identification of a specific tyrosine residue in bryodin 1 distinct from

Protein Science (1998). 7318-324. Cambridge University Press. Printed in the USA. Copyright 0 1998 The Protein Society

Identification of a specific tyrosine residue in Bryodin 1 distinct from the active site but required for full catalytic and cytotoxic activity

DANIEL K. FRYXELL, SUSAN L. GAWLAK, ROBERT W. DODGE, AND CLAY B. SIEGALL Molecular Immunology Department, Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98 121

(RECEIVED July IO, 1997; ACCEPTED August 13, 1997)

Abstract

Bryodin 1 (BDl) is a type I ribosome-inactivating protein (RIP) with low inherent animal toxicity. It has been cloned recently and the recombinant protein (rBD1) has been produced and crystallized. To gain insight into the relationship of rBDl structure and function, we investigated the role of sequences in a region (residues 128-156) that exhibits homology with membrane interactive sequences and is not part of the enzymatically defined active site. Progressive deletions representing a-helical turns within these residues were generated; mutant rBDl proteins were expressed in Escherichia coli and demonstrated increasing losses of enzymatic activity. Point mutations were also generated within this region to replace Y140, Y141, and Y142 with either alanine or lysine. Mutants at position 140 or 142 retained full enzymatic activity, whereas A141 and K141 mutants were > 19-fold less potent. In cytotoxicity assays, the rBDl point mutants at Y141 were >80-fold less potent than either rBDl or mutants at residues 140 or 142. However, when introduced into the anti-CD40 single-chain immunotoxin rBDIG28-5 sFv, the A140 and A141 point mutations led to decreased cytotoxicity toward CD40 positive cell lines. These data indicate that Y141 plays an important role in the enzymatic activity of BDI and that Y140, although not essential for catalytic activity, is required for full BDI function. Because residues 140 and 141 are distinct from residues implicit in the active site, they may be involved in ribosomal and/or membrane interactions or in intracellular trafficking of the toxin and immunotoxin.

Keywords: bryodin; immunotoxin; membrane interaction; mutagenesis; ribosome-inactivating protein

Bryodin-1 (BDI), a type 1 RIP isolated from Bryonia dioica (Stirpe et al., 1986), may have a therapeutic advantage over other RIPs due to its lower toxicity in vivo (Siegall et al., 1994). Type 1 RIPs have a common N-glycosidase activity, a basic isoelectric point, and molecular mass of 26-30 kDa (Barbieri et al., 1993). Type I RIPs from different plants show high sequence and structural homology with each other as well as with the A chain of the type 2 RIPs ricin and abrin. Alignment of sequences from BDl and other RIPs indicates five regions of highest homology, several of which are clustered around the active site cleft (Shaw et al., 1992). The role of sequences in other regions has not yet been elucidated.

In order to better understand the function of BDl sequences in one of the undescribed regions that was also interesting as a potential mediator of membrane interactions, a mutational analysis was performed within BDI residues 128-156. This region is on the solvent surface of BDl according to the X-ray crystal structure (H. Klei & H. Einspahr, pers. comm.) and is not thought to be part of the active site of RIPs (Katzin et al., 1991). Local secondary structure consists of two approximately antiparallel a-helices sep-

Reprint requests to: Clay B. Siegall, Seattle Genetics, Inc., 22215 26th Ave. SE, Bothell, WA 98021; e-mail: [email protected].

arated by a turn containing three consecutive tyrosines. One, two, or all three of these tyrosines are conserved among many RIPs.

To investigate the contribution of this domain to rBDl activity, three progressive deletions were produced as well as point mutations of the individual tyrosines to either alanine (to retain hydrophobic character) or lysine (to introduce a charge). Mutant rBDl proteins were constructed, expressed in E. coli, and characterized for their ability to inhibit protein synthesis in cell-free and cytotoxicity assays. Based on these results, two point mutations were introduced independently into a rBDI-based anti-CD40 single- chain immunotoxin (rBDI-G28-5 sFv) and tested for activity against CD40 positive cell lines. These studies suggest an integral role for Y 141 in the catalytic and cytotoxic activity of BDl and a potential role for Y140 in intracellular trafficking.

