Protein engineering studies of A-chain loop 47-56 of Escherichia coli heat-labile enterotoxin point...

Molecular Microbiology (1 996) 20(4), 823-832

Protein engineering studies of A-chain loop 47-56 of Escherichia coli heat-labile enterotoxin point to a prominent role of this loop for cytotoxicity

lngeborg K. Feil,”’ Raghava Red~Jy,~ Lolke de Haan,’ Ethan A. Merritt,’ Focco van den Akker,2,4 Daniel R. Storm3 and Wim G. J. HoI’~’~~.~* ‘Howard Hughes Medical Institute, University of Washington, Box 357742, Seattle, Washington

Departments of “Biological Structure, 3Pharmacology and 4Department of Biochemistry, University of Washington, Seattle, Washington, USA. ’iaboratory of Physiological Chemistry, University of Groningen, Groningen, The Netherlands.

98195-7742, USA.

Summary

Heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, is a close relative of cholera toxin (CT). These two toxins share approximately 80% sequence identity, and consists of one 240-residue A chain and five 103-residue B subunits. The B pentamer is responsible for Gml receptor recognition, whereas the A subunit carries out an ADP-ribosylation of an arginine residue in the G protein, G,,, in the epithelial target cell. This paper explores the importance of specific amino acids in loop 47-56 of the A subunit. This loop was observed to be highly mobile in the inactive R7K mutant of the A subunit. The position of the loop in wild-type protein is such that it might require considerable reorganization during substrate binding and is likely to have a crucial role in substrate binding. Five single-site substitutions have been made in the LT-A subunit 47-56 loop to investigate its possible role in the enzymatic activity and toxicity of LT and CT. The wild-type residues ThrdO and Val-53 were replaced either by a glycine or by a proline. The glycine substitutions were intended to increase the mobility of this active-site loop, and the proline substitutions were intended to decrease the mobility of this same loop by restricting the accessible conformational space. Under the hypothesis that mobility of the loop is important for catalysis, the glycine-substitution mutants T50G and V53G would be expected to exhibit activity equal to or greater than that of the wild-type

Received 11 August, 1995; revised 25 January, 1996; accepted 8 February, 1996. *For correspondence. E-mail sue@ blackhook.bmsc. washington edu; Tel. (206) 685 7044, Fax (206) 685 7002.

0 1996 Blackwell Science Ltd

A subunit, while the proline substitution mutants T50P and T53P would be less active. Cytotoxicity assays showed, however, that all four of these mutants were considerably less active than wild-type LT. These results lend support for assignment of a prominent role to loop 47-56 in catalysis by LT and CT.

Introduction

Heat-labile enterotoxin (LT) produced by enterotoxigenic fscherichia coli is the causative agent of travellers’ diar- rhoea and is also responsible for the death of a large number of young children in developing countries (Black, 1986; Wanke eta/., 1987). LT, and its close relative cholera toxin (CT), are heterohexameric molecules consisting of one A subunit and five identical B subunits (Finkelstein, 1988; Gill and Coburn, 1987; Merritt and Hol, 1995). The B pentamer is responsible for the attachment of the toxin to its receptor, ganglioside GMl, which is present on mucosal- cell surfaces. The A subunit is activated by reduction of a single disulphide bond and proteolytic cleavage to yield the fragments A1 and A2. The A1 fragment contains ADP- ribosylating activity and its primary substrate in the target cell is the G-protein Gsm. G,, is activated by binding GTP, and the G,,:GTP complex stimulates adenylate cyclase to convert ATP into cyclic AMP (CAMP). Normally, G,, is deactivated by its own intrinsic GTPase activity; i.e. after hydrolysis of the bound GTP to GDP, G,, no longer stimulates adenylate cyclase. The ADP-ribosylation of G,, by LT destroys its GTPase activity, which results in the per- manent stimulation of adenylate cyclase and, therefore, in increased CAMP levels in the cells (Spangler, 1992), which ultimately leads to a large efflux of water and ions from the target epithelial cells.

