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Structural diversity of antibody catalysts
Journal of Immunological Methods 269 (2002) 157 – 171 www.elsevier.com/locate/jim
Structural diversity of antibody catalysts Be´atrice Golinelli-Pimpaneau * Laboratoire d’Enzymologie et Biochimie Structurales, CNRS Baˆt. 34, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France Received 19 December 2001; accepted 2 January 2002
Abstract The structural diversity of the immune response may be considerably restricted by the structure of the hapten used to elicit catalytic antibodies. The ligand-binding mode and the shapes of the binding pockets of hydrolytic antibodies induced to different transition-state analogs that contain an unsubstituted arylphosphonate group are very similar. Moreover, antibodies elicited against a single transition state analog evolve from a single germline gene or different precursors, depending on the nature of the hapten. Germline antibodies seem to adopt multiple conformations with antigen binding, together with somatic mutation stabilizing the conformation with optimum complementarity to antigen. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Catalytic antibodies; X-ray structure; Transition-state analog; Catalytic mechanism; Structural diversity; Affinity maturation
1. Introduction Several strategies are now available to generate catalytic antibodies that can accelerate a wide range of chemical reactions. The most frequently used method relies on antibodies elicited against a hapten which mimics the transition state of a given reaction (Schultz and Lerner, 1995; Blackburn et al., 1998). Many structures of catalytic antibodies generated by this method validate this strategy and show that these antibodies catalyze their reaction by binding and stabilizing the transition state of the reaction through electrostatic, van der Waals’ and hydrogen bonding
Abbreviations: Fab, fragment antigen binding; CDR, complementarity-determining region; VL, variable light chain; VH, variable heavy chain. * Tel.: +33-1-6982-4235; fax: +33-1-6982-3129. E-mail address: [email protected] (B. Golinelli-Pimpaneau).
interactions (Mac Beath and Hilvert, 1996; Charbonnier et al., 1997a; Wade and Scanlan, 1997; Hilvert et al., 1998; Golinelli-Pimpaneau, 2000; Tantillo and Houk, 2001). However, it is only by chance that the antibody catalysts possess a chemically active residue, which explains their low catalytic efficiency. In order to elaborate new, more efficient catalytic antibodies, some questions arise. Is the transition-state analog strategy really suitable for selecting a nucleophilic or base-catalyzed mechanism? Is the low frequency of this event linked to a poor diversity of the immune response when it is probed to elicit antibody catalysts? Are there other immunization approaches more appropriate to generate functional residues? To answer these questions, the structural diversity of the immune response is addressed at different levels in this article. The X-ray structures of antibodies generated against different phosphonate transition-state analogs and catalyzing different ester
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hydrolysis reactions are first compared to see how the structure of the hapten dictates the shape of the binding pocket. The diversity of the immune response is then studied through the comparison of structurally characterized antibodies, elicited against a single transition-state analog (Charbonnier et al., 1997b; Buchbinder et al., 1998; Gigant et al., 1999). The comparison of the structures of the hapten-bound germline and affinity-matured catalytic antibodies then reveals how the somatic mutations alter the structure of the combining site and affect catalysis (Wedemayer et al., 1997a; Mundorff et al., 2000; Yin et al., 2001). Finally, alternative methods to the transition-state analog approach appear to lead to combining sites that precisely position a crucial catalytic residue while accommodating structurally diverse substrates.
2. The esterolytic antibodies elicited against unsubstituted aryl phosphonate haptens share a common deep Hydrophobic combining site 2.1. General mechanism of hydrolytic antibodies: the transition-state analog approach induces a nucleophile only fortuitously At present, X-ray structures of six families of hydrolytic antibodies have been determined: CNJ206 (Charbonnier et al., 1995), D23 (Charbonnier et al., 1997b), 17E8 (Zhou et al., 1994), 48G7 (Wedemayer et al., 1997a,b), 43C9 (Thayer et al., 1999), 6D9 (Kristensen et al., 1998). All these antibodies were elicited against an aryl phosphonate or phosphonamidate transition-state analog and catalyze the hydrolysis of arylesters (Scheme 1). First, compar-
Scheme 1. Phosphonate or phosphonamidate haptens used to generate the structurally characterized hydrolytic antibodies and sulfide oxidase antibody 28B4 and the corresponding substrates.
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ison of the X-ray structures of the antibody– hapten complex of CNJ206 with that of 17E8 (Charbonnier et al., 1995), then with that of 48G7 (Mac Beath and Hilvert, 1996) and D23 (Charbonnier et al., 1997a) has revealed that a common structural motif has been induced (Fig. 1A) and that the antibodies present a similar binding surface (Fig. 2A – D). In this ‘canonical’-binding mode, the aryl end of the hapten is buried deep in the binding pocket. The phosphonate moiety contributes to the major part of the binding energy in the antibody– hapten interaction. Residues of the combining sites, forming a recurring motif, donate hydrogen bonds to the negatively charged oxygens of the phosphonate group and are believed to stabilize the oxyanion intermediate in the ester hydrolysis (Table 1). The rate enhancement (kcat/knon = 103 – 105) roughly parallels the number of hydrogen bonds to the phosphonate and reflects what can be achieved with an oxyanion hole mechanism alone. The low efficiency is also associated with flexibility in the active site for 48G7 germline (k cat /k non = 10 2 ) and perhaps for CNJ206 (kcat/knon = 103) although the movement of the CDR H3 loop that occurs upon hapten binding may result from crystallographic contacts (GolinelliPimpaneau et al., 1994; Charbonnier et al., 1995). In the D23 family, a ligand-induced conformational change leads to a 30– 170-fold increase in the affinity towards the transition-state analog and hence to higher catalytic rates (Lindner et al., 1999). But in D23, a preequilibrium between two conformations of the antibody’s active site exists and the rate of catalysis is limited by the substrate-induced isomerization of the less active conformer to the more active one. 2.2. The role of the residues forming the oxyanion hole has been addressed by site-directed mutagenesis The effect of mutations in the residues that bind the phosphonate part of the hapten (Table 1) has been tested both on the activity and on ligand binding to understand whether these residues have a catalytic and/or a structural role. The question has been asked whether His H35, which forms part of the combining site in most catalytic antibodies (Fig. 1A,B, Table 1), could play the role of a general base or a nucleophile in catalysis. Indeed, mutagenesis studies of 48G7 and 17E8 indicate that His H35 clearly plays a significant role in catalysis: The HisH35-Glu mutant of 48G7 is