Results

Selection of rBD1 sequences for deletion studies

The regions of rBDl that share highest sequence homology with other RIPs and the region analyzed in this study are depicted in Figure 1A (Robertus, 1992; Gawlak et al., 1997); little structural

318

Tyrosines in Bryodin 1 required for full activity

A

v 4 26 60 98109 148 169 186 I I 1

247 I

M A A m d h

I

k c

Fig. 1. A: Schematic diagram of rBDl including locations of deletion mutations, point mutations, and alignment with other RIP sequences. TCS, trichosanw, LUFa, luffm-a; MOM, momorcharin, RTA, ricin A chain, ATA, abrin A chain; GEL, gelonin; MAP, Miribilis antiviral protein; SO6, saporin-6; BPSl, barley inhibitor. Degree of sequence conservation is indicated by shaded boxes (high), open boxes (low), or no boxes (intemedi- ate). B: Location of deletion and point mutations in the crystal structure model of rBD1. D139-146 is shown in yellow, D136-149 in yellow and orange, and D128-156 in yellow, orange, and red. The three consecutive tyrosines (shown in yellow with side chains) in the turn between the a-helices were substituted with either alanine or lysine. Analogous ricin A chain residues implicated in active-site involvement are depicted as triangles in A and in green with side chains in B.

319

information has been reported concerning regions of lesser homology in RIPS. BD1 residues 128-156 were identified with the Kyte- Doolittle algorithm as being hydrophobic and long enough to traverse a lipid bilayer. This region is located at the solvent surface of rBDl and is comprised of two roughly antiparallel a-helices, named a-helices D and E in RTA (Katzin et al., 1991), separated by a turn of approximately six amino acids (Fig. 1B). It is unusual for a hydrophobic patch to be present at the surface of a protein, sug- gesting a potential involvement in membrane interaction during cellular intoxication.

This region does not include any of the active site residues indicated by triangles in Figure 1A. The stretch from 134 to 149 is also remarkable in its high proportion of hydroxylated amino acids (1 1 of 16) that may be important in maintaining solubility of this domain. It is possible that, although the primary sequence is not highly conserved in this region, especially between diverse mem- bers of the RIP family, secondary or tertiary structures may have common elements that could be important for certain aspects of RIP function. We therefore focused on residues 128-156 in rBDl for mutagenic and structural studies.

Cell-free activity of rBDl deletion mutants

Three deletion mutants of rBDl (D139-146, D136-149, and D128- 156) were constructed as diagrammed in Figure 1B. Figure 2 shows decreased enzymatic potency of the deletion mutants relative to rBD1, with EC50 values of 8 nM, 124 nM, and 559 nM versus 33 pM for rBDl (Table 1). Thus, certain structures located within amino acids 128 and 156 may contribute either directly or indi- rectly to the enzymatic activity of rBD1.

Generation and characterization of rBDl point mutants

With the indication that rBDl residues between 128 and 156 may influence enzymatic activity, we narrowed the study to focus on point mutation of individual amino acids within the smallest deletion (D139-D146). We focused on the three tyrosine residues (140-142) located in the turn between the two a-helices, with each being mutated to either an alanine or lysine residue. An SDS-

.- C 0) c

a 2

140

120

100

80

60

40

- 2 w

6 20 8 n 0.1 1 IO 100 1000 10' 105 lo6

Concentration of Toxin, pM

Fig. 2. Cell-free protein translation inhibition by rBDl deletion mutants. Data presented were obtained in quadruplicate and are representative of three independent experiments. rBDl (X); D139-146 (+); D136-149 (m); D128-156 (0).

