Combinatorial mutagenesis of three major groove-contacting residues of EcoRI: single and double amino acid replacements retaining methyltransferase-sensitive activities

Gene. 106 (1991) 7-12 CC:1991 Elsevier

GENE

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Publishers

B.V. All rights reserved.

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0378-I 119/91/$03.50

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Combinatorial mutagenesis of three major groove-contacting residues of EcoRI: single and double amino acid replacements retaining methyltransferase-sensitive activities (DNA-protein

interactions;

enzyme

specificity;

polymerase

chain reaction;

protein

engineering)

Joel Osuna, Humberto Flares and Xavier Soberh Cerltvode Investigaci6nsobre Ingenieria GenCticay Biotecnologiu, UniversidadNational Aminoma de MPxico, Apdo. Postal510-3, Cuernavaca, Morelos 622 71 (Mexico) Received by F. Bolivar: 6 May 1991 Revised/Accepted: 20 May/23 July 1991 Received at publishers: 29 July 1991

SUMMARY

A library of mutant ecoRIR genes encoding EcoRI restriction endonuclease was generated using trinucleotide blocks and a combination of recombinant DNA procedures, including primer extension and the polymerase chain reaction. Codons corresponding to three amino acids (E’44, R’45 and R2”’ ), previously implicated in the specific recognition of the DNA substrate, were combinatorially mutated so as to generate a library that potentially contains all 203 possible single, double and triple aa replacements, in a balanced distribution. Inspection of the phenotypes of Escherichiu colt’ colonies bearing the mutant genes showed that several of them retained activities that were deleterious to the cells but were still protected by the EcoRI methyltransferase. These included new enzyme variants, including non-conservative single (Thr or Val for Glui4”) and double (Val for G~u’~~ and Thr for Argi4’) replacements.

INTRODUCTION

In the study of the structure-function relationship of proteins, as well as for the generation of proteins with novel properties, methods for the site-directed mutagenesis of the corresponding genes have become an indispensable tool. There are several stages, conceptual and methodological, to be taken into account when designing a rational muta-

Correspondence

Cot. Miraval

tot Dr. X. Soberon, CIIGB/UNAM, Apdo. Postal 510-3, 62271, Cuernavaca, Morelos (Mexico)

Tel. (52-73) 172-399; Fax (52-73) 172-388. aa, amino acid(s); bp, base pair(s); ecoRIR,gene encoding

Abbreviations: /&RI;

proRIM,

gene encoding

clease;

EtdBr,

ethidium

pyranoside;

M. EcoRI;

bromide;

kb, 1000 bp; Km, kanamycin;

M. EcoRI, EcoRl

MTase;

MTase,

ENase,

IPTG,

restriction

endonu-

isopropyl-P-D-thiogalacto-

LB, Luria-Bertani

methyltransferase;

(medium);

nt, nucleotide(s);

oligo, oligodeoxyribonucleotide; PCR, polymerase chain reaction; PolIk, Klenow (large) fragment of E. coli DNA polymerase I; u, resistance; ss, single-strand(ed);

wt, wild type.

genesis strategy: first, even when there is a known threedimensional structure for the protein, it is extremely difficult to predict which changes are necessary to achieve the desired property of the protein. This type of prediction is still harder in the event that more than one residue change is needed. Second, frequently the residues one wishes to alter are far away from each other in the primary sequence (although they may be close together in the active conformation of the protein), and the combinatorial alteration of such residues is highly desirable (Wells, 1990). Third, as the number of target residues increases, the use of codons as mutagenic units becomes important in order to obtain a large number of useful alleles within a manageable mutant population (Sirotkin, 1986). We propose here a method for combinatorial, saturation mutagenesis, with codons as mutagenic units. In order to test a mutagenesis method incorporating the considerations mentioned above, we chose the restriction endonuclease EcoRI as a model protein. The first report on the structure of the protein-DNA complex, to 3A resolution

8 (McClarin et al., 1986), led to the proposition that three aa (E 144 , R’45 and R200) directly contact the purine bases at the major groove of the substrate DNA molecule. A recent revision of the structure (Kim et al., 1990) reveals additional regions of the protein that contact the pyrimidine bases, also at the major groove, in a new model which involves

the sequence. Second, through the use of trinucleotides we have aimed at a non-biased collection of replacements, I