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60 times less active that the native antibody (Patten et al., 1996) and the HisH35-Ala mutation abolishes catalysis of 17E8 (Baca et al., 1997). However, the HisH35-Gln mutation has little effect on catalysis for 48G7 and decreases kcat/Km of 17E8 only 13-fold. This suggests that the catalytic role of His H35 is simply to stabilize the negatively charged transition state. For 43C9, the substitution of His H35 with either Asn or Phe results in a loss of catalytic activity but also in decreased hapten binding (Stewart et al., 1994), which argues for a structural role for His H35 in this case. The X-ray structures have shown that the nonprotonated nitrogen Ny1 of His H35 is involved in a conserved hydrogen bond with Trp H47 and therefore it is the protonated hydrogen of His H35 Nq2 that points towards the hapten. Hence, it is more likely that His H35 is a simple hydrogen bond donor that stabilizes the oxyanion of the transition state rather than a base or a nucleophile, which necessitates breaking the H-bond and flipping the imidazole side chain. Three hydrolytic antibodies 17E8, 43C9 and 48G7 contain arginine at position L96 which hydrogen bonds with one oxygen of the phosphonate hapten (Table 1). Site-directed mutagenesis studies have revealed that this residue is important for catalysis; the activity of a variant of 17E8 that contains the ArgL96-Trp substitution is reduced by a factor of 20, even though the hapten is bound 5-fold tighter (Baca et al., 1997). The ArgL96-Gln mutant of 43C9 is not catalytic and shows a 20-fold decrease in hapten binding while binding of products that do not incorporate the phosphonamidate functionality is conserved (Roberts et al., 1994). However, the ArgL96Gln mutation in 48G7 leads to only a 3-fold drop in kcat/Km (Patten et al., 1996). Nevertheless, all the results are consistent with the idea that when an arginine is present at position L96, it stabilizes the anionic tetrahedral transition state by electrostatic interactions. It has been suggested that ArgL96 could also stabilize the charge that develops on the leaving aryl group in 17E8 (Baca et al., 1997). From mutagenesis studies on tyrosine residues whose side-chain hydrogens bind to the phosphonate part of the hapten, it is not clear whether these residues play an essential role in the catalytic mechanism. The mutation of Tyr-H33 to Phe in 48G7
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resulted in only a 5-fold reduction in kcat/Km (Patten et al., 1996) and the TyrH95-Phe mutant of 43C9 possessed appreciable catalytic activity (Stewart et al., 1994). The role of the tyrosines may be to assist in substrate orientation within the active site. In the first structurally characterized hydrolytic antibody, 17E8, the nucleophilic role for Ser H95 that was originally proposed (Zhou et al., 1994) was abandoned when it was shown that the SerH95-Ala mutant is fully active (Baca et al., 1997). Moreover, antibody 7G11 that was obtained during the same immunization as 17E8 (see below), in which the Ser H95 is substituted for Ala, has a similar catalytic activity as 17E8 (Buchbinder et al., 1998). In conclusion, all the site-directed mutagenesis experiments have confirmed the general mechanism of catalysis inferred from the crystallographic studies; it consists of the nucleophilic attack of the ester bond by a water molecule or a hydroxide ion and the stabilization of the oxyanion intermediate by residues of the active site of the antibody. 2.3. The shape of the binding pocket depends on the nature of residue H100c of CDR H3 The antigen-binding sites of antibodies are constructed from six loops (CDRs) between strands of the
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h-sheets in the variable domains of the light (VL) and heavy chains (VH). CDR H3 is much more variable in length, sequence and conformation than the other antigen-binding loops because it falls in the region of the V – D – J join in the assembly of the immunoglobulin heavy chain domain. Several mechanisms contribute to generate its diversity, such as selection of VH, D and J gene segments and alternative splicing patterns. CDR H3 is located at the center of the antibody-combining site and makes numerous contacts with the antigen (Fig. 2). Its C-terminal region significantly influences the shape of the bottom of the binding pocket. The conformational flexibility of the H3 loop makes H3 particularly sensitive to its environment, and thus prediction of the H3 structure extremely difficult (Morea et al., 1998). The presence of a small residue at position H100c of CDR H3 allows the hapten to be deeply buried in a hydrophobic pocket constructed essentially from framework residues (Figs. 1A and 2A – C) (GolinelliPimpaneau, 2000). The p-nitrophenyl groups of the haptens occupy the bottom of the cleft. When a bulky residue (Phe, Met) occupies position H100c, the H3 loop has the region proximal to the framework in the common h-bulge, or kinked conformation at position H101. This bulged conformation is most often stabilized by an ionic interaction between the side chains of
Fig. 1. The atom-coloring scheme for the ligand is as follows: carbons in a color close to that of the antibody (Fig. 1) or yellow (Fig. 2), oxygen in red, nitrogen in blue, phosphorus in magenta, fluoride in violet, chloride in maroon. The structures are that of the Fab – hapten complexes except for 43C9 which is a complex of the Fab – product complex. Comparison of the combining site residues of hydrolytic antibodies. The active site residues are a different color for different Fabs in the same figure. The a carbons of residues of the framework of the Fabs (L1 – L25, L33 – L49, L53 – L90, L97 – L107, H1 – H25, H36 – H51, H57 – H93, H103 – H111) are superimposed. (A – C) are presented in the same orientation. (A) Comparison of the combining site residues of hydrolytic antibodies. Fabs 48G7 germline (yellow), CNJ206 (blue; hapten in ˚ (48G7 cyan), D23 (red; hapten in pink) have been superimposed on 17E8 (green). The root mean squared deviations from 17E8 are 1.31 A ˚ (CNJ206), 1.53 A ˚ (D23). The aryl substituent of the haptens maximizes hydrophobic contacts with the antibody at the germline), 1.42 A hydrophobic bottom of the cavity, which is made of conserved residues of the framework (Trp H103, Phe L98, Trp H47, Val/Ile H37). This leads to a common shape for the bottom of the combining sites. The more variable residues in position L34, L36, L89, H35 and L91 form the walls of the cavity. (B) Comparison of the combining sites of hydrolytic antibodies 48G7, 48G7 germline and 43C9. Fabs 48G7 (brown), 48G7 germline ˚ (48G7), 1.31 A ˚ (yellow), 43C9 (gray; product in light gray) have been superimposed on 17E8. The root mean squared deviations are 1.26 A ˚ (43C9). The p-nitroaryl ring of the hapten occupies very different locations in the combining sites of 48G7 germline (48G7 germline), 1.73 A and 48G7 mature, due essentially to a Ser L34-Gly somatic mutation. In 43C9, the two water molecules that are replaced by the phosphonamidate oxygen atoms of the docked hapten in the antibody – hapten model are indicated in cyan. (C) Comparison of the combining sites of hydrolytic antibodies 7C8, 6D9 and of the sulfide oxidase antibody 28B4. Fabs 7C8 (yellow, hapten in salmon), 6D9 (red, hapten in pink), have been superimposed on sulfide oxidase Fab 28B4 (blue, hapten in cyan) which also possesses a bulky residue at position H100c of ˚ (7C8), 1.51 A ˚ (6D9). These antibodies possess a regular bulged conformation of the CDR H3. The root mean squared deviations are 1.54 A CDR H3 C-terminus that results in the same location for residue H100c at the bottom of the binding pocket (Morea et al., 1998), thereby preventing the haptens contacting buried Trp H103. The shapes of the pockets are more diversified and the haptens are bound more shallowly than in the other esterolytic antibodies. The aromatic rings of the acyl side chain are oriented differently in 7C8 and 6D9: towards the exterior of the cavity in 7C8 and into the combining site, for 6D9. The two aromatic rings of 6D9 and the p-nitrophenyl ring of 7C8 are sandwiched at the mouth of the combining site between residues in position H95 and H50 while the phosphonates are highly exposed.