One of the many interesting properties of LT is its strong mucosal immunogenicity and ability to act as an adjuvant to increase the immune response to unrelated antigens when they are co-administered to mucosal surfaces (Elson and Ealding, 1984; Clements et al., 1988). Origin- ally, these properties were believed also to be characteristic of the B pentamer alone (Nashar et al., 1993), but, recently, several groups have reported a requirement for ADP-ribosylation activity in order to demonstrate effective adjuvanticity (Czerkinsky, 1989; Lycke et a/., 1992). The molecular basis for such dependence of adjuvant efficacy

824 1. K. Feil et al.

on ADP-ribosylation activity is not known, although the presumptive targets are the Peyers Patch cells in the intestinal system (Wilson et a/., 1993). LT is known to ADP-ribosylate a variety of membrane and soluble proteins (Moss and Vaughan, 1977), and it is not necessarily the case that the adjuvant effect is mediated through modi- fication of the same intracellular target as the toxic effect. Therefore, it may be possible to modify the native toxin structure using protein-engineering techniques so as to dif- ferentially alter the toxicity and the adjuvanticity of the modified protein. That is, affinity for G,, as a substrate might be reduced, while affinity for a hypothetical second target in Peyers Patch cells might be retained or increased. This, in turn, would make it more reasonable to use LT as a vaccine carrier protein or as an adjuvant for use in immu- nizing humans.

When the LT crystal structure became available (Sixma et a/., 1991; 1993), many biochemical properties of the cholera toxin family could be analysed from a structural point of view (Hol eta/., 1995). In particular, the possibility of identifying enzymatically relevant residues in the three- dimensional structure of the toxin should assist the modifi- cation of its enzymatic activity through structure-based protein engineering.

Structural comparisons of the A1 domain of LT with exotoxin A from Pseudomonas aeruginosa and with diphtheria toxin have revealed the location of the active site of LT (Sixma et a/., 1991; 1993). Mutational studies by other groups have identified several essential residues in this region, including Glu-110 and Glu-112 (Tsuji eta/., 1990), Ser-61 (Harford et a/., 1989), His-44 (Jobling et a/., 1993) and Arg-7 (Burnette eta/., 1991). Structural studies of wild-type LT and the A subunit Arg7Lys mutant (van den Akker eta/., 1995) suggest that a high degree of conformational flexibility of the loop consisting of residues 47-56 may be crucial for binding NAD by the A subunit of LT. Because the amino acid sequence of the loop 47-56 is

Fig. 1. SDS-PAGE of heat-labile enterotoxin and its mutant toxins before and after tryptic digestion. For each toxin, the left lane represents the non-trypsinized form, and the right lane represents the trypsinized form. Protein samples were not boiled before applying to the gel, leaving the assembled toxin 6 pentamer intact. The outermost lanes contain low-molecular-weight markers.

identical for LT and CT, except for the residue Tyr-55 (His-55 in CT) (Dykes et a/., 1985), this probably holds for all members of the cholera toxin family.

In order to unravel the importance of flexibility of loop 47-56, we used protein-engineering techniques to create mutants in which this loop becomes intrinsically either less or more flexible than that found in the wild-type toxin. The basic rationals for four of these mutations was that a non- glycine to glycine substitution would render the loop more flexible, while non-proline to proline substitutions would decrease loop flexibility. The fifth substitution, G47R, was also intended to decrease flexibility. The formation of a salt bridge from the arginine side-chain to one of the aspar- tates in positions 43, 56 or 57 could constrain the loop to a closed conformation. We now report the characterization of these five mutations in the A subunit of LT involving residues Gly-47, Thr-50 and Val-53.

Results

Recovery of engineered toxin mutants

The expression plasmids encode the natural leader pep- tides of the A and B subunits (Dykes et a/., 1985), which guarantee secretion of the mutant A subunits into the peri- plasmic space of f. coliwhere they assemble with the wild- type B subunits to form the AB5 holotoxin. The mutant LT genes are expressed under the control of the thermoinducible lambda pR promoter in the E. coli strain MC1061. Fractionation of the induced sonicated cells by centrifugation, and subsequent analysis of the supernatant by SDS- PAGE and G,,,,,-ELISA showed that wild-type and mutant toxins were in the soluble fraction.