320 D.K. Fryxell et al.

Table 1. Comparison of protein synthesis inhibition EC50 values for rBDI and rBDI mutantsa

Cell-free potency

Fold-reduction cell-free

JAR cell potency

Fold-reduction whole-cell potency

rBDl D139-146 D136-149 D128-156 A140 A141 A142 K140 K141 K142

33 pM 8 nM

124 nM 559 nM

37 pM 1,033 pM

107 pM 15 pM

630 pM 96 pM

1 253

3,755 16,894

31 1.1

3.2 0.4

19 2.9

52 nM N D ~ ND ND

70 nM >7.052 nM

177 nM 88 nM

4. I63 nM 77 nM

1 ND ND ND

1.4 >I37

3.4 1.7

80.8 1.5

'Data presented are means of three or more independent experiments and were not taken directly from Figures 2 and 4. bNot determined.

PAGE analysis of the purified proteins is shown in Figure 3. These six rBDl point mutants were tested in the cell-free protein synthesis assay for comparison with rBDl. rBDl mutations A141 and K141 reduced protein synthesis by 31- and 19-fold, respectively (Fig. 4A). Mutants A140 and K140 exhibited similar activity compared to parent rBD1, and mutants A142 and K142 had marginally reduced activity (Table 1). These data indicate that Y141 is required for full rBDl catalysis.

Cell-based activity of rBDI point mutants

We next tested the tyrosine point mutants in cytotoxicity experiments to determine if the position 141 effect observed in cell-free activity would also be apparent in whole-cell studies. The differ- ence in potency of type 1 RIPS in these two assays of roughly 1,000-fold commonly has been attributed to the inefficient process by which R I P S are internalized by cells and translocate into the cytosol (Barbieri et al., 1993). The 19-31-fold reduction in po-

- kD 250 -

98 - 64 - 50 - 36 - 30 - 16 -

1 2 3 4 5 6 7 8 Fig. 3. 12% SDS-PAGE of purified rBDl and point mutants. Lane 1, molecular weight markers; lane 2, rBDI; lane 3, A140; lane 4, A141; lane 5 , A142; lane 6, K140; lane 7, K141; lane 8, K142.

tency of A141 and K141 mutants in the protein synthesis assay might, therefore, be masked by the requirement of having to use such high concentrations of toxin. However, alanine and lysine mutations at amino acid 141 reduced the cytotoxicity by greater than 137- and 80-fold, respectively (Fig. 4B). The loss of whole-cell activity was actually greater than for cell-free catalytic activity, although the precise reasons are not clear. The data suggest that Y 141 may also play a role in the intracellular trafficking of BDl. TheAl40, K140, and K142 mutants demonstrated similar potency, whereas the A142 mutant was approximately threefold less potent (Table 1).

CD analysis of rBDI mutants

The primary nucleotide sequence of each rBDl mutation was verified by DNA sequencing, and the purified proteins were assessed by SDS-PAGE and immunoblot. However, mutations in rBDl could have reduced activity due to alterations in secondary structure. To more completely define the biophysical integrity of rBDl point mutants, each was studied by CD. The spectra of the deletion mutants were considerably different from that of rBDl (Fig. 5A), indicating major structural changes. Alternatively, none of the six point mutations led to a significant change in the shape of the spectra (Fig. 5B).

Mean residue molar ellipticity was determined based on theo- retical extinction coefficient values and may have led to variations in estimated protein concentration. Each of the point mutations involved loss of a single tyrosine, which is reflected in the e280 value only as number of tyrosines present. Other alterations to the extinction coefficient due to possible changes in peptide bond conformations were not taken into account. When the CD spectra for the point mutants were normalized for maximum deflection along the molar ellipticity axis, they overlaid precisely. Therefore, there was no change in the qualitative curve shape of any of the rBDl point mutants that might indicate protein secondary structure alterations.