162 B. Golinelli-Pimpaneau / Journal of Immunological Methods 269 (2002) 157–171 Fig. 2. The binding surface of the hydrolytic antibodies and of oxidase antibody 28B4. The molecular surfaces of the combining sites, in pink for the light chain and light yellow for the heavy chain, were obtained using the program GRASP. They are shown in a similar orientation with residues H35, Trp H47, Trp H103 and the H3 loop (residue H92 – H103, shown as a worm) indicated in white. Residue H100c is cyan. For 28G7 and 28B4, the position of the hapten in the germline – hapten complex is orange. Antibodies 6D9, 7C8, 28B4, 48G7 and 48G7 germline possess a bulge at residue H101, which positions H100c at the same location in the combining site. (A) 17E8, (B) CNJ206, (C) 48G7, (D) D23, (E) 43C9. The p-nitrophenyl of the product of antibody 43C9 lies at the surface of the combining site compared to the same part of the haptens of the other hydrolytic antibodies. If one assumes that the p-nitrophenyl of the phosphonate hapten of 43C9 occupies the same position as the p-nitrophenyl of the product (this is the case in antibody D23), this indicates that the hapten of 43C9 is not much buried in the hydrophobic pocket. Phe in position H100c partially hinders contact with Trp H103 although Phe H100c of 43C9 is not superimposable to Phe H100c of antibodies with the bulged conformation of CDR H3. (F) 7C8, (G) 6D9, (H) 28b4.
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Fig. 2 (continued ).
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Table 1 Sequence comparison of the active site residues of the structurally characterized hydrolytic antibodies (A) Hydrolytic antibodies with the ‘canonical’-binding pocket and amidase antibody 43C9 Residue
Location
48G7 48G7ger
17E8 29G11
D23 D24 D25
CNJ206
43C9
L32 L34 L36 L49 L89 L91 L96 L98 H33 H35 H37 H47 H50 H93 H95 H96 H97 H100 H100a H100b H100c H101 H103
CDR1 CDR1 FR2 FR2 CDR3 CDR3 CDR3 FR4 CDR1 CDR1 FR2 FR2 FR2 FR3 CDR3 CDR3 CDR3 CDR3 CDR3 CDR3 CDR3 CDR3 FR4
Tyr Gly Ser Leu Tyr Leu Tyr Arg Phe Tyr His Val Trp Arg Ala Tyrb – – – – Tyr Gly Ile Trp
Tyr Gly Tyr His Leu Tyr Arg Phe Ala Val His Val Trp Tyr Lys Ser Gly Tyrb Tyr Ser Arg Ser Tyr Asn Val Asp Trp
Tyr Asn Asn Ser Leu His Tyr Tyr Val Gly Tyr Phe Trp His Val Leu Val Trp Arg Thr Thr Val Trp Gly Phe Asp Tyr Val Ala Val Met Met Leu Asp Trp
Tyr Ser Leu Tyr Leu Tyr Tyr Phe Gly His Val Trp Tyr Ala Gly Aspb Tyrb Gly Ser Arg Gly Ala Trp
Tyr Ala Tyr Tyr Gln Hisa Arg Phe Asn His Val Trp Met Val Tyr Gly Tyr Gly Asp Arg Phe Ser Trp
(B) Antibodies with a bulky residue at position H100c and a bulged conformation of CDR H3 Residue
Location
7C8
6D9
28B4 28B4ger
L27d L32 L34 L36 L49 L89 L91 L94 L96 L98 H33 H35 H37 H47 H50 H52 H53 H93 H95 H100a H100b H100c
CDR1 CDR1 CDR1 FR2 FR2 CDR3 CDR3 CDR3 CDR3 FR4 CDR1 CDR1 FR2 FR2 FR2 FR2 CDR2 FR3 CDR3 CDR3 CDR3 CDR3
Asn Lys His Tyr Lys His Thr Arg Leu Phe Tyr Asn Leu Trp Trp Tyr Gly Ala Tyra Glu Gly Phe
His Tyr Asp Phe Tyr Phe Gly Val Pro Phe Ala Ser Val Trp Ser Ser Gly Ala Val Trp Tyr Phe
His Tyr Glu Tyr Tyr Phe Gly Val Arg Phe Tyr Asn Ser Ala Val Trp Phe Arg Lys Asn Ala Trp Asp Tyr Ala Met
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Asp H101 and Arg or Lys at position H94 as well as by a hydrogen bond between the carbonyl oxygen of residue H100c and the side chain of Trp H103 (Morea et al., 1998). This positions the bulky H100c residue at the bottom of the combining site, which makes the binding site shallower (Figs. 1C and 2F – H). The hapten is bound high in the binding pocket and the antibodies adopt different cavity shapes. This was also observed for nonhydrolytic catalytic antibodies (Golinelli-Pimpaneau, 2000). However, in D23, the C-terminal region of CDR H3 is unusual in that it continues the antiparallel hsheet structure of the antibody framework despite the presence of a bulky Met at position H100c, Arg at position H94 and Asp at position H101. The occurrence of the unpredictable extended conformation of the H3 loop instead of the bulged one prevents Met H100c occupying the bottom of the binding pocket and allows the formation of the deep ‘canonical’binding pocket (Figs. 1A and 2D). The structural convergence observed towards the ‘canonical’-binding mode may be linked to the ligand-induced conformational change of D23 (see above) (Lindner et al., 1999). Crystallographic studies are necessary to find out if the nonactive conformer possesses the bulged conformation of the H3 loop. The 48G7 germline and the mature antibody have the same conformation as CDR H3. The bulged conformation positions Gly H100c at the bottom of the binding pocket. Nevertheless, the small size of this residue allows the p-nitrophenyl group of the germline precursor of 48G7 to lie in the ‘canonical’ pocket, whereas in mature 48G7 it is rotated to occupy the bottom of a hydrophobic cleft formed between CDRs L1 and H3 (Fig. 2C). 2.4. Why is 43C9 the only hydrolytic antibody that is also able to hydrolyze an aromatic amide? Residues that play the role of a nucleophile or a base in catalysis have rarely been found in antibody-
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combining sites as a result of the chemical diversity of the immune response. An exception is antibody 43C9 which catalyses the hydrolysis not only of esters but also of aromatic amides with an outstanding acceleration of 2.5 105. The combining site of 43C9 possesses a histidine residue in position L91 that presumably plays the role of a nucleophile. Substitution of HisL91 by Gln produced a mutant with no catalytic activity, but its affinity for ligands was nearly the same as that of the wild type (Stewart et al., 1994). This catalytic residue appeared fortuitously during the immunization process rather than the result of an elaborate strategy. A model of the antibody complexed to the transition-state analog has been built from the X-ray structure of the antibody complexed to the p-nitrophenol product (Thayer et al., 1999), assuming that the p-nitrophenyl ring occupies the same position in both cases. This hypothesis was demonstrated in the case of antibody D23 for which X-ray structures of both the antibody – hapten complex and the antibody– product complex have been determined (Gigant et al., 1997). The model of 43C9 complexed to the transition-state analog indicates that the oxyanion would be stabilized by H-bonds with His H35 and Arg L96 and that the nucleophilic attack of His L91 would be assisted by tyrosines L36 and H95 through a hydrogen bonding network. No residue would play the role of a general acid that protonates the leaving group. The hydrolysis of the acylenzyme intermediate by a hydroxide ion would limit the rate of the reaction. The crystal structure of antibody 43C9 complexed to the product has been compared to that of the other hydrolytic antibodies (Thayer et al., 1999; Tantillo and Houk, 2001). It shows that, instead of binding in the specific ‘canonical’ hydrophobic pocket, the pnitrophenol lies flat at the mouth of the combining site pocket between CDR H3 and L1 (Figs. 1B and 2E) (Thayer et al., 1999). The absence of a distinct deep pnitrophenol-binding pocket is correlated with the presence of Phe in position H100c of CDR H3 although
Notes to Table 1: The three residues preceding H101 are denoted H100a, H100b, H100c and the H3 supervariable loop was aligned according to Morea et al. (1998). This table can be compared to Table 2 of Golinelli-Pimpaneau (2000) which concerns nonhydrolytic antibodies. Common residues forming the combining site are indicated in bold in the residue column. Residues that hydrogen bind to the phosphonate oxygens and are capable of stabilizing the oxyanion intermediate in ester hydrolysis are indicated in bold. a Nucleophile. b H-bond occurring through the main chain NH group.