It has previously been reported by Clements and Finkel- stein (1979) that LT has an affinity to agarose-based chromatography columns, presumably as a result of the interaction of the B pentamer with the column material. Mutations in the A subunit are not expected to interfere

0 1996 Blackwell Science Ltd, Molecular Microbiology, 20, 823-832

Importance of A subunit 47-56 loop of Escherichia coli enterotoxin 825

Table 1. Analysis of changes in CHO cell morphology after toxin treatment.

Toxin Effect on CHO cell morphology

LT +++ T50G ++ T50P ~

V53G -

V53P + Plus signs indicate degree of cell elongation: +++, 100%; ++, more than 50%; i , less than 50%. Minus indicates no cell elongation.

with sugar binding by the toxin B pentamer, and, as expected, all five mutant toxins bound to a Biogel A5m column (see the Experimental procedures). The purity of the mutant toxins after the chromatography step was esti- mated to be >98% on SDS-PAGE (Fig. 1). The typical yield of purified and assembled protein was 5 mg per litre of culture.

The mutation A G47R showed only assembled B pentamer on the SDS-PAGE gel stained with Coomassie brilliant blue, while gels of the other four mutants (T50G, T50P, V53G and V53P) showed the presence of both A and B subunits (data not shown). Figure 1 shows the SDS-PAGE gels of the unnicked and nicked form of LT-A and three mutant A subunits. It is clear that the mutations in positions 50 and 53 did not alter susceptibility to trypsin treatment, as the tryptic fragmentation pattern of the mutants matches the wild-type pattern exactly. Thus, the enzymatic and cytotoxic activity of the mutant toxins is not expected to be reduced due to incomplete proteolytic activation.

Morphological changes on CHO cells caused by toxin treatment

LT and CT induce a well-known elongation of cultured Chinese hamster ovary (CHO) cells that is probably caused by changes in the cytoskeleton (Guerrant, 1974). Table 1 lists the effects of wild-type and mutant toxins on the morphology of CHO cells. Whereas untreated control cells grow into a confluent monolayer, cells treated with wild- type toxin grow at a lower density and show the typical elongated, spindle-shaped morphology. Cells treated with mutant toxins also grow at lower densities, but the morphology of T50P and V53G treated cells more closely resembles that of the control cells (data not shown). How- ever, some morphological differences compared to the control cells are detectable, which might be caused by the B subunit. It has been reported that the B subunit has a mitogenic effect on some target cells (Spiegel, 1989; Mitsui eta/ . , 1991). Another reason for the minor changes in morphology might be a residual toxicity of the mutant toxins, in particular in the case of T50G (see below).


Effects of LT and mutant toxins on CAMP conversion in CHO cells

Activation of adenylate cyclase leading to an increase in intracellular cAMP in LT-treated cells represents a key biochemical event associated with the cytotoxicity of this toxin. The initial tests on cAMP conversion were performed with sterile filtrates of the cell-free culture superna- tants of LT and of all mutant-toxin expression cultures. At this stage, no 3-isobutyl-1 -methylxanthine (IBMX) orforsko- lin was added and the results indicated a twofold increase in cAMP conversion of the LT-treated cells, while cells treated with the mutant toxins showed no differences when compared to the control cells (data not shown).

In order to rule out the possibility of an effect of the B subunit on the cAMP conversion, recombinant purified LT-B was added to CHO cells to a final concentration of 0.5 pg ml-’. No increase in cAMP conversion was observed (data not shown). Determination of the minimal LT concentration that causes maximal toxic effect showed that a 50 ng ml-’ wild-type-toxin final concentration causes the maximum increase in cAMP concentration under the chosen conditions.

The results of the assay with values divided by the protein concentration are shown in Fig. 2A. The basal levels of cAMP conversion after mutant-toxin treatment do not differ significantly from the basal level observed for the untreated control cells. After stimulating adenylate cyclase activity with forskolin, the mutant-toxin-treated cells showed 2-3.5-fold higher cAMP conversion compared to the control cells. The cAMP conversion caused by the most active mutant toxin, V53P, reached only one third of the level observed in the wild-type-toxin assay (Fig. 2A). This result was obtained even though mutant toxins were added in 20-fold excess over the wild-type toxin.