Analysis of single-chain anti-CD40 immunotoxins containing mutant rBDI

It was next of interest to determine whether the reduced cytotoxic activity observed in rBDl mutants at position 141 would result in

Tyrosines in Bryodin 1 required for full activity 321

a loss of activity in single-chain immunotoxin form. Immunotoxins are more potent than type 1 RIPS in killing cells because they include binding subunits. The antibody component of immunotoxins mediates cell surface binding and can modify the intracellular mute taken by rBDl compared to the RIP alone. To investigate their effect, the A140 and A141 mutations were introduced into the single-chain rBD1-based immunotoxin targeted to CD40, rBD1- (328-5 sFv. Both rBDI(A140)-G28-5 sFv and rBDl(A141)-G28-5 sFv bound to immobilized CD40-Ig in a fashion similar to unmod- ified rBDl-G28-5 SFV (Table 2). The three rBD1-based anti-CD40 immunotoxins were tested for protein synthesis inhibition activity in a cell-free system and found to have ECso values of 63, 134, and 1,103 pM, respectively (Table 2). These data indicate that the A141 mutant was decreased similarly in catalytic activity, whether as an isolated toxin or in immunotoxin form.

The three anti-CD40 immunotoxins were also tested for cytotoxicity against CD40 positive and negative cell lines. rBD1- G28-5 sFv killed all three CD40 positive cell lines, with comparable ECS0 values of 0.5-3.0 nM. The A141 mutation in immunotoxin format was less active on the three CD40 positive cell lines, with

0.1 1 10 100 1000 1 o4 Concentration of Toxin, pM

B v) 140

120

.- v)

5 2 100 C

C .- c 0) 80 2 a 60 -

40 0

C

6 20 8

0 1 10 100 1000 1 o4

Concentration of Toxin, nM

Fig. 4. Activity of rBDl point mutants. A: Cell-free protein synthesis inhibition activity. Data presented were performed in quadruplicate and represent 3-8 independent experiments. B: Cytotoxicity analysis on JAR choriocarcinoma cells. rBDl (X); A140 (+); A141 (m); A142 (0); K140 (0); K141 (0); K142 (0). Data presented are from one experiment performed in triplicate, representative of three experiments.

A

""-

200 210 220 230 240 250 260 nm

B .- L. 0 .- .n 0 w 2 -5 IO6

c.

- - - 2 a -1 io7

z cn-1.5 1 o7 2 5 -2 10'

=-2.5 107

J

a,

200 210 220 230 240 250 260 nm

Fig.5. CD spectra of rBDl mutants. A: rBDI (bold line), D139-146 (solid line), D136-149 (dashed line), D128-156 (dotted line). B: rBDl (bold line); A140, A141, A142 (dashed lines); K140, K141, K142 (solid lines). Data were obtained as described in Materials and methods.

ECsO values from 10.6 to >18 nM (Table 2, Fig. 6). Interestingly, the rBDl(Al40)-G28-5 sFv was several-fold less potent on Raji and HS-Sultan cell lines, but similar in the case of T51 cells (Table 2, Fig. 6). Even so, the A141 mutation was more detrimental to cytotoxicity than was the A140 mutation in all cases exam- ined. CD40 negative cells (HPB-ALL) were not killed by any of the rBDI-G28-5 sFv proteins. These data indicate that Y141 is integral to BDl function, whether by itself or in the context of a single-chain immunotoxin. In contrast, mutations at residue 140 have minimal effect on BDl activity when by themselves but reduce specific cytotoxicity when in immunotoxin form.

Discussion

RIPS inhibit protein synthesis by enzymatically removing a specific adenine (A4324) on 28s rRNA by N-glycosidation (Endo et al., 1987). Sequence alignment of BD1 with other R I P S shows that there are invariant or highly conserved amino acid residues found in the active site cleft that mediate substrate binding, including N78, Y80, Y123, R134, 4173, E177, R180, E208, N209,


Table 2. Antigen binding and translation inhibition activity of rBDl-G28-5 sFv and mutantsa

Cell-based EC50 (nM)h CD40-Ig Cell-free ECSo binding (PM ) T5 1 Raji HS-Sultan HPB-ALL

rBDI-G28-5 sFv + c 63 0.5 3.0 0.5 >18.2 rBDI(A140)-G28-5 sFv + 134 1 .I >18.2 6.5 >18.2 rBDl(A141)-G28-5 sFv + 1,103 10.6 > 18.2 >18.2 >18.2

aData are means of three independent experiments performed in triplicate and were not taken directly from data presented in

'T51, Raji, and HS-Sultan cells express surface CD40; HPB-ALL is CD40 negative. 'Positive CD40-Ig binding indicates EC5o range of 15-66 pg/mL as determined by ELISA.