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Phe H100c is not located at the bottom of the binding pocket but lies much higher than expected, as the Cterminal region of CDR H3 does not adopt the bulged conformation (Table 1, Fig. 2E). This variation from the ‘canonical’-binding mode seems to be linked to the structure of the hapten used to generate 43C9 which contains two different hydrophobic groups (Scheme 1). The use of well-defined, distinct combining sites to accommodate these two groups would position the cleavable bond precisely near His L91. This is not the case for the other hydrolytic antibodies where the aryl group is buried in the active site, whereas the second lateral chain is linear, flexible or short and is exposed to the solvent (Fig. 2). It has been proposed that the combination of a nucleophile and of a combining site that immobilizes the two ends of the substrate but does not bury deeply the p-nitrophenyl group of the hapten confers on 43C9 its capacity to hydrolyze amides (Thayer et al., 1999). 2.5. Use of a structurally different hapten in the 6D9 family leads to more diversified binding pockets Antibodies 6D9 and 7C8 were raised against a single chloramphenicol phosphonate which also contains two aromatic groups (Scheme 1). They are the more efficient antibodies of the family that catalyze the hydrolysis of the corresponding chloramphenicol ester. Their X-ray structures have shown that these antibodies do not bind the hapten in the commonly found hydrophobic pocket, as do the other hydrolytic antibodies (Kristensen et al., 1998; Gigant et al., 1999) (Figs. 1C and 2F,G). While the use of similar haptens containing both a phosphonate and a single aryl group restricts the shape of the combining site pocket as do other similar hydrophobic hapten structures (Fig. 1A) (Golinelli-Pimpaneau, 2000), the use of a hapten that contains two different hydrophobic groups expands the structural diversity of the immune response, as shown previously for 43C9. In contrast to the other esterolytic antibodies, in the 6D9 family, position H100c is occupied by a phenylalanine residue that prevents the hapten burying itself deep into the combining site (Table 1, Figs. 1C and 2F,G). In addition, the H35 residue that makes part of the combining site of the esterolytic antibodies described previously, is not the commonly encountered histidine but either a serine (6D9) or an aspar-
agine (7C8) (Table 1). These two sequence differences have also been found in 28B4, which has also been induced by a hapten containing both a phosphonate and a p-nitroaryl moiety (Scheme 1). Indeed, the hapten-binding modes of these three antibodies show a great similarity (Fig. 1C).
3. Structural convergence or divergence inside a family of antibodies? Comparison of the structures of different antibodies elicited against a single hapten during a single immunization protocol (defined as belonging to a ‘family of antibodies’) has also given insight into the diversity of the immune response. 3.1. Diverse structural solutions in the 6D9 family Hydrolytic antibodies from the 6D9 family usually possess a high sequence identity and therefore probably a high structural homology (Miyashita et al., 1994; Fujii et al., 1995). However, antibody 7C8 differs from the other antibodies. It presents only 50% sequence identity to 6D9 (Table 1) and it uses a very different hapten-binding mode with the aromatic ring of the acyl side chain oriented towards the exterior of the cavity in 7C8 (Figs. 1C and 2F) while, for 6D9, it is oriented into the combining site (Fig. 2G). Moreover, the two phosphonate groups occupy very different positions at the surface of the antibody combining sites. The hapten used to generate 6D9 and 7C8 appears to bear multiple epitopes, which explains the fact that the germline precursors of 6D9 and 7C8 are very different. Consequently, the specificities, the catalytic residues, the rate-limiting steps and the catalytic mechanisms are different in the two antibodies. 3.2. Imprecise positioning of the catalytic residues in the 17E8 family The structures of antibodies 17E8 and 29G11 that have been induced to the same norleucine-phosphonate transition-state analog (Scheme 1) and that possess the highest level of catalytic activity towards the norleucine phenyl ester have been compared (Buchbinder et al., 1998). Despite their great sequence similarity (eight substitutions in the heavy chain
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variable region, including four in the hapten-combining site; Table 1), the same length of CDR H3, the presence of the same catalytic groups in the combining site and the similar location and orientation of their hapten, these antibodies have their catalytic ˚ . The groups positioned with differences of 1– 2 A different positioning of residues in the hapten-binding pocket comes from a very different association of the VH and VL domains in the two antibodies. The lack of subtle structure conservation, inside a family of antibodies, contrasts with natural enzymes that have evolved active sites with precise positioning of catalytic and substrate-binding residues. It has been proposed that the generally lower efficiencies of catalytic antibodies compared to enzymes may reflect a relatively inaccurate alignment of the bond-making and bond-breaking apparatus (Buchbinder et al., 1998). A more precise arrangement of the catalytic residues in antibody-combining sites is obtained by the ‘bait and switch’ and ‘reactive immunization’ strategies (see below). 3.3. A same hapten-binding mode despite different conformations of CDR H3 in the D23 family The structures of three antibodies D23, D24 and D25, which are the most efficient antibodies of the family to catalyze the hydrolysis of p-nitrobenzylesters, have been compared (Charbonnier et al., 1997b). These antibodies were identified by screening the entire hybridoma repertoire for catalysis (Tawfik et al., 1993). The amino acid sequences of the variable domains of D24 and D25 differ from D23 in 16 and 14 positions, respectively (Table 1). These differences come from somatic mutations from the same germline precursor (Kim et al., 1997). The sequence of CDR H3 of D24 possesses four differences and one insertion compared to D23 resulting from a difference in the recombination at the junction of the D and J segments. This leads to a very different conformation of this loop for the two antibodies. Although the antibodies have different conformations of the H3 loop, they bind their hapten in a similar mode with Tyr H100a and residue L34 hydrogen binding to one of the phosphonate oxygens and Trp H95 to the other. In the structure of D23 complexed to an amide inhibitor, the presence of a single hydrogen bond between the carbonyl oxygen of the substrate analog
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and Tyr H100a has identified Tyr H100a and residue L34 as the oxyanion hole (Gigant et al., 1997). As the H-bond to one of the phosphonate oxygens that occurs with Asn L34 in D23 is replaced by a water mediated H-bond with Ser L34 in D25, the catalytic motif that constitutes the oxyanion hole is similar.