Enzymatic activity

Of the mutant toxins that were recovered as fully assembled AB5 holotoxin, only the V53G substitution retained a substantial fraction of wild-type activity against the substrate diethylamino-benzylidine-aminoguanidine (DEABAG) (Fig. 28). All other mutants had significantly reduced activity, which was virtually the same as the control without toxin, and approximated the ADP-ribosylation detection limit of the highly sensitive DEABAG assay. The standard deviations of the triplicate determinations were within the range of 10% of the absolute values.

Discussion

The active site in the LT-A subunit is remote from the subunit interface with the B pentamer (Fig. 3), and single amino acid substitutions in the loop 47-56 near the active


Effect of LT mutant toxins on cAMP production in CHO cells A

% of enzymatic activity relative to wild-type LT

T

ctrl LT V53P T50P V53G T50G notoxin LT V53P T50P V53G T50G Toxin

Legend

Basal

0 Forskolin

Fig. 2. Activity of mutant toxins. A. Effect of heat-labile enterotoxin and mutant toxins on cAMP conversion in CHO cells. The measured activities are shown with error bars corresponding to the range of values measured in three replications of the assay. The percentage of cAMP conversion is caiculated as follows:

cAMP (counts) x 100 ATP pool (counts) x 30 + cAMP (counts) x mg protein

% cAMP conversion =

B. Enzymatic activity of wild-type and mutant LT. Toxins were assayed for ability to ADP-ribosylate the substrate DEABAG, as described in the text. Toxin containing the V53G substitution retained approximately 40% of wild-type activity; the remaining three substitutions effectively abolished activity against this substrate.

site (Fig. 4) were not expected to interfere with AB, holotoxin assembly or stability. Both the guanidyl-substrate and NAD' must bind to the toxin in order for the ADP-ribosylation reaction:

NAD' + guanidyl-substrate + nicotinamide + ADP-ribose- guanidyl-substrate + H+

to occur, but details of binding interactions are not known for either substrate. Nevertheless, previous structural analysis of the wild-type E. coli LT toxin and of mutant toxins had led to a hypothesis that the flexibility of the loop consisting of residues 47-56 in the A subunit is important for catalytic activity (van den Akker et a/., 1995). The conformation seen for the 47-56 loop in the wild-type LT structure partially overlaps the NAD-binding site, as deduced by structural homology to a complex of ApUp

with diphtheria toxin (Choe eta/., 1992), which is another catalyst of ADP-ribosylation. Structural homology can also be observed after superimposing AMP and nicotinamide, of which the binding sites have been structurally analysed in exotoxin A (Li et a/., 1995), in the proposed NAD-binding cavity of the A subunit (Fig. 5). The AMP- and nicotinamide-binding mode agrees well with mutagenesis data carried out on neighbouring residues shown in Fig. 5. Substitutions of residues Arg-7 (Burnette et a/., 1991; Lobet eta/., 1991), Asp-9 (Pizza eta/., 1994), Ser- 61 (Harford eta/., 1989), Ser-63 (Fontana et a/., 1995), and Glu-112 (Tsuji eta/., 1991) have resulted in lossof catalytic activity. The postulated binding mode of NAD in LT demonstrates the need for loop 47-56 to be displaced in order to accommodate the substrate in the active site. A structurally rigid loop would interfere with NAD binding, and hence prevent catalytic activity.

0 1996 Blackwell Science Lid, Moiecutar Microbioiogy, 20, 823-832


Fig. 3. Schematic structural representation of the E. coli heat-labile enterotoxin (LT) as seen crystallographically for the wild-type holotoxin (Sixma et a/., 1991 ; 1993). The tryptic- cleavage site between the A1 and A2 fragments of the A subunit is indicated, as are the three mutation sites investigated in this study.

Fig. 4. Close-up in stereo of the loop consisting of residues 47-56 at the NAD-binding site in the LT-A subunit. The catalytically important active-site residues and the mutation sites are labelled. The conformation of loop 47-56 shown in grey is that seen crystallographically in the wild-type toxin structure (Sixma eta / . , 1991; 1993). In the substitution mutant R7K, this !oop becomes substantially destabilized and is not visible crystallographically (van den Akker e ta / . , 1995). Also shown are the side-chains of residues Glu-110 and Glu-112, which are believed to lie at the catalytic site.



