same aa exchange in mouse DHFR (J. Thillet, pers. commun.). Mutant protein T63Q was produced more slowly and to a lower plateau level than wt (Fig. 3). This may reflect slower folding of the mutant protein, or more rapid turnover. At the 18-h time point used to collect wt DHFR, W21L yielded no protein at all (Fig. 3). Mutant protein W21L was found to be produced transiently after induction, with a peak level of

The first goal of all protein NMR work is the assignment of resonance peaks to particular aa. The 2D NMR allows a high proportion of peaks to be assigned for proteins up to 80 aa in size. Larger proteins present increasingly greater difficulty. Isotopic labelling has been used to highlight resonances due to the aa chosen, since they will stand out over the lesser peak intensity due to natural frequencies of deuterium or i3C. Our standard 1~1857 producing host K12dHldtrp grew very poorly in minimal medium, and it was desirable to assess all 20 auxotrophic strains. This goal has been achieved by expressing L. casei dhfr from the I pL promoter of pMAC 5-14, and providing kZ857 from a second plasmid, ~~1857 (Remaut et al., 1983) with which it is compatible. We established the complete set of E. cofi auxotrophic strains, and showed that the system yields 6 y0 of total cell protein as wt L. casei DHFR in acetate or succinate minimal medium with kanamycin and ampicillin selection, or about 7.5 mg/l.

223

224

I

I

I

TIME

I

AFTER

I

I

INDUCTION

I

I

I

/-:-(H)

Fig. 3. Synthesis of wt and mutant L. casei DHFRs. DHFR levels are expressed as y0 of total protein, plotted against time after induction (growth at 40°C). Blackened circles correspond to wt clone pMT702. Blackened triangles correspond to pMT702-T63Q. Open circles are pMT702-W21L. Amounts of DHFR relative to standards were calculated by laser densitometer scanning of SDS-PAGE gels. The other mutants are similar to wt.

(f) Conclusions (1) Expression of the L.caseidhfr gene in E.colirequired the removal of nt sequences flanking the coding region. (2) The L.casei mutant DHFRs, W21L, D26E, D26N, R57H, R57K and T63Q were synthesised from a temperature-inducible 2 p,_ promoter system in E.coli at levels allowing loo-150 mg of pure protein to be obtained from lo- to 40-liter cultures by affinity chromatography. (3) W21L was stable enough in vitro after affinity purification for NMR experiments to be carried out. (4) A two-plasmid I p,-based inducible system combined with the set of 20 auxotrophic E.coli strains was developed to allow isotopic labelling of each aa to expedite NMR studies of new and larger proteins.

ACKNOWLEDGEMENTS

We thank E. Remaut and H.-J. Fritz for the gift of strains and plasmids, G. Ostler and J. Thomas for help with the purification of mutant proteins and G.C.K. Roberts, J. Feeney and B. Birdsall for help with the design of the various mutations. This work was supported by a Medical Research Council U.K. project grant to R.W.D., and by a subsequent MRC post-doctoral fellowship to J.A.

REFERENCES Andrews, J., Clore, G.M., Davies, R.W., Gronenborn, A.M., Gronenborn, B., Kalderon, D., Papadopoulos, P.C., Schafer, S.,

Sims, P.F.G. and Stancombe, R.: Nucleotide sequence ofthe dihydrofolate reductase gene of methotrexate-resistant Lacrobacillus casei. Gene 35 (1985) 217-222. Andrews, J., Fierke, C.A., Birdsall, B., Ostler, G., Feeney, J., Roberts, G.C.K. and Benkovic, S.J.: A kinetic study of wild-type and mutant dihydrofolate reductases from Lactobacillus casei. Biochemistry 28 (1989) 5743-5750. Birdsall, B., Andrews, J., Ostler, G., Tendler, S.J.B., Feeney, J., Roberts, G.C.K., Davies, R.W. and Cheung, H.T.A.: NMR studies of differences in the conformations and dynamics of ligand complexes formed with mutant dihydrofolate reductases. Biochemistry 28 (1989) 1353-1362. Blakley, R.L.: Dihydrofolate reductase. In: Blakley, R.L. and Benkovic, S.I. (Eds.), Folates and Pterins, Vol. 1. Wiley, New York, 1985, pp. 191-253. Dann, J.G., Ostler, G., Bjur, R.A., King, R.W., Scudder, P., Turner, P.C., Roberts, G.C.K., Burgen, A.S.V. and Harding, N.G.L.: Large-scale purification and quantitation of dihydrofolate reductase from a methotrexate-resistant strain of Laciobacillus casei. Biochem. J. 157 (1976) 559-571. Davies, R.W. and Gronenbom, A.M.: Molecular cloning of the gene for dihydrofolate reductase from Lactobacillus casei. Gene 17 (1982) 229-233. Fierke, C.A., Johnson, K.A. and Benkovic, S.J.: Construction and evaluation of the kinetic scheme associated with dihydrofolate reductase from Escherichia coli. Biochemistry 26 (1987) 4085-4092. Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. and Pease, L.R.: Sitedirected mutagenesis by overlap extension using the polymerase chain reaction. Gene (1989) 5 l-59. Jimenez, M.A., Arnold, J.R.P., Andrews, J., Thomas, J.A., Roberts, G.C.K., Birdsall, B. and Feeney, J.: Dihydrofolate reductase: control ofthe mode of substrate binding by aspartate 26. Prot. Engin. 2 (1989) 627-631. Kramer, W., Drutsa, V., Jansen, H.-W., Kramer, B., Pflugfelder, M. and Fritz, H.-J.: The gapped duplex DNA approach to oligonucleotidedirected mutation construction. Nucleic Acids Res. 12 (1984) 9441-9456. Masui, Y., Mizuno, T. and Inouye, M.: Novel high-level expression cloning vehicle: 104-fold amplification of Escherichia coli minor protein. Bio/Technology 2 (1984) 81-85. Mullis, K.B. and Faloona, F.A.: Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155 (1987) 335-350. Peterson, G.L.: Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Anal. Biochem. 100 (1979) 201-220. Remaut, E., Stanssens, P. and Fiers, W.: Plasmid vectors for high et% ciency expression controlled by the p,_ promoter of coliphage lambda. Gene 15 (1981) 81-93. Remaut, E., Tsao, H. and Fiers, W.: Improved plasmid vectors with a thermo-inducible expression and temperature-regulated runaway replication. Gene 22 (1983) 103-113. Stanssens, P., Opsomer, C., McKeown, Y.M., Kramer, W., Zabeau, M. and Fritz, H.-J.: Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers. Nucleic Acids Res. 17 (1989) 4441-4454. Zoller, M.J. and Smith, M.: Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a singlestranded DNA template. DNA 3 (1984) 479-488.