4. How does the mature antibody structurally differ from its germline precursor? 4.1. Conformational flexibility of the active site of the germline antibody During the process of affinity maturation, somatic mutations are introduced that increase the binding affinity of the antibody to the antigen. To determine the effects of affinity maturation on binding and catalysis, the structures of the hapten-bound and free germline precursor and affinity-matured antibodies have been determined for three catalytic antibodies: esterolytic antibody 48G7 (Wedemayer et al., 1997a), oxy Cope antibody AZ-28 (Mundorff et al., 2000) and sulfide oxidase antibody 28B4 (Yin et al., 2001). In all cases, the germline antibodies show significant structural changes upon hapten binding, in particular a significant rearrangement of CDR H3 that improves packing interactions with the hapten. In contrast, in the affinity-matured antibody, somatic mutations have led to a lock-and-key binding mode in which the antibody-combining site is pre-organized for hapten binding. It has been proposed that the conformational flexibility seen in the germline antibodies significantly enhances the binding potential of the primary antibody repertoire (Wedemayer et al., 1997a). In contrast, the structure of the unliganded germline precursor to Diels Alderase antibody 39-A11 is very similar to that of the 39-A11 hapten complex (Romesberg et al., 1998). Hence, neither somatic mutation nor ligand binding seems to result in substantial conformational changes in the active site for antibody 39-A11. This is not unexpected as the germline precursor differs from the mature antibody by only two somatic mutations. One of them, SerL91Val, increases the affinity of the antibody to the hapten by eliminating a cavity near the combining site, thereby improving the packing between the hapten and the antibody. Despite the small sequence differ-
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ences between the germline and mature antibodies, the germline antibody possesses a polyspecific-combining site, unlike the mature antibody. It was suggested that the selection of polyspecific germline genes could be a general mechanism for rapid generation of antibodies of moderate to high affinity for a broad range of antigens that could expand the binding potential associated with the structural diversity of the primary antibody repertoire (Romesberg et al., 1998). 4.2. Have the germline and mature antibodies the same hapten-binding mode? In antibody AZ-28, the hapten in the germline complex is bound in a conformation similar to that seen in the affinity-matured antibody complex. Five out of the six somatic mutations appear to increase the binding affinity of the mature antibody predominantly through secondary sphere hydrogen-bonding interactions while the SerL34-Asn mutation, localized in the combining site, reduced the catalytic rate by fixing the transition-state analog in an unfavorable conformation. Here, the decreased degree of flexibility in the CDR H3 in the mature antibody was unresponsive to catalysis. Mutations introduced into the germline antibody during the affinity maturation process may also lead to differences in the hapten-binding mode. In contrast to antibody AZ-28, in 48G7 and 28B4, the haptens are bound in different conformations in the germline and mature antibody (Wedemayer et al., 1997a; Yin et al., 2001) (Figs. 1B and 2C,H). Antibodies 48G7 and 28B4 have been induced against haptens containing both a p-nitrophenyl ring and a phosphonate part. The germline antibodies bind the p-nitrophenyl ring of haptens in an orientation significantly different from that seen in the affinity-matured antibodies, whereas the phosphonate moiety is bound in a similar mode. It is very surprising to see that for 48G7germline, the p-nitrophenyl part of hapten is fitted in the commonly found orientation in the hydrophobic pocket, while for the affinity-matured antibody the p-nitrophenyl ring occupies a different position in the combining site, lying flat, nearer to the mouth of the combining site (Wedemayer et al., 1997a) (Figs. 1B and 2C). A single somatic mutation near the combining site, Ser L34-Gly, is essentially responsible for this huge effect. The other eight somatically mutated residues do not directly
contact the bound hapten but likely play an important role in organizing the conformation of residues that make up the active site and in limiting flexibility in the germline antibody. In this way, an additional H-bond occurs in the mature antibody complex relative to the germline complex between residue Tyr H33 and one of the phosphonate oxygens. In 28B4, three out of the nine replacement mutations occur at the hapten-binding site (Table 1) but the new orientation of the bound hapten is caused mainly by one of the mutations, AspH95-Trp in CDR H3. The presence of this mutation is a prerequisite for other somatic mutations to enhance the binding affinity for the hapten like the AsnH53-Lys mutation that introduces two new hydrogen bonds between the antibody and the phosphonate moiety. Affinity maturation allows antibody 48G7 to avoid burying the hapten deep into the hydrophobic-combining site pocket thereby enhancing the catalytic activity. On the contrary, the p-nitrophenyl group of the hapten is buried further in mature 28B4 than in the germline antibody. As the phosphonate group of the haptens is bound in a similar fashion in the germline and affinitymatured antibodies, the catalytic residues are conserved but the H-bonds and the complementarity of the hapten to the antibody are optimized during the evolution of the germline antibody. It is likely that if the antibodies had been screened directly for catalytic efficiency, there would be more evolution of the catalytic residues. 4.3. How is the transition-state analog strategy translated into the antibodies? The increase of affinity for the transition-state analog that comes with the affinity maturation most often leads to an increase of the catalytic activity. This confirms that the major part of catalysis comes from the complementarity to the transition state. However, in a few cases, the catalytic constants and the hapten affinities do not agree with the transition-state theory. Compared to 17E8, the affinity of 29G11 for the transition-state analog is actually 19 times better while the activity is four times less. Moreover, the improvement of the affinity to the transition-state analog did not lead to mutants with a better catalytic efficiency (Baca et al., 1997).
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The germline precursor to AZ-28 catalyzes the reaction with a 35-fold higher rate despite a 40-fold lower binding affinity for the transition-state analog because affinity maturation fixes the transition state in a catalytically unfavorable conformation (Mundorff et al., 2000). As the increase of affinity to the transitionstate analog does not always translate into higher efficiency and as the transition-state analog approach is not appropriate to program a chemically active residue in the combining site, other strategies have been thought to lead more active antibodies.
5. Strategies different from the transition-state analog approach lead to broad substrate tolerance The ‘bait and switch’ strategy uses a charged hapten to induce a complementary charged residue at a predetermined location with respect to the hapten. The crystallographic structure of an antibody, in which the general base carboxylate residue was induced by this strategy, has validated this approach (Golinelli-Pimpaneau et al., 2000). The ‘reactive immunization’ method aims at generating a residue that accelerates the reaction through covalent catalysis. It involves the formation of a stable complex between the hapten and the functional residue of the antibody, that can be easily monitored to allow the selection of the catalytic clones (Wagner et al., 1995; Barbas et al., 1997; Karlstrom et al., 2000). The appearance of the covalent bond during the immunization procedure hinders further evolution of the immune response. These two methods result in broad substrate tolerance and lead to antibodies analogous to the polyspecific germline antibodies. It remains to be studied if the broad substrate tolerance is linked to a high conformational flexibility.