Figure 6.

and W211 (Katzin et al., 1991; Ready et al., 1991; Gawlak et al., 1997). The only report of noncatalytic cleft amino acids being implicated in cell-free inhibition of protein synthesis identified serine 203 of ricin A chain (Gould et al., 1991), which is located within an e-helix near the active site. Studies of BDl residues 128-156 or similar regions of other RIPS have not been described to date.

As discussed above, these sequences were highlighted as being potentially involved in membrane interactions thought to be nec- essary in the cytotoxic mechanism of this family of toxins. We now

A 120

100

80

60

40

20

0

C

present data indicating that a specific tyrosine residue (141) in BDI outside of the active site is required for full catalytic activity. Because the cytotoxic activities of Y141 mutants are more reduced than their enzymatic activities, this residue may also contribute to BDl functions other than catalysis.

The three-dimensional structure of rBDl superimposes very closely onto that of ricin A chain, a related ribosome-inactivating protein with which many structural studies have been performed. Each active site residue listed above is identical to the amino acid in the analogous position of rBDl as determined by crystal model

B L 4

1

I . . ......, . . . . . . "I . . . . . . . I . . ..-

D

0.001 0.01 0.1 1 10 100

Immunotoxin Concentration, nM Fig. 6. Cytotoxicity of A140 and A141 mutant immunotoxins on human cell lines. A: T51. B: Raji. C: HS-Sultan. D: HPB-ALL. rBDl-Gz8-5 SFV (x); rBDl(A140)-G28-5 sFv (+); rBDl(A141)-G28-5 sFv (H). Data were performed in triplicate and are representative of three independent experiments.

Tyrosines in Bryodin I required for full activity 323

superimposition. Because of these and other similarities between the two proteins, it is reasonable to make use of existing ricin A chain information to aid in understanding BDl structure and function. When the above-listed ricin A chain residues are mapped to the rBDl crystal model, they identify a cleft that does not include the mutated residues described in this report (Fig. IB).

The importance of Y141 to rBDl catalytic activity may be due to contact of this residue with the ribosome at a site near but not immediately adjacent to A4324. The existence of such an “extend- ed site” has been hypothesized previously (Could et al., 1991; Katzin et al., 1991; Mlsna et al., 1993). The importance may be to stabilize the interaction between toxin and ribosome, thereby con- tributing to ribosomal inactivation without actually being involved in the catalytic event. Tyrosine 141 has its side chain oriented along the surface of the crystal with the face of its aromatic ring exposed to the solvent. The ability of rBDl to inhibit cell-free translation was more sensitive to replacement of this tyrosine with alanine than with lysine (31- versus 19-fold). The polar character of the tyrosine may be relevant in this potential interaction and would have been lost with the A141 mutation.

Substitution of Y 140, Y141, or Y142 did not alter the secondary structure content of the protein as determined by CD analysis (Fig. 5). This verification was important to rule out the possibility that the A141 and K141 mutants had more extensive structural differences that could be responsible for the activity changes observed. That all six point mutants had essentially wild-type structure indicates that the variance in potency is most likely due to substitution of the amino acid side chains at those particular positions and not to a backbone rearrangement that could sterically alter the active site.

