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Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA
Reprint requests to: Jack F. Kirsch, Department of Molecular and Cell Biology, 229 Stanley Hall, University of California, Berkeley, CA 94720, USA; e-mail: jfkirsch{at}uclink4.berkeley.edu; fax: (510) 642-6368.
(RECEIVED April 8, 2002; FINAL REVISION June 28, 2002; ACCEPTED July 2, 2002)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0209102.
1 Present address: Rinat Neuroscience, 3155 Porter Drive, Palo Alto, CA 94304, USA. 
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Keywords: Alanine scanning mutagenesis; antigen; antibody; epitope mapping; hen egg-white lysozyme; scFv HyHEL-10; scFv F9.13.7; protein-protein interaction
Abbreviations: CDRs, complementarity-determining regions • Fab, antigen-binding fragment • GEL, guinea fowl lysozyme • HEWL, hen (chicken) egg-white lysozyme • HyHEL-10 and F9.13.7, monoclonal antibodies raised against HEWL • scFv, single-chain variable fragment • subscripts L and H, antibody light and heavy chain, respectively • WT, wild type
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Introduction |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsReferences |
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Can a given epitope drive the formation of multiple paratopes of unrelated sequence? The answer appears to be yes, because the HyHEL-10/lysozyme epitope is also recognized by the F9.13.7 Fab (Lescar et al. 1995). HyHEL-10 and F9.13.7 are two independently isolated high-affinity antibodies that bind nearly the same epitope of hen egg-white lysozyme (HEWL), but they have different, nonhomologous, CDRs.
The crystallographic structures of HyHEL-10 in complex with HEWL (Padlan et al. 1989; Kondo et al. 1999) and F9.13.7 with guinea fowl lysozyme (GEL; Lescar et al. 1995) show that they interact with essentially the same conserved epitope residues (Table 1
). The buried surface area totals 
1600 Å2 in both complexes. This contacting area is nearly equally shared between the heavy and light chains of HyHEL-10; however, it is unevenly distributed for F9.13.7 (570 Å2 for the heavy and 220 Å2 for the light chain). The HyHEL-10 complex features 14 intermolecular hydrogen bonds and one salt bridge, whereas the F9.13.7 complex contains 12 hydrogen bonds and three salt bridges. The polar and charged groups are distributed similarly in the two antibody combining sites. These antibodies show different cross-reactivity patterns with related avian lysozymes (Smith-Gill et al. 1984; Tello et al. 1990). Association of either antibody with HEWL inactivates the enzyme by insertion of a CDR loop into the active site.
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The F9.13.7/lysozyme interaction, by comparison, is largely unexplored. No systematic site-directed mutagenesis study has focused on this complex. The above-mentioned cross-reactivity results, the crystal structure, and a thermodynamic comparison of the energetics of binding of F9.13.7 and other specific antibodies with lysozyme constitute what is known about this complex.
We report here the construction and expression of a synthetic scFv gene for F9.13.7, the quantitative analysis of the contribution of epitope residues to the stability of the complex by both alanine scanning mutagenesis and conservative mutations, and a comparison with the HyHEL-10/HEWL complex. These two structurally and sequence diverse antibodies, which bind to the same epitope, offer a unique probe to explore multiple modes of protein–protein recognition and to elucidate further relationships between affinity and specificity.
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Results and Discussion |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsReferences |
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2.8 times greater for the Fab complex with lysozyme (scFv: 8.0 ± 0.5 x 10-4 sec-1; Fab: 22 ± 2 x 10-4 sec-1). The calculated KD values (scFv: 0.57 ± 0.05 nM; Fab: 1.6 ± 0.2 nM) show that scFv binds 2.5 times more tightly to HEWL than does the corresponding Fab. These results show that the synthesized scFv is fully functional. Increased affinity of scFv over Fab has been reported elsewhere (Iliades et al. 1998). The KD values obtained here by homogeneous kinetics are very close to the results obtained with the F9.13.7 Fab by calorimetry (1.3 ± 0.6 nM; Schwarz et al. 1995).
Alanine scanning mutagenesis of F9.13.7
The epitope of the GEL–Fab F9.13.7 complex is identified in the crystal structure (Lescar et al. 1995). The guinea fowl and chicken lysozymes differ by only 11 amino acids in sequence; however, all residues in the epitopes recognized by HyHEL-10 and F9.13.7 are conserved. The RMSDs for C
atoms in equivalent positions are <0.9Å. All non-glycine HEWL residues in contact with F9.13.7 in the complex were individually replaced with alanine for comparison with the previously obtained data with the HyHEL-10 Fab (Rajpal et al. 1998) and HyHEL-10 scFv (this work). The KD and 
G values are collected in Table 1
.