6. Conclusion The broad diversity initially present in the primary repertoire is reduced to a small set of solutions when the immune system is challenged to bind haptens that contain an aryl group. Indeed, the Xray structures of antibodies selected for tight binding to arylphosphonate transition-state analogs have revealed that the majority share a common deep
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hydrophobic-binding pocket. Structural convergence is even evident in antibodies raised against unrelated hydrophobic haptens (Golinelli-Pimpaneau, 2000). There is a strong selective pressure to bind the hydrophobic portion of the hapten that could be more easily removed if the antibodies were selected directly for catalysis rather than for hapten binding (Tawfik et al., 1993). The transition-state analog approach generates an oxyanion hole constituted from H-bond donating residues but does not program a nucleophile or a general base that accelerates the reaction. Comparison of antibodies resulting from a single immunization has shown that the transition-state analog approach actually can lead not only to an imprecise arrangement of the catalytic residues inside the binding pocket but also to completely different locations of the combining site residues themselves. The inadequacy of the transition-state analog approach in programming a precisely positioned functional residue gives little hope for obtaining a sophisticated antibody that operates, for example, via a catalytic dyad by this strategy. The diversity of the primary immune response appears to be significantly enhanced when antibodies adopt more than one binding site conformation. The structural changes that occur on binding of hapten to the germline antibody are further optimized by affinity maturation which stabilizes one of the conformers and decreases the structural flexibility intrinsic to the germline antibody to enhance the antibody –hapten complementarity. Hence, the hapten binds the mature antibody in a ‘lock-and-key’ fashion. This contrasts to enzymatic catalysis where conformational flexibility is expected to improve catalytic activity. However, an example where conformational restriction of the antibody limits its activity has been provided. To mimic the polyspecificity of the germline antibodies and obtain more structurally diversified active sites, more diversified hapten structures should be used. References Baca, M., Scanlan, T.S., Stephenson, R.C., Wells, J.A., 1997. Phage display of a catalytic antibody to optimize affinity for transitionstate analog. Proc. Natl. Acad. Sci. U. S. A. 94, 10063. Barbas III, C.F., Heine, A., Zhong, G., Hoffman, T., Gramatikova,
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S., Bjo¨rnestedt, R., List, B., Anderson, J., Stura, E.A., Wilson, I.A., Lerner, R.A., 1997. Immune versus natural selection: antibody aldolases with enzymic rates but broader scope. Science 278, 2085. Blackburn, G.M., Datta, A., Denham, H., Wentworth, P., 1998. Catalytic antibodies. Adv. Phys. Org. Chem. 31, 249. Buchbinder, J.L., Stephenson, R.C., Scanlan, T.S., Fletterick, R.J., 1998. A comparison of the crystallographic structures of two catalytic antibodies with esterase activity. J. Mol. Biol. 282, 1033. Charbonnier, J.-B., Carpenter, E., Gigant, B., Golinelli-Pimpaneau, B., Tawfik, D., Eshhar, Z., Green, B.S., Knossow, M., 1995. Crystal structure of the complex of a catalytic antibody Fab fragment with a transition state analog: structural similarities in esterase-like catalytic antibodies. Proc. Natl. Acad. Sci. U. S. A. 92, 11721. Charbonnier, J.-B., Gigant, B., Golinelli-Pimpaneau, B., Knossow, M., 1997a. Similarities of hydrolytic antibodies revealed by their X-ray structures: a review. Biochimie 79, 653. Charbonnier, J.B., Golinelli-Pimpaneau, B., Gigant, B., Tawfik, D., Chap, R., Schindler, S.H., Kim, S.H., Green, B.S., Eshhar, Z., Knossow, M., 1997b. Structural convergence in the active site of a family of catalytic antibodies. Science 275, 1140. Fujii, I., Tanaka, F., Miyashita, H., Tanimura, R., Kinoshita, K., 1995. Correlation between antigen-combining site structures and functions within a panel of catalytic antibodies generated against a single transition state analog. J. Am. Chem. Soc. 117, 6199. Gigant, B., Charbonnier, J.-B., Eshhar, Z., Green, B.S., Knossow, M., 1997. X-ray structures of a hydrolytic antibody and of complexes elucidate catalytic pathway from substrate binding and transition state stabilization through water attack and product release. Proc. Natl. Acad. Sci. U. S. A. 94, 7857. Gigant, B., Tsumuraya, T., Fujii, I., Knossow, M., 1999. Diverse structural solutions to catalysis in a family of antibodies. Structure 7, 1385. Golinelli-Pimpaneau, B., 2000. Novel reactions catalysed by antibodies. Curr. Opin. Struct. Biol. 10, 697. Golinelli-Pimpaneau, B., Gigant, B., Bizebard, T., Navaza, J., Saludjian, P., Zemel, R., Tawfik, D.S., Eshhar, Z., Green, B.S., Knossow, M., 1994. Crystal structure of a catalytic antibody Fab with esterase-like activity. Structure 2, 175. Golinelli-Pimpaneau, B., Goncß alves, O., Dintinger, T., Blanchard, D., Knossow, M., Tellier, C., 2000. Structural evidence for a programmed general base in the active site of a catalytic antibody. Proc. Natl. Acad. Sci. U. S. A. 97, 9892. Hilvert, D., MacBeath, G., Shin, J.A., 1998. The structural basis of antibody catalysis. In: Hecht, S.M. (Ed.), Bioorganic Chemistry: Peptides and Proteins. Oxford Univ. Press, NewYork, p. 355. Karlstrom, A., Zhong, G., Rader, C., Larsen, N.A., Heine, A., Fuller, R., List, B., Tanaka, F., Wilson, I.A., Barbas III, C.F., Lerner, R.A., 2000. Using antibody catalysis to study the outcome of multiple evolutionary trials of a chemical task. Proc. Natl. Acad. Sci. U. S. A. 97, 3878. Kim, S.-H., Schindler, D.G., Lindner, A.B., Tawfik, D.S., Eshhar, Z., 1997. Expression and characterization of recombinant sin-