Fig. 5. Schematic representation of the AMP and nicotinamide moieties of NAD in the active site of LT. To generate this model, LT residues 5-9, 14-16, 21 -24, 59-72, 83-89, 94-96, and 11 1-1 16 have been superimposed onto structurally equivalent residues 438-442, 448-450, 454-457, 468-481, 496-502, 541 -543, and 552-557, respectively, in exotoxin A. The resulting superposition yields a root mean square deviation of 1.7 A for the 42 C' atoms. Also highlighted in this figure are residues that have been shown, by site-directed mutagenesis, to affect ADP-ribosylation, as described in the text. The loop 47-56 is shown in grey and its start and end points are highlighted. It can be seen in this Fig. that the positions of AMP and nicotinamide, as observed in exotoxin A, confirm the need of the loop to move out of the way, because there are several short distances between the loop 47-56 and AMP, as well as between the nicotinamide and loop 47-56, which are 0.6 and 1 . 5 A , respectively. The loop 46-56 in its native conformation leaves virtually no space for the second phosphate-ribose moiety that bridges AMP and nicotinamide in the complete NAD substrate. However, it should be noted that the model of this ternary complex indicates the need for some small structural adjustments of the NAD and the protein, because there are several VDW distances of the two moieties with the residues listed above which are 3.OA or even shorter

Substitutions at residues 50 and 53

Residues Thr-50 and Val-53 of the wild-type LT toxin were identified as sites that are likely to tolerate substitutions by either glycine or proline (Fig. 4). The +/$torsion angles of these residues lie in the region favoured by proline (Thr-50 + = - 75^, $ = 130"; Val-53 + = - 65", $ = 143"). The hydro- xyl group of Thr-50 was not observed to participate in hydrogen bonding in the wild-type toxin, so loss of hydrogen-bonding energy was not predicted to destabilize mutants containing a substitution at this site. Energy mini- mization of a modelled T50P mutant resulted in a shift of 1 .O A in the location of the residue Ca, with a slight increase in the amount of exposed hydrophobic surface for the residue. Val-53 exhibits, in the wild-type toxin, hydrophobic interactions with lle-76. Modelling of the V53P mutation appeared to improve the local packing of the protein, and no worsening of energy terms resulted during energy mini- mization. Both residues at positions 50 and 53 were therefore selected for engineered mutations, with the intent that substitutions of a glycine at either site would increase the flexibility of the 47-56 loop, while substitution of a proline would decrease the flexibility. Our corresponding predic- tion was that the mutant toxins containing glycine substitutions would exhibit no loss of activity compared to the wild-type toxin, while the proline mutants would exhibit

decreased toxic activity. These predictions were necessarily tentative, given that the precise site of substrate binding and protein conformation during catalysis by the toxin remains unknown. A similar line of argument was proposed by Pizza et a/. (1994), who also investigated substitutions at residues in the 47-56 region of LT. These authors reported that substitution of Val-53 by Asp or Glu results in the loss of toxicity, and proposed that stabilization of the wild-type-loop position made the active site unavailable for substrate binding.

The results presented here show that, in reality, both X + Gly and X i Pro substitutions at residues 50 and 53 lead to reduced enzymatic activity and a reduction in cytotoxicity. The V53G substitution retains a substantial fraction of wild-type enzymatic activity (40%), whereas it has no effect on the accumulation of intracellular CAMP. One interesting explanation for these intriguing observations could be that the natural substrate G,, cannot be bound and ADP-ribosylated by the V53G mutant, whereas the synthetic DEABAG substrate maintains some binding affinity and catalysis occurs at a reduced rate.

The G47R mutation

The backbone conformation at residue 47 (+ = -81", $= 165 ) is compatible with substitution by a larger residue.


lmportance of A subunit 47-56 loop of Escherichia coli enterotoxin 829

Gly-47 in the wild-type toxin is solvent accessible, and modelling indicated that substitution by a charged residue would be possible. The G47R mutation reported here was selected because the energy-minimized model structure exhibited acceptable energy terms, including likely favourable salt bridges from the newly introduced side-chain nitrogen atoms to residues Asp-43, Asp-56, and Asp-57. These interactions were likely to constrain the flexibility of the 47-56 loop as compared to the wild type, as would the presence of the much bulkier arginine side- chain.