The A141 and K141 mutants were at least 137- and 80-fold less potent than wild-type rBDl in whole-cell cytotoxicity. As was true for cell-free protein synthesis inhibition activity, alanine substitution was more detrimental than lysine substitution. In addition, because the cytotoxic effects were several-fold greater than the cell-free effects (for both mutants of Y141), there may be a step in the cell intoxication process other than ribosomal interaction that is affected with alanine or lysine at this position.

Y141 may be important as a contact residue with a potential cellular receptor or in interaction with lipid bilayers. If this residue could assist in bringing BDI into close proximity with a membrane, the remainder of the hydrophobic 128-156 region might be involved in mediating actual membrane translocation. Alterna- tively, the mutants may be more sensitive to intracellular proteases than the wild type, leading to a decreased concentration of active toxin.

The changes at positions 140 or 142 had no or minimal effect (with rBDl alone on JAR cells), pointing again to the specific importance of residue 141. Tyrosines 140 and 142 are relatively buried within the protein and may be less accessible for promoting interactions with other molecules. Evidence for membrane translocation-associated residues in ricin A chain within amino acids 245-256 has been reported previously (Simpson et al., 1995).

When incorporated into rBDl-G28-5 sFv, the A141 mutant reduced the cell-free and cell-based activity of the immunotoxin in a fashion similar to that seen with A141 in BDl itself. In addition, the substitution of Y140 with alanine reduced the activity of the anti-CD40 immunotoxin against both Raji and HS-Sultan cell lines, even though a similar effect was not seen in the cytotoxicity assays of rBDl(A140) against JAR cells. One explanation for this differ- ence may be in the cytoplasmic delivery mechanism of rBDl

compared to rBDl-G28-5 sFv, although variations between cell lines may exist in intracellular processing activities. Type 1 RIPs do not contain binding subunits and are generally thought to enter cells through nonspecific fluid-phase endocytosis (Barbieri et al., 1993). The immunotoxin binds CD40 on the cell surface and is internalized through a distinct pathway that may require a tyrosine at position 140 for full activity. Continued structure-function analysis of rBDl and other RIPs will be needed to further elucidate the process by which they intoxicate cells.

Materials and methods

BD1 mutagenesis

BD1 deletions were generated by PCR with mutagenic primers complementary to 18 bases on either side of the target region. Amino acids LYYYTASS (D139-146), ITTTLYYYTASSAAS (D136-149), or GLPALDSAITTLYYYTASSAASALLVLIQ (D128-156) were removed for the three deletions. BDI sequences 5’ and 3‘ to the mutation were amplified independently in a first- round reaction. Products were combined and annealed at overlap- ping complementary sequences flanking the mutation for full- length amplification of mutant rBDl with T7 promoter and terminator primers. These PCR products were digested with Nco 1 and EcoR 1 for ligation into the expression vector pSE 13.0 (Caw- lak et al., 1997).

Point mutations were generated as above except that the mutagenic primers were mismatched only for the codon to be changed. For construction of mutant rBDl-G28-5 sFv, an internal fragment containing the point mutation at position 140 or 141 was produced by restriction digestion of the mutant rBDl cDNA with Pst 1 and Xho 1. This fragment was purified and introduced in place of the analogous fragment from pSE 151.0 containing rBDI-G28-5 sFv (Francisco et al., 1997). All constructs were transformed into com- petent E. coli (DH5a) and verified by DNA sequencing.

Recombinant protein expression and purification

Recombinant BDl proteins were produced in E. coli strain BL21 (ADE3) carrying the desired construct, as described previously (Gawlak et al., 1997). Briefly, log phase cultures were induced with IPTG for 2 h, and inclusion bodies were isolated, washed, and denatured in 7 M guanidine-HCI. Denatured proteins were refolded in PBS containing 400 mM L-arginine (for rBDl and all rBDl point mutants) or 20 mM Tris, pH 8.0, containing 400 mM L-arginine (for rBDl-G28-5 sFv and mutants). Deletion mutants of rBDl were denatured in 8 M urea and refolded in PBS containing 2 M urea.