TABLE Variant

enzyme

phenotypes

-____ EcoRI mutants”

differences in chain connectivity. The lethal activity of the enzyme in vivo, together with the existence of a corresponding MTase, which inhibits DNA cleavage by the ENase, has permitted the design of sufficiently sensitive in vivo assays for the detection of mutant ENases with either normal or altered activity and/or specificity for the canonical site (Heitman and Model, 1990; Oelgeschlager et al., 1990). In this paper, we report the generation of a library of DNA fragments, potentially containing all of the 8000 possible aa combinations at three positions that face the DNA major groove (E’44, R14’ and R200). We used trinucleotide blocks in the oligo synthesis and obtained a fragment library which resulted in the expected distribution of replacements (single, double and triple), with no obvious bias and no stop codons. Using this library and a colony phenotype assay, we have isolated new partially active mutants, including a double aa replacement, that further strengthen the notion of the importance of DNA conformation (Lesser et al., 1990), and/or additional contacts at the bases (Heitman and Model, 1990), on the recognition process.

RESULTS

AND

DISCUSSION

(a) Combinatorial mutagenesis of aa 144, 145 and 200 In order to have access to all 203 possible combinations of aa at these three positions, we designed an experiment in which we would introduce, via synthetic DNA, a fragment consisting of 50 y0 wt codon, 50 y0 of an equimolar mixture of codons for the 20 aa, at each of the three positions (see Fig. 1 legend). In the ideal case (e.g., with equal coupling efficiencies of the trinucleotides and no other artifacts during chemical and enzymatic manipulations), a binomial distribution would predict the composition of the fragment to be 12.5% wt in all three positions, 37.5% single, 37.5Y0 The number of double and 12.5”< triple replacements. clones sequenced so far (as shown in Table I) is still too low to tell how close the actual distribution of changes is, compared to the ideal. Nonetheless, our results do show that we have a collection with a variety of replacements, in all three positions, which has already provided mutants with interesting phenotypes (see section b). This mutagenesis scheme goes beyond previous reports on combinatorial mutagenesis (Reidhaar-Olson and Sauer, 1988; Dunn et al., 1988; Sartorius et al., 1989). First, we have targeted residues that are far away from each other in

-__-Plating

aa“ __ R’“”

phenotype’

without

M EcoRI”

- IPTG

-

-

wt E144C

+ IPTG

+ ++

R200C

K -

R200K

-

R200N

-

R200H

-

H

+++++

+ + + t+++ +++++ +++++ +++ + +t++t *++++ +++++

R200Q’

-

ER-VT’

T

Q -

ND +++++

ND tf

ER-KY

Y

-

+++++

t+t++

ER-PF ER-YW’

F

-

+++++

+++++

W -

-

ND

ND

E

T

G -

ND +++++

ND +++++ +++++

E144T E144V El44G E144F E144W R145K

(ASN

149)

ER-DE’ ERR-VTG ERR-DIH’

K I

ERR-RHT

H

ERR-IKL

(LEU 203)

,’ Designations

for variant

the aa present

in the mutant,

+++++ ++++t ++t++ +++++ +++++ +++A+

C

+++

K N

+++++

t++++

+++++

ENase indicate

ND +++++

ND

H ‘T

+++++

the original aa at the left and

at the right, separated

occurred.

omitted

they can be derived from columns

for simplicity,

in parentheses genesis

denote

Numbers

by the aa number

where the replacement

mutations

present

outside

are

2-4. Numbers

the intended

muta-

window.

” Sequencing

was performed

derived from mutant were also recloned phenotypes

on the entirety

colonies,

but similar colonies;

size;

visually. Colonies were grown

of 50 pg/ml. Where indicated,

plates

with IPTG to a final concentration of 1 mM. appearing colony; + + + + , lower density colonies

+ + + and

enzyme).