While the four mutants at position 50 and 53 all assembled to form complete AB5 holotoxin, the G47R mutant failed to do so. This is a quite puzzling result, but studies in other groups have yielded similar results, i.e. they also observed that mutations in the A subunit quite far away from the A-B interface led to loss of assembly (e.g. Pizza et al., 1994; Okamoto et a/., 1995). Most likely the assembly and/or secretion apparatus of f. coli is rather sensitive to alterations of certain residues in the LT-A subunit. Certain mutations might interfere with the secretion of the mutant A chain into the periplasm or might be detri- mental for the assembly into the hexameric structure in the periplasm.

Role of the 47-56 loop in catalysis

In the absence of a crystal structure of LT/CT in complex with the substrates NAD and G,,, several possible explanations for the results presented here have to be considered. Our initial hypothesis was that the 47-56 loop played a passive role in the catalytic action of the toxin and its flexibility would allow substrates to bind in the active-site. Whereas the active-site cleft as seen in the wild-type structure is too narrow to contain the known substrates, the site is more accessible in the R7K mutant structure (van den Akker et al., 1995) because of the increased flexibility of this loop. We, and others (Dome- nighini et a/., 1994), have considered a model in which this loop was simply displaced during substrate binding. However, the findings presented here that all four X to Pro and X to Gly substitutions in the 47-56 loop lead to a decrease in the toxicity of LT suggest that this loop plays a prominent role in the toxicity of LT and CT, most likely through direct interaction with bound NAD.

In addition to our four less-active mutants described in this study, other reports of decreased activity due to mutations in the 47-56 loop have appeared. Pizza eta/. (1 994) showed that the substitutions of Val-53 by Asp or Glu result in loss of toxicity, and proposed that stabilization of the wild-type loop position makes the active site unavailable for substrate binding.

Our results show that both the creation of a less-flexible as well as a more-flexible loop decreases activity, and we


also know from the structure of LT R7K (van den Akker et a/., 1995) that the 47-56 loop has been proven to be cap- able of changing its conformation drastically. However, decreasing as well as increasing loop flexibility lead to similar results: less activity. These results, taken together with the known inhibitor- and substrate-fragment-binding sites in diphteria toxin and exotoxin A (Fig. 5), indicate that residues of the loop 47-56 play a prominent role in the toxicity of LT and CT. Probably several of the residues in the region are involved in the correct positioning of substrates, most likely NAD (Fig. 5) but possibly also the protein substrate Gsm. This latter possibility is tentatively suggested by the fact that our V53G mutant retains signi- ficant activity with respect to the synthetic DEABAG substrate but loses virtually all toxicity. There are obviously other possible explanations for the lack of toxicity of the V53G mutant ranging from lack of ability to cross membranes to interaction with one of the numerous proteins in the cytoplasm such that recognition of the cellular substrate is prevented. Clearly, further studies are necessary to decide which of those, or yet other, factors are the major cause of lack of toxicity in the V53G mutant.

Even though numerous structures of LT have been reported by our laboratory (van den Akker et al., 1995; Merritt et a/., 1994; 1995; Sixma et a/., 1991; 1992; 1993), the active form of the toxin, in complex with substrates, products or inhibitors, still remains to be determined. It will be most intriguing to see exactly how the active site of the cholera toxin family adapts itself to binding substrates. Once that knowledge has been obtained, the current protein-engineering results can be explained in their proper context. In the meantime, however, we have created a number of mutants with decreased toxicity, which may be of relevance in unravelling the relationship between adjuvanticity and cytotoxicity in this intriguing family of bacterial toxins.