The mutations Y20A, R73A, and K96A each destabilizes the complex by >4 kcal/mole. Thus residues Y20, R73, and K96 are defined as hot spots. Both Y20 (OH) and K96 (NH3+) interact with the carboxyl group of D52H in the CDR-H2 loop, forming a hydrogen bond and a salt bridge, respectively. Although most of the important F9.13.7 interactions with HEWL involve the heavy chain, there is a single significant contact residue in HEWL that is made with the F9.13.7 light chain. Mutation of R73, which makes hydrogen bonds with (Y32L) in CDR-L1 and Y92L in CDR-L3, causes the largest destabilization effected by alanine substitution (G = 7.0 kcal/mole).
Moderate destabilization, 1 < G < 4 kcal/mole (warm spots), is seen in complexes formed from HEWLs with alanine mutations at H15, R21, W63, K97, S100, and D101. H15 and K97 form the two remaining salt bridges of the complex, H15/D54H and K97/E50H, respectively. The W63 side chain only contacts the antibody through CD2 of T104H and the S105H backbone; however, the indole group makes close contacts with the hot spot K97 as well as with epitope residues W62 and L75, which may buttress R73. The same role has been attributed to some of the HyHEL-10 epitope warm spots (Rajpal and Kirsch 2000). Thus the effect of the W63A mutation may be on the lysozyme structure itself. The R21, S100, and D101 residues each contributes hydrogen bonds to the CDR-H3 loop: that is, to Y96H, T100H, and S100(A)H, respectively. The remaining contact residues in the crystal structure, R14, N77, and N93, destabilize the complex by <1 kcal/mole when mutated to alanine, and are thus null spots.
F9.13.7 versus HyHEL-10: Conservation of residues
The relative importance of alanine epitope mutations for both complexes is diagramed in Figure 1. All three epitope hot-spot residues in the HyHEL-10/HEWL complex (Y20, K96, and K97) are also important in the corresponding F9.13.7 complex. R73 provides an additional hot spot in the latter complex, but contributes negligibly to the stability of the HyHEL-10/HEWL complex. The definition of largely coincident hot spots for the two complexes indicates that certain residues/features are strongly preferred for antibody recognition within a given epitope.
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The relative ranking for the alanine scanning destabilizations of the two complexes is F9.13.7: R73A > Y20A > K96A > K97A = R21A > D101A > H15A > W63A > S100A. HyHEL-10: K96A > K97A > Y20A |L: D101A > L75A > R21A (see Table 1). The positions of the HEWL hot-, warm-, and null-spot residues in both antibody complexes are shown in Figure 2. Although the important residues are essentially the same in both complexes, the topologies differ. In HyHEL-10 there is a clear center of interaction with its three hot spots (Y20, K96, and K97), whereas in F9.13.7 the hot spot R73 is separated from the other hot spots, Y20 and K96, by a patch of warm spots. The percentages of warm plus hot spots in the epitopes are 75% and 46% in the F9.13.7 and HyHEL-10 complexes, respectively.
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, which shows the losses in free energies of formation in HyHEL-10/HEWL complexes affected by epitope alanine substitutions compared with the effects in the F9.13.7/HEWL complexes. The mutations that fall on the line of unit slope are equally important in determining the affinity in both complexes. Those that are found in the upper, dark-shaded triangle are more important for the interaction with HyHEL-10, and those located in the lower triangle more significantly destabilize the F9.13.7/HEWL complex. This plot does not include the contributions of L75, R14, and N77, which are not common to both complexes (see Table 1). What is meant by specificity? It may be considered a function of an antibody's power of discrimination between similar antigens. Quantitatively, a specificity determinant may be defined by eliciting a G for association with an antigen that is greater than an arbitrary threshold level in response to a given replacement of that determinant in the antigen. The more specific antibody will show such a response G for a larger number of amino acid substitutions in the antigen. As documented above, there are a significantly greater number of important energetic determinants in the F9.13.7 than in the HyHEL-10/HEWL complex; therefore, F9.13.7 shows greater specificity despite its 20-fold lower affinity for HEWL (KD for HEWL/HyHEL-10 = 30 pM; KD for HEWL/F9.13.7 = 600 pM).
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compares the G with respect to the wild-type complex for each of these mutants. These results emphasize the necessity of including mutations in addition to alanine to illuminate fully the details of the contributed interactions.