Cl When

the mutant

colonies

had a normal gene. Under

(see Fig. 2). u Data obtained

pKGS to verify the observed

were assessed

+ +, reduced

$_, flat, small and translucent

the parent

fragments,

in Fig. 2).

on Km plates at a final concentration were suplemented + + + + +, normal

of Purl-Hind111

cloned on M 13mplY. The same fragments

in PstI-HindIII-digested

(shown

’ JM 101 colony phenotypes

ecoRIR

for multiple replacements

~, no growth. genes

were

phenotype, induction

size and visibly translucent

colonies

(similar

appearance

as

ND, not determined. expressed including

conditions

from direct cloning

in a MTase colonies

only the parent

of the Bg/II-PstI

context,

bearing

all

the parent

gene is lethal

fragment

in Ml3

vectors. ’ In this case, the ecoRIM gene was complemented both in 1rcln.7, in a compatible plasmid, and in cir, with the emRIM gene cloned in the same plasmid

as the ecoRiR

gene. In both cases, there was a protective

effect

of the MTase on the lethality of the mutant ENase under induction conditions. The rescue was more evident in the cells containing the rcoRIM

gene present

in truns (see Fig. 2).

9 SSDNA Ml3mpl8HPECO t200

ss bNA Ml3mpl8HPECO t 144-145 mutagenic primer+ PolIk MluI

mutagenic primer tPalIk EcoRI

t complementary oligo t DNA ligose

Gel purification

i

I

Primer 5’ond primer 3’tPCRtgel

purification

BglIi t PstI digestion+gel purificutiin,claning

Fig, 1. Combinatorial denote mutagenic experiments

mutagenesis

were carried

1979) as recipient a TcR derivative

strategy.

regions). Hatched

out with Ml3mpl8,

strain. Plasmid of pSClO1,

on a DNA synthesizer

The short, solid lines indicate

pKGS,

containing

(Systec

M13mpl9

which carries

oligos used as mutagenic

(Yanisch-Perron

the ecoRIM gene (Betlach

1460A) using the phosphoroamidite the introduction

20 different,

trinucleotides,

phosphotriester

et al., 1985) or pKGS

of a cocktail synthesized

for one aa, as follows: Ala, GCT; Arg, CGT; Asn, AAC; Asp, GAC;

of the inducible

of trinucleotides,

was prepared

Gln, CAG;

Oligos were synthesized

containing

Gly, GGT;

equimolar

His, CAC; Be, ATC; Leu, CTG; corresponding

valve, with a solution

a total of 20 pmol trinucleotides sequence.

Purification

for heterogeneity as described coding

from the cocktail

of oligos was performed

of migration

(Su and El-Gewely,

These oligos introduced

was

of Ito et al. (1982), through in 20?, polyacrylamide-8

of convenient

an M13mp19

employed

as a single

silent mutations

creating

to the ecoRIR gene ofpKGS

the HindHI-PslI

template.

plasmid,

substituting

verification,

to codons selected

to the aa present

mutagenesis

made with

in the wt EcoRI

was performed

insert, which corresponds

We simultaneously

added

all three

5’-TTAACAACGCCTCCATCTGGTC,

BglII, XbaI and MIuI sites, at positions

was chosen and, after sequence

corresponding

sites, oligo-directed

clone that carries stranded

injection

M urea gels. Large pieces were sliced from the gel to allow

restriction

5’-GACCCCTCTAGAAAAAGGACGT,

A clone scoring positive for all three mutations

a manual

above and 20 pmol of the trinucleotide

by gel electrophoresis

1988). M13mpl9HPEC0,

for aa 68 to 206 of the ENase

bearing the mutations,

described

of the mixed oligos. For the creation

5’-GCCATTAGATCTTGATCTCCTC, primer.

using the procedure

of

to a codon

for replacements,

oligo was reacted,

(Adams

amounts

corresponded

Lys, AAA; Met, ATG; Phe, TTT; Pro, CCG; Ser, TCT; Thr, ACT; Trp, TGG; Tyr, TAC and Val, GTA. At the positions the growing

(Messing,

and pSClOlmeth,

pore glass as support

et al., 1980). Each trinucleotide

Cys, TGT; Glu, GAA;

(thickenings

Methods: All cloning

lacUV5,

(UCSF).