Experimental procedures

Mutagenesis and expression of mutant toxins

The gene encoding LT from porcine-derived E. coli originated from plasmid EWD299. A BamHl site was introduced just in front of the ATG start codon using the 5'-3' primer 5'-ACG- GATCCATGAAAAATATAACTTTC-3' and a fst l site was introduced in the non-coding part of the sequence by a point mutation using the primer 5'-AAGCTTGCCCCTGCAGCC- TAGCTTAGTT-3' by conventional polymerase chain reaction (PCR) methods. The nucleic acid sequence of the complete LT gene from EWD299 reported by Yamamoto and Yokota (1 983) was used as the basis for sequence alterations. Muta- genesis was performed with the mutagenesis kit from Promega. Briefly, the pLT gene was cloned into PALTER (Pro- mega) at the BarnHl and f s t l sites and single-stranded DNA (ssDNA) was produced using the helper phage M13K07. The oligonucleotides coding for the substitutions were

830 1. K. Fed et al

phosphorylated and annealed in combination with the ampicillin-repair oligonucleotide. After completion of the synthesis reaction, mutated plasmids were transformed into f. coli BMH 71-18 mutS, selected for resistance to ampicillin and then retransformed into JM109. Single colonies were grown up and the plasmid DNA was sequenced by the chain-termi- nation method (Sanger eta/., 1977) in order to verify the incor- poration of the desired mutation.

The following oligonucleotide sequences were constructed: wild type, 5’-TCACGCGAGAGGAACACAAAC-3‘; G47R, 5‘- TCACGCGAGACGTACACAAAC-3; T50G, 5‘-GAGGAAC- ACAAGGTGGCTTTG-3‘; T50P, S’GAGGAACACAACCCG- GCTTTG-3; V53G, 5-AACCGGCTTTGGCAGATATGA-3; V53P, 5’-AACCGGClTTCCCAGATATGA9’.

After sequence verification, the mutated genes were cloned into the thermoinducible expression vector PROFIT and transformed into €. co/i strain MC1061. Cultures were grown in Luria-Bertani (LB) medium supplemented with 50 pg ml-’ kanamycin at 28‘C until the ODeo0 reached levels of 0.7-0.8 and then expression was induced by shifting the temperature to 42’C. After a minimum of 5 h induction, cells were recovered by centrifugation.

Protein purification

Cell pellets from 1 I cultures were resuspended in 3-4ml of lysis buffer (20mM Tris-HCI, 0.1 mM DTT, 0.2mM EDTA; pH 7.5) and sonicated until complete cell lysis occurred. The cell fragments were pelleted and the supernatant subjected to a 30% ammonium sulphate precipitation in 20mM Tris- HCI buffer (pH 7.5). Each mutant protein remained in the soh- ble fraction and the buffer was changed using a PDlO column (Pharmacia), equilibrated with TEAN (50 mM Tris-HCI, pH 9.7, containing 1 mM EDTA, 3 mM NaN3 and 200 mM NaCI). The protein fraction was applied to a Biogel A-5m column (Bio- Rad), with dimensions of 12 x 6 cm, which was equilibrated with the same buffer. The column was washed with 2x column volumes of TEAN buffer and then the mutant protein was eluted with TEAN buffer containing 200 mM D-galactose. Frac- tions were assayed for the presence of the expressed toxin by using a GM,-ELISA as described by Amin and Hirst (1994). The mutant toxin-containing fractions were pooled and con- centrated in an Amicon cell using PM10 membranes. The protein concentration was determined using the method described by Bradford (1 976).

As expressed in f. coli, the A1 and A2 fragments of the toxin A subunit form a continuous polypeptide chain that must be proteolytically nicked in order to activate the toxin. To evaluate changes in mobility or band intensity of the A subunit after such proteolytic nicking, samples of the mutant proteins T50G, T50P, V53G, and V53P, each containing approximately 250 ng protein, were incubated with 5 pg of trypsin at 37 C for 30 min. The unnicked and nicked forms of the mutant proteins were analysed on a 12% SDS-PAGE gel and stained with Coomassie brilliant blue (Fig. 1).

Test of morphological changes on CHO cells

CHO cells were seeded at an initial density of 2 x 1 O4 cells per ml in 6-well cell-culture plates (Corning) with 2 ml per well in

complete Dulbecco’s Modified Eagle Media (DMEM, Gibco- BRL) containing 10% bovine calf serum and 100 units per ml penicillin G and 100 pg ml-’ streptomycin sulphate. To each well, 2004 of dilute wild-type toxin or mutant toxin was added resulting in final concentrations for the wild type of 50ngml-’ and 1-1.5pgmI-’ for the mutant toxins. In the control wells, 200 pl of sterile phosphate-buffered saline (PBS) was added. The cell cultures were grown for 5 d and then cells were fixed in methanol and stained with a mixture of water:giemsa:ctystal violet (37.5:2:0.5) for 60 min. After washing the cells three times with water, cells were evaluated for their degree of elongation.