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G = 0.06 ± 0.03 kcal/mole), indicating that all of the important contacts with this residue are located in the ring. However, this conservative mutation is strongly disruptive in the F9.13.7/HEWL complex (3.6 ± 0.09 kcal/mole). These large F9.13.7 complex destabilizations correspond to the loss of the hydrogen bond between the Y20 (O
) and D52H (O
1). The semiconservative mutation Y20L, which presumably retains an intermediate amount of hydrophobic contacts (intermediate between alanine and phenylalanine), shows a moderate destabilization value (2.7 ± 0.1 kcal/mole) for the interaction with HyHEL-10. This mutation also yields a major destabilization of the F9.13.7 complex (5.2 ± 0.1 kcal/mole).
The K96M mutation results in a >4 kcal/mole destabilization of both antibody complexes. These values are similar to those observed in the K96A complexes; therefore, the important interaction is due almost entirely to the K96 
amino group, which forms a hydrogen bond with N31L (HyHEL-10) or a salt bridge with D52H (O2) (F9.13.7). The hydrogen bond K96/N31L contributes 5.2 kcal/mole to the stability of the HyHEL-10 complex (Pons et al. 1999). The present results show that the salt bridge K96/D52H is also very important for the interaction between F9.13.7 and HEWL.
Both antibodies form salt bridges with HEWL at position K97 (HyHEL-10, K97/D32H and F9.13.7, K97/E50H). K97 mutations in lysozyme show different effects on the stabilization of the two antibody complexes. K97A shows a twofold greater free energy of destabilization of the HyHEL-10 complex, compared with the K97A/F9.13.7 complex (6.2 vs. 3.3 kcal/mole, respectively). The K97M mutation restores most of the lost interaction in the K97A complex with HyHEL-10 (1.1 kcal/mole), but equally destabilizes the F9.13.7 complex (3.6 kcal/mole). These data show that the side-chain interactions of K97 in the HyHEL-10 complex are more important than the salt bridge, but the converse is true for the F9.13.7 complex. It has been shown that the entire salt bridge can be replaced by chain-length conservative mutations in the HyHEL-10 interaction (K97M/D32HN) with a very small destabilization, 0.3 ± 0.1 kcal/mole (Pons et al. 1999).
The R73 mutations are silent in their effects on the stabilities of the HyHEL-10 complexes (-0.3 kcal/mole); however, they define R73 as the most important residue for the stabilities of the F9.13.7 complexes (>7 kcal/mole). The guanidino nitrogen atoms participate in three hydrogen bonds: R73 (N)/Y92L (O); R73 (NH1)/S100H(A) (O); and R73 (NH2)/Y32L (O). The fact that the R73A and R73M mutations are equally destabilizing isolates the guanidino moiety as the major contributing factor.
Polar interactions are more important for the higher-specificity antibody
There are more energetically important polar contacts observed in the F9.13.7 complex than in that formed with HyHEL-10. Conversely, two out of three HEWL hot spots interact mainly by side-chain van der Waals contacts in the HyHEL-10 complex. Although about half of the amino acids found in proteins have side chains with the potential to participate in hydrogen bonds or salt bridges, such interactions require specific complementary functional groups and geometrically constrained participation from the associating partner protein. Nonpolar interactions are not similarly constricted. Thus, few replacements will maintain polar interactions, whereas a greater number of substitutions are allowed to maintain the strength of a nonpolar interaction. And therefore, antibodies that obtain most of the complex stabilization energy from nonpolar interactions will be less specific than antibodies with a high number of significant polar contacts. The same relation between nonpolar interactions and cross-reactivity have been described in receptor–hormone and other protein–protein interactions (DeLano et al. 2000).
How is specificity defined in antibody–antigen interactions
An antibody is generally considered specific if it cross-reacts with only a few natural variants of the antigen. The lysozyme antibody literature provides a rich data source for comparative purposes. To date, most specificity studies have focused on cross-reactivity reactions of antibodies with closely related natural antigens. The results of these investigations may, however, suffer from bias owing to the phylogenetic variability of the epitope. That is, antibodies directed to conserved epitopes will be highly cross-reactive, and are therefore categorized as nonspecific, whereas those targeted to variable epitopes will not cross-react, and are thus termed specific. Thus, specificity as so defined does not address the intrinsic capacity of different antibodies to detect possible variations in the antigen. It follows that investigation of cross-reactivity with naturally occurring variants will not serve to define the potential specificity of antibodies properly. To illustrate, D1.3 and D11.15 are two highly affine monoclonal antibodies raised against HEWL from the same mouse. The epitopes of these two antibodies partially overlap, but each shows a different cross-reactivity pattern. D1.3 cross-reacts only with bobwhite quail lysozyme and, therefore, has been termed a highly specific antibody, whereas D11.15 cross-reacts with all the tested avian lysozymes (Harper et al. 1987) and was categorized as a nonspecific antibody. The crystal structures of D1.3 complexed with HEWL (Amit et al. 1986; Fischmann et al. 1991) and D11.15 complexed with pheasant egg lysozyme (Chitarra et al. 1993) explain the differences in cross-reactivity patterns between these closely related antibodies. Q121, which is present only in chicken and bob white quail lysozymes, is an essential component of the D1.3 epitope. On the other hand, the centrally located residues in the D11.15 epitope are strictly conserved among all avian lysozymes. Therefore, the observed difference in cross-reactivity between these antibodies is the result of fortuitous variability of the natural antigens. For example, just the opposite specificity classification would have been concluded if lysozyme Q121 were conserved and the essential residues for the interaction with D11.15 were variable among avian lysozymes.