gle-chain Fv and Fv fragments derived from a set of catalytic antibodies. Mol. Immunol. 34, 891. Kristensen, O., Vassylyev, D.G., Tanaka, F., Morikawa, K., Fujii, I., 1998. A structural basis for transition-state stabilization in antibody-catalyzed hydrolysis: crystal structures of an abzyme at ˚ resolution. J. Mol. Biol. 281, 501. 1.8 A Lindner, A.B., Eshhar, Z., Tawfik, D.S., 1999. Conformational changes affect binding and catalysis by ester-hydrolysing antibodies. J. Mol. Biol. 285, 421. Mac Beath, G., Hilvert, D., 1996. Hydrolytic antibodies: variation on a theme. Chem. Biol. 3, 433. Miyashita, H., Hara, T., Tanimura, R., Tanaka, F., Kikuchi, M., Fujii, I., 1994. A common ancestry for multiple catalytic antibodies generated against a single transition state analog. Proc. Natl. Acad. Sci. U. S. A. 91, 6045. Morea, V., Tramontano, A., Rustici, M., Chothia, C., Lesk, A.M., 1998. Conformations of the third hypervariable region in the VH domain of immunoglobulins. J. Mol. Biol. 275, 269. Mundorff, E.C., Hansin, M.A., Varvak, A., Ulrich, H., Schultz, P.G., Stevens, R.C., 2000. Conformational effects in biological catalysis: an antibody-catalyzed oxy-Cope rearrangement. Biochemistry 39, 627. Patten, P.A., Gray, N.S., Yang, P.L., Marks, C.B., Wedemayer, G.J., Boniface, J., Schultz, P.G., 1996. The immunological evolution of catalysis. Science 271, 1086. Roberts, V.A., Stewart, J.D., Benkovic, S.J., Getzoff, E.D., 1994. Catalytic antibody model and mutagenesis implicate arginine in transition-state stabilization. J. Mol. Biol. 235, 1098. Romesberg, F.E., Spiller, B., Schultz, P.G., Stevens, R.C., 1998. Immunological origins of binding and catalysis in a Diels-alderase antibody. Science 279, 1929. Schultz, P.G., Lerner, R.A., 1995. From molecular diversity to catalysis: lessons from the immune system. Science 269, 1835. Stewart, J.D., Roberts, V.A., Thomas, N.R., Getzoff, E.D., Benkovic, S.J., 1994. Site-directed mutagenesis of a catalytic antibody: an arginine and a histidine residue play key roles. Biochemistry 33, 1994. Tantillo, D.J., Houk, K.N., 2001. Canonical binding arrays as molecular recognition elements in the immune system: tetrahedral anions and the ester hydrolysis transition state. Chem. Biol. 8, 535. Tawfik, D.S., Green, B.S., Chap, R.M.S., Eshhar, Z., 1993. Efficient and selective p-nitrophenyl-ester hydrolyzing antibodies elicited by a p-nitrobenzyl phosphonate hapten. Proc. Natl. Acad. Sci. U. S. A. 90, 373. Thayer, M.M., Olender, E.H., Arvai, A.S., Koike, C.K., Canestrelli, I.L., Stewart, J.D., Benkovic, S.J., Getzoff, E.D., Roberts, V.A., 1999. Structural basis for amide hydrolysis catalyzed by the 43C9 antibody. J. Mol. Biol. 291, 329. Wade, H., Scanlan, T.S., 1997. The structural and functional basis of antibody catalysis. Annu. Rev. Biophys. Biomol. Struct. 26, 461. Wagner, J., Lerner, R.A., Barbas III, C.F., 1995. Efficient aldolase catalytic antibodies that use the enamine mechanism of natural enzymes. Science 270, 1797. Wedemayer, G.J., Patten, P.A., Wang, L.H., Schultz, P.G., Stevens, R.C., 1997a. Structural insights into the evolution of an antibody combining site. Science 276, 1665.
B. Golinelli-Pimpaneau / Journal of Immunological Methods 269 (2002) 157–171 Wedemayer, G.J., Wang, L.H., Patten, P.A., Schultz, P.G., Stevens, R.C., 1997b. Crystal structures of the free and liganded form of an esterolytic catalytic antibody. J. Mol. Biol. 268, 390. Yin, J., Mundorff, E.C., Yang, P.L., Wendt, K.U., Hanway, D., Stevens, R.C., Schultz, P.G., 2001. A comparative analysis of
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the immunological evolution of antibody 28B4. Biochemistry 40, 10764. Zhou, G.W., Guo, J., Huang, W., Fletterick, R.J., Scanlan, T.S., 1994. Crystal structure of a catalytic antibody with a serine protease active site. Science 265, 1059.
Structural diversity of antibody catalysts Be´atrice Golinelli-Pimpaneau * Laboratoire d’Enzymologie et Biochimie Structurales, CNRS Baˆt. 34, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France Received 19 December 2001; accepted 2 January 2002
Abstract The structural diversity of the immune response may be considerably restricted by the structure of the hapten used to elicit catalytic antibodies. The ligand-binding mode and the shapes of the binding pockets of hydrolytic antibodies induced to different transition-state analogs that contain an unsubstituted arylphosphonate group are very similar. Moreover, antibodies elicited against a single transition state analog evolve from a single germline gene or different precursors, depending on the nature of the hapten. Germline antibodies seem to adopt multiple conformations with antigen binding, together with somatic mutation stabilizing the conformation with optimum complementarity to antigen. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Catalytic antibodies; X-ray structure; Transition-state analog; Catalytic mechanism; Structural diversity; Affinity maturation
1. Introduction Several strategies are now available to generate catalytic antibodies that can accelerate a wide range of chemical reactions. The most frequently used method relies on antibodies elicited against a hapten which mimics the transition state of a given reaction (Schultz and Lerner, 1995; Blackburn et al., 1998). Many structures of catalytic antibodies generated by this method validate this strategy and show that these antibodies catalyze their reaction by binding and stabilizing the transition state of the reaction through electrostatic, van der Waals’ and hydrogen bonding
Abbreviations: Fab, fragment antigen binding; CDR, complementarity-determining region; VL, variable light chain; VH, variable heavy chain. * Tel.: +33-1-6982-4235; fax: +33-1-6982-3129. E-mail address: [email protected] (B. Golinelli-Pimpaneau).
interactions (Mac Beath and Hilvert, 1996; Charbonnier et al., 1997a; Wade and Scanlan, 1997; Hilvert et al., 1998; Golinelli-Pimpaneau, 2000; Tantillo and Houk, 2001). However, it is only by chance that the antibody catalysts possess a chemically active residue, which explains their low catalytic efficiency. In order to elaborate new, more efficient catalytic antibodies, some questions arise. Is the transition-state analog strategy really suitable for selecting a nucleophilic or base-catalyzed mechanism? Is the low frequency of this event linked to a poor diversity of the immune response when it is probed to elicit antibody catalysts? Are there other immunization approaches more appropriate to generate functional residues? To answer these questions, the structural diversity of the immune response is addressed at different levels in this article. The X-ray structures of antibodies generated against different phosphonate transition-state analogs and catalyzing different ester
0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 ( 0 2 ) 0 0 2 4 0 - 5
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hydrolysis reactions are first compared to see how the structure of the hapten dictates the shape of the binding pocket. The diversity of the immune response is then studied through the comparison of structurally characterized antibodies, elicited against a single transition-state analog (Charbonnier et al., 1997b; Buchbinder et al., 1998; Gigant et al., 1999). The comparison of the structures of the hapten-bound germline and affinity-matured catalytic antibodies then reveals how the somatic mutations alter the structure of the combining site and affect catalysis (Wedemayer et al., 1997a; Mundorff et al., 2000; Yin et al., 2001). Finally, alternative methods to the transition-state analog approach appear to lead to combining sites that precisely position a crucial catalytic residue while accommodating structurally diverse substrates.