1981) and controlled

a mixture

(Broka

products.

promoter

gift from P.J. Greene

and Caruthers,

as described

or as ligation adapters

(Kuhn et al., 1986) as vector DNA and JMlOl

et al., 1976), were a generous method (Beaucage

primers

Dashed lines depict the extension

a full length ecoRIR gene under the control

et al., 1983). In some steps, those involving fully protected,

synthetic

double lines denote the M 13mp I8 ss DNA template.

corresponding

mutagenic

plus the Ml3

primers ‘universal

to aa 135, 169 and 187, respectively.

used as a source of DNA to return the HindIII-PsrI

for the original region. Combinatorial,

essentially

to the fragment

site saturation

mutagenesis

fragment,

at aa positions

144, 145 and 200 ofEcoR1 endonuclease was approached by the following strategy: M13mplSHPEC0, an M13mp18 clone containing the HindIII-PstI insert of the ecoRtR gene was used to infect E. coli strain JMlOl. The ss DNA was purified and used as template for primer extension using the two mutagenic oligos (5’-ATCAAGATCTAATGGCTGCTGGTAATGCTATCXXXXXXTCTCATAAGA, TCTGGTATATTAAATXXXTTAGATCGAC, where the X denote positions mutagenized in the manner mutagenic

oligos were phosphorylated

by T4 polynucleotide

with PolIk for 1 h at 37°C. After inactivating the oligo used in the extension. autoradiographic complementary another,

The ss DNA fragments

aided by 0.3 pmol of an adaptor

gel purified primers,

exposure.

and subsequently

and performed

the polymerase

The ss DNA fragment

to the regions of aa 144-145 amplified

kinase using [y-3ZP]ATP. by heating

produced

by 20% polyacrylamide-8

of 161 and 99 nt (corresponding were gel purified.

to the expected

lengths

The fragments

(approx.

oligo (5’-TTAACAACGCGTCCATCTGGTC)

in a 20 ~1 reaction.

for 1.5, 1.5 and 3 min, respectively.

unexpected

difficulty to digest with BgfII at the site located 4 bp from the end of the fragment)

was cloned

either in pKGS,

the wt with the mutant

region as a cassette,

of the fragments

generated

on

of the band by with the oligos

0.03 pmol each) were then ligated to one The expected

fragment

and then digested

or in M13mp19,

with M[uI or EcoRI depending

260 nt ss DNA product

and 5’CTAGAGTCGACCTGCAGTTA

The amplified

The

to the ss DNA and treated

M urea gel after visualization

by PCR. We used 5’-ATCAAGATCTAATGGCTGCTGGTAA

30 cycles at 95, 55 and 65°C replacing

oligos were annealed

at 65°C for 10 min, the DNA was digested

was purified

and 200, respectively)

The radioactive

5’-CGCGTTGTTAATCTTGAGTATAATdescribed above) in separate experiments,

for direct

was ligated

to adapters

was as

(due to the

with BglII + PstI. The puritied fragment sequencing.

10 containing a suitable distribution of triple, double and single replacements, with no stop codons; with methods based on synthesis with mononucleotides, the resultant mutant library would be expected to be poorer, due to inherent limitations imposed by the degeneracy of the genetic code as well as the fact that the triplet is the coding unit. As the advantage of using triplets for mutagenesis, although significant (Sirotkin, 1986) is probably offset by the general difftculty of producing and using such synthetic units, we are currently working at further advancing these methods, adapted to more widely available synthetic chemistries. Our efforts are also stimulated by the versatility attainable by the use of PCR. (b) Phenotypes of bacteria bearing the mutant ecoRZR gene5 The transformation mixture of the mutant fragment cloned in pKGS was plated in conditions in which the gene is repressed (no IPTG added). The ecoRZR gene present in this plasmid carries a mutation that results in a protein product, altered at one aa (E16” + D) (Kuhn et al., 1986) and with lower activity under repressing conditions (note that all of the mutants reported in the present study contain this replacement as well). The presence of this variant of the ecoRZR gene, under the repressed conditions, is tolerated but confers a translucent, flat appearance to the E. coli JMlOl colony bearing it. With the mutant collection, we observed colonies with at least three distinctly different phenotypes: normal, intermediate, and wholly flat-translucent (Fig. 2). Furthermore, when these colonies were replica-plated to media containing the inducer, we could

1 no IPTG 1

again observe differences among them, growth, poor growth and no growth (see I). TO verify the correspondence between the observed phenotype, plasmid DNA used both for retransformation and as sequencing.

namely: normal Fig. 2 and Table the sequence and was purified and a source for the