The preceding analysis shows the necessity to investigate unbiased epitopes, which are accessible only by site-directed mutagenesis, to quantify antibody specificity. Alanine scanning mutagenesis is a method for systematic screening of the entire epitope. We have shown, through the application of alanine scanning mutagenesis of a system where two antibodies bind the same structural epitope, that antibodies with lower affinity for one epitope can at the same time detect more variations and therefore be more specific. This shows that affinity and specificity will not always correlate.
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Materials and methods |
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F9.13.7 scFv gene synthesis
An scFv gene with the identical variable domain sequences of the crystallized Fab of F9.13.7 (Lescar et al. 1995) was constructed by sequential subcloning of nine small fragments (160 bp) into the Pichia pastoris pPIC-9 plasmid (Invitrogen). Each fragment was generated by PCR fill-in of both overhang single-stranded portions of two large oligonucleotides (90 bp) that were annealed together with a complementary region (25 bp) located at the 3` end of the top oligonucleotide and the 5` end of the bottom oligonucleotide. The final gene contains in the following order from the N terminus: (1) the heavy-chain variable domain (VH), (2) a linker sequence (SSASSGGGGSGGGGSGGGGS), (3) the light-chain variable domain (VL of the 
family), and (4) a penta-his tag.
The final F9.13.7 scFv protein sequence is: VSLEKRQVQ LQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQGP GQGLEWIGEIDPSDSYPNYNEKFKGKATLTVDKSSSTAYM QLSSLTSEDSAVYYCASLYYYGTSYGVLDYWGQGTSVTV SSASSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTI SCRASQDISNYLNWYQKKPDGTVKLLIYYTSRLHSGVPSRF SGSGSGTDYSLTIRNLEQEDIATYFCQQGYTLPYTFGGGTK LEIKRHHHHH.
Expression and purification
The scFvs were purified as described in Pons et al. (1999), and HEWL as described in Matsumura and Kirsch (1996). The purities of the samples were verified by SDS-PAGE. The absence of glycosylation for all the proteins and the correct mass for mutants was confirmed by electrospray mass spectrometry (facility of the Department of Chemistry, University of California, Berkeley). No dimers of scFvs were detected by HPLC gel filtration (Bio-sil-sec 250 Column, Bio-Rad).
Site-directed mutagenesis
Some HEWL mutant genes were available from previous investigations of this laboratory (Rajpal et al. 1998; Taylor et al. 1998). Additional mutations were produced by PCR with either the megaprimer method (Landt et al. 1990; Pons et al. 1997) or by recombinant PCR using two mutagenic oligonucleotides (Innis et al. 1995). All mutants were fully sequenced.
Determination of kinetic parameters for complex formation and dissociation
Both HyHEL-10 and F9.13.7 occlude the active site of HEWL. The complex retains only 5% of the catalytic activity of free HEWL; therefore, HyHEL-10 binding with HEWL can be determined in homogeneous solution by monitoring the rate of loss of activity of HEWL following addition of scFv as described by Taylor et al. (1998). The values of kon were determined from nonlinear regression of the data using:
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0.995 were accepted.
The dissociation rate constants, koff, were determined by monitoring the rate of recovery of active HEWL from a preformed scFv/HEWL complex. The nascent free antibody was sequestered with an excess of E35Q HEWL, an inactive mutant. The data were fitted to the following equation (Rajpal et al. 1998),
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The kinetics are too fast to monitor by the above methods, where KD values are >3 nM (100 x KD WT); therefore, only the values of KD determined by equilibrium methods (Kam-Morgan et al. 1993) are given.
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Acknowledgments |
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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