CAMP accumulation

Intoxication of cells by LT normally results in elevated levels of intracellular cAMP as a result of the stimulation of adenylate cyclase by ADP-ribosylated Gsx. Therefore, in order to moni- tor the intracellular effect of the mutant toxins, changes in intracelldar cAMP levels were measured by determining the ratio of [3H]-cAMP to a total ATP, ADP, and AMP pool in [3H]-adenine-loaded cells as described by Wong et a/. (1991). Although this assay system allows rapid and extre- mely sensitive measurements of relative changes in intracellular cAMP levels in response to various effectors, the absolute numbers for cAMP accumulation generally show some varia- tion between experiments using different sets of cells (Federman eta/., 1992; Choi eta/., 1993). it is important to em- phasize, however, that relative changes in cAMP were highly consistent between experiments. CHO cells were seeded and treated with toxin as described above. Control cells were treated identically without the addition of toxin. Confluent cells in 6-well plates were initially incubated in DMEM containing [3H]-adenine (2.0pCi ml-’, ICN) for 16-20 h, washed once with 150mM NaCI, and incubated at 37°C for 30min in DMEM containing 1 .O mM IBMX (Sigma) and 25 pM forskolin (Sigma). Reactions were terminated by aspiration, washing cells once with 150mM NaCl, and adding 1.0ml of ice-cold 5% trichloroacetic acid (TCA) containing 1 .O pM CAMP. Cul- ture dishes were maintained at 4°C for 1-4 h and acid-soluble nucleotides were separated by ion-exchange chromatography as described by Salomon eta/. (1974). Reported data are the average of triplicate determinations.

Enzymatic activity

The enzymatic activity of the wild-type and mutant toxins was assayed against the artificial substrate diethylamino-benzylidine-aminoguanidine (DEABAG) (Narayanan et a/., 1989; A. A. Platas et a/., unpublished). For each assay, 750ng of trypsinized toxin was added to 200 pI of 200 mM phosphate buffer at pH7.5 containing 20mM DTT, 0.1 mgml-’ BSA, 0.1% Triton X-100, and 1.75mM DEABAG. The ADP-ribosylation reaction was started by adding 25 pI of 40 mM NAD. After 2 h of incubation at 30”C, unreacted DEABAG was removed by adsorption to treated DOWEX resin (Bio-Rad) and 500 pI of supernatant was then assayed for fluorescence at 440 nm, characteristic of the ADP-ribosylation product.

Molecular modelling of 47-56 loop mutants

We set out to alter the loop flexibility by making single point

0 1996 Blackwell Science Ltd, Molecular Microbiofogy, 20, 823-832


mutations at loop residues. Possible mutations in the 47-56 loop were first evaluated by using interactive computer gra- phics to inspectdhe crystal structure of the native LT holotoxin refined at 1.95A resolution (Sixma eta/ . , 1993). The design goal for the X + Pro mutants was to decrease loop flexibility whilst retaining the wild-type conformation. Therefore, substitution of proline was only considered for residues whose observed conformation was consistent with the restricted range of the torsion angles 4 r-. -60" energetically favourable for proline residues. Substitutions of non-glycine residues by glycine does not impose any restrictions on permitted b/@ conformations. Mutations which would disrupt hydrogen- bonding interactions observed in the native structure were ranked less desirable than those which would make fewer such disruptions. Prior to actual mutagenesis, the proposed substitutions were modelled and energy minimized using the BIOGRAF program (Mayo et a/., 1990) to de tx t possible steric clashes, internal strain, or other energetically unfavourable interactions.

Acknowledgements

This research was supported by the National Institute of Health (Grant A134501), the University of Washington Royalty Research Fund, and by an equipment grant from the Murdock Charitable Trust. We offer special thanks to Alanna Hoeper and to Christophe Verlinde for useful discussions.

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Protein engineering studies of A-chain loop 47-56 of Escherichia coli heat-labile enterotoxin point...

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