Out of the limited number of colonies analyzed so far (about 103, from several transformation experiments), some preliminary observations could be noted. Some lo’?;, of the colonies displayed phenotypes indistinguishable from those carrying the parent gene, under repressing conditions. Sequencing experiments from several of such colonies rcvealed the presence of the expected silent mutations (coming from codons present in the trinucleotide mixture used to synthesize the oligo, which are synonymous, but different from the original). Less than 1% of the colonies showed the intermediate phenotype; sequencing of several such clones revealed only the CYS’~~ and Cys200 variants. From a few hundred colonies that were replica plated to inducing conditions, about 2% manifested a distinguishably poorer growth (E ‘44---f V, E144 -+ T, R’45 -+ K, and E’44R’45 -+ VT were identified this way). Overall, therefore, there is an indication that less than 5% of the variant EcoRI altered in one or more of these three residues confers a phenotype to the cells carrying them (as detected in our assay system). Sensitivity to the presence of the M. EcoRI was tested for some of the mutants either by transforming the corresponding plasmid DNA into JMlOl cells bearing the ecoRZM gene in a compatible plasmid or by replacing the mutant fragment in a plasmid that contains both the ecoRIR

1 + IPTGl

Fig. 2. Colony phenotypes of variant ENases. Colonies expressing EcoRI mutant ENases are as follows: a, parent enzyme; b, E14” + C; c. E14’ + T: d, EM4 + G; e, E’44R’45 + VT, f E’44R’45 --t VT + M. EcoRI (present in cis, see Table I); g, R“‘5 + K; b, RZoo + C; i, R”” + K; and j, R’“” 4 N. Left plate, , 7 no IPTG inducer, right plate, 1 mM IPTG. Each plate is divided in two columns containing three independently transformation. Colonies in the left and right columns come from host strains lacking and containing the M. EcoRI,

isolated colonies respectively.

from a fresh

11 and the ecoRIM genes. All mutants tested MTase-sensitive phenotype in one or more

showed a conditions

activity and specificity, reinforces the experimental support of the notion that other aa play a significant role in the

(Fig. 2). It was striking to observe variants with remanent activity that are still MTase-sensitive, especially those containing entirely non-conservative (e.g., E’44 + V) as well as double (El44 --+ V + R145 -+ T) aa replacements. Our results are in general agreement with recent reports

recognition and cleavage processes. Work under way is aimed at the screening of a sufficiently large number of clones in order to score the phenotypes of other multiple replacements as well as extending the mutagenesis window. We believe that our approach, applied to all residues involved in hydrogen bonding interactions, as interpreted

(Heitman and Model, 1990; Needels et al., 1989; Alves et al., 1989) showing that some replacements at aa 144, 145 and 200 of EcoRI generate ENases with reduced activities, but are still specific towards the GAATTC sequence. The use of combinatorial mutagenesis, together with our assay system and, possibly, the utilization of the mutant at position 160, permitted the identification of the double replacement variant that retains activity against the canonical site. It is noteworthy that the phenotypes conferred by some of the mutations we isolated differ from those previously reported. For instance, mutants E’44 -+ T and E’44 + V have detectable in vivo activities in our hands, whereas Heitman and Model (1990) listed them as inactive. On the other hand, we observed a null phenotype for E’44 + G, listed as marginally active by the same authors. Since our detection system is different and uses a different strain from those previously reported, it is possible that mutations that result in mechanistically different activities (e.g., nicking, dissociation prior to hydrolysis of the second strand, etc.) stand out in one assay, but not in the other. We are currently working on the isolation of the mutations at aa 144, 145 and 200 from the lesion at position 160, which is also a conceivable cause of the discrepancies noted above. Due to the amendments on the crystal structure of EcoRI, there has been caution in interpreting recent studies ofEcoR1 in structural terms (Lesser et al., 1990). Based on the description from the article reporting the revised, refined, coordinate set (Kim et al., 1990) as well as on our own observations using the available a-carbon coordinates (Brookhaven PDB entry lRlE), we infer that the side chains of aa 145 and 200 are within hydrogen bonding distance of the bases at the major groove of the DNA molecule, with aa 144 also nearby. Therefore, although our results substantiate the notion that additional contacts are involved in the determination of specificity, they clearly pertain to the protein-DNA interface.

from the new structure, should be valuable in providing information with regard to the possible overdetermination of the specificity of this enzyme.

ACKNOWLEDGEMENTS

We are grateful to Paul Gaytan for oligo synthesis, Maria Elena Munguia for technical assistance, Sonia Caro for secretarial help and Mario Zurita for critically reading the manuscript. J.O. is supported by a Ph.D. scholarship from Consejo National de Ciencia y Tecnologia, Mexico.

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