Refolded proteins were dialyzed extensively into 20 mM Tris, pH 7.2 (for rBDl and point mutants) or pH 8.0 (for rBDl-G28-5 sFv and mutants); deletion mutants were purified without dialysis. rBDl and mutant rBDl proteins were purified by adsorption and removal of residual bacterial proteins with Sepharose C M B fol- lowed by binding to CM-Sepharose FF and elution with a linear gradient from 0 to 300 mM NaCI. rBDlG28-5 sFv and mutant immunotoxins were batch purified on Blue Sepharose 6 FF fol- lowed by TSK 2000SW size-exclusion chromatography. Homo- geneity of each protein was verified by SDS-PAGE; noncovalent multimeric complexes may have been present that would not have been detected by this analysis.


Assay of rBDl catalytic activity

Inhibition of cell-free translation activity of rBDl mutants was tested by using a rabbit reticulocyte lysate assay as described previously (Gawlak et al., 1997). except that the assay was adapted for 96-well plate format to increase sample throughput. Following precipitation with 25% trichloroacetic acid and 2% casamino acids, tritiated proteins were collected with a Tomtek 96-well plate har- vester (Orange, Connecticut) on filter mats designed for protein retention. Samples were tested at least three times in quadruplicate.

Assay of rBDl and rBDI-G28-5 sFv imunotoxin cytotoxicity

JAR choriocarcinoma cells (ATCC, Rockville, Maryland) were plated at lo4 cells/well, exposed in triplicate to rBDl or mutant rBDl for 48 h, incubated in leucine-free medium for 30 min, pulsed with 3H-leucine for 6 h, and harvested. Data are presented as percentage of tritiated protein incorporation relative to medium treated controls. rBDl-G28-5 sFv and mutant forms were tested as above except that CD40 positive cell lines T51 (lymphoblastoid, gift from Dr. Paul Gladstone Bristol-Myers Squibb Seattle, Washing- ton), Raji (Burkitt's lymphoma, ATCC), HS-Sultan (plasmacyto- ma, ATCC), and the CD40 negative T cell leukemia line HPB-ALL (gift from Roberta Connelly, Bristol-Myers Squibb Seattle, Wash- ington) were exposed for 16 h instead of 48 h.

CD analysis

CD in the far-UV region was used to assess the secondary structure of the parent and mutant rBDl (Woody, 1995). CD measurements were performed on an Aviv model 62A DS CD spectrophotometer (Lakewood, New Jersey) at 4 "C and with a 0.4-cm pathlength quartz cuvette (Stama Cells, Atascadero, California). Protein in 20 mM Tris, pH 7.2, buffer was scanned every 0.5 nm from 260 to 200 nm; five scans of each sample were averaged to obtain each spectrum.

The UV absorbance from 400 to 200 nm was measured immediately prior to the CD measurement on a Hewlett Packard 8452A diode array spectrophotometer (Palo Alto, California). Concentra- tions of the protein solutions were determined using a calculated molar extinction coefficient for each protein ( f l r p X 5,540) + (#'Qr X 1,480) + (#disulfide bonds X 134) (Mach et al., 1992). The CD spectra were converted to mean residue molar ellipticity with the relationship [Q] = (Q X 100)/(#amino acids X molar concentration X cell pathlength in cm).

Assay of G28-5 binding to CD40 antigen

Correct folding of the antigen binding component of rBDl-G28-5 sFv and mutant immunotoxins was determined in an ELISA format

by using immobilized human CD40-Ig as described previously (Francisco et al., 1997). Rabbit polyclonal anti-BD1 antiserum and goat anti-rabbit IgG conjugated to horseradish peroxidase were used for detection.

Acknowledgments

We thank Drs. J. Francisco, H. Klei, H. Einspahr, and M. Neubauer for suggestions and guidance and J. Cook, T. Youngman, and B. Bear for their expert assistance in preparation of oligonucleotides and DNA sequencing.

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identification of a specific tyrosine residue in bryodin 1 distinct from

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