2. The esterolytic antibodies elicited against unsubstituted aryl phosphonate haptens share a common deep Hydrophobic combining site 2.1. General mechanism of hydrolytic antibodies: the transition-state analog approach induces a nucleophile only fortuitously At present, X-ray structures of six families of hydrolytic antibodies have been determined: CNJ206 (Charbonnier et al., 1995), D23 (Charbonnier et al., 1997b), 17E8 (Zhou et al., 1994), 48G7 (Wedemayer et al., 1997a,b), 43C9 (Thayer et al., 1999), 6D9 (Kristensen et al., 1998). All these antibodies were elicited against an aryl phosphonate or phosphonamidate transition-state analog and catalyze the hydrolysis of arylesters (Scheme 1). First, compar-
Scheme 1. Phosphonate or phosphonamidate haptens used to generate the structurally characterized hydrolytic antibodies and sulfide oxidase antibody 28B4 and the corresponding substrates.
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ison of the X-ray structures of the antibody– hapten complex of CNJ206 with that of 17E8 (Charbonnier et al., 1995), then with that of 48G7 (Mac Beath and Hilvert, 1996) and D23 (Charbonnier et al., 1997a) has revealed that a common structural motif has been induced (Fig. 1A) and that the antibodies present a similar binding surface (Fig. 2A – D). In this ‘canonical’-binding mode, the aryl end of the hapten is buried deep in the binding pocket. The phosphonate moiety contributes to the major part of the binding energy in the antibody– hapten interaction. Residues of the combining sites, forming a recurring motif, donate hydrogen bonds to the negatively charged oxygens of the phosphonate group and are believed to stabilize the oxyanion intermediate in the ester hydrolysis (Table 1). The rate enhancement (kcat/knon = 103 – 105) roughly parallels the number of hydrogen bonds to the phosphonate and reflects what can be achieved with an oxyanion hole mechanism alone. The low efficiency is also associated with flexibility in the active site for 48G7 germline (k cat /k non = 10 2 ) and perhaps for CNJ206 (kcat/knon = 103) although the movement of the CDR H3 loop that occurs upon hapten binding may result from crystallographic contacts (GolinelliPimpaneau et al., 1994; Charbonnier et al., 1995). In the D23 family, a ligand-induced conformational change leads to a 30– 170-fold increase in the affinity towards the transition-state analog and hence to higher catalytic rates (Lindner et al., 1999). But in D23, a preequilibrium between two conformations of the antibody’s active site exists and the rate of catalysis is limited by the substrate-induced isomerization of the less active conformer to the more active one. 2.2. The role of the residues forming the oxyanion hole has been addressed by site-directed mutagenesis The effect of mutations in the residues that bind the phosphonate part of the hapten (Table 1) has been tested both on the activity and on ligand binding to understand whether these residues have a catalytic and/or a structural role. The question has been asked whether His H35, which forms part of the combining site in most catalytic antibodies (Fig. 1A,B, Table 1), could play the role of a general base or a nucleophile in catalysis. Indeed, mutagenesis studies of 48G7 and 17E8 indicate that His H35 clearly plays a significant role in catalysis: The HisH35-Glu mutant of 48G7 is
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60 times less active that the native antibody (Patten et al., 1996) and the HisH35-Ala mutation abolishes catalysis of 17E8 (Baca et al., 1997). However, the HisH35-Gln mutation has little effect on catalysis for 48G7 and decreases kcat/Km of 17E8 only 13-fold. This suggests that the catalytic role of His H35 is simply to stabilize the negatively charged transition state. For 43C9, the substitution of His H35 with either Asn or Phe results in a loss of catalytic activity but also in decreased hapten binding (Stewart et al., 1994), which argues for a structural role for His H35 in this case. The X-ray structures have shown that the nonprotonated nitrogen Ny1 of His H35 is involved in a conserved hydrogen bond with Trp H47 and therefore it is the protonated hydrogen of His H35 Nq2 that points towards the hapten. Hence, it is more likely that His H35 is a simple hydrogen bond donor that stabilizes the oxyanion of the transition state rather than a base or a nucleophile, which necessitates breaking the H-bond and flipping the imidazole side chain. Three hydrolytic antibodies 17E8, 43C9 and 48G7 contain arginine at position L96 which hydrogen bonds with one oxygen of the phosphonate hapten (Table 1). Site-directed mutagenesis studies have revealed that this residue is important for catalysis; the activity of a variant of 17E8 that contains the ArgL96-Trp substitution is reduced by a factor of 20, even though the hapten is bound 5-fold tighter (Baca et al., 1997). The ArgL96-Gln mutant of 43C9 is not catalytic and shows a 20-fold decrease in hapten binding while binding of products that do not incorporate the phosphonamidate functionality is conserved (Roberts et al., 1994). However, the ArgL96Gln mutation in 48G7 leads to only a 3-fold drop in kcat/Km (Patten et al., 1996). Nevertheless, all the results are consistent with the idea that when an arginine is present at position L96, it stabilizes the anionic tetrahedral transition state by electrostatic interactions. It has been suggested that ArgL96 could also stabilize the charge that develops on the leaving aryl group in 17E8 (Baca et al., 1997). From mutagenesis studies on tyrosine residues whose side-chain hydrogens bind to the phosphonate part of the hapten, it is not clear whether these residues play an essential role in the catalytic mechanism. The mutation of Tyr-H33 to Phe in 48G7
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resulted in only a 5-fold reduction in kcat/Km (Patten et al., 1996) and the TyrH95-Phe mutant of 43C9 possessed appreciable catalytic activity (Stewart et al., 1994). The role of the tyrosines may be to assist in substrate orientation within the active site. In the first structurally characterized hydrolytic antibody, 17E8, the nucleophilic role for Ser H95 that was originally proposed (Zhou et al., 1994) was abandoned when it was shown that the SerH95-Ala mutant is fully active (Baca et al., 1997). Moreover, antibody 7G11 that was obtained during the same immunization as 17E8 (see below), in which the Ser H95 is substituted for Ala, has a similar catalytic activity as 17E8 (Buchbinder et al., 1998). In conclusion, all the site-directed mutagenesis experiments have confirmed the general mechanism of catalysis inferred from the crystallographic studies; it consists of the nucleophilic attack of the ester bond by a water molecule or a hydroxide ion and the stabilization of the oxyanion intermediate by residues of the active site of the antibody. 2.3. The shape of the binding pocket depends on the nature of residue H100c of CDR H3 The antigen-binding sites of antibodies are constructed from six loops (CDRs) between strands of the
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h-sheets in the variable domains of the light (VL) and heavy chains (VH). CDR H3 is much more variable in length, sequence and conformation than the other antigen-binding loops because it falls in the region of the V – D – J join in the assembly of the immunoglobulin heavy chain domain. Several mechanisms contribute to generate its diversity, such as selection of VH, D and J gene segments and alternative splicing patterns. CDR H3 is located at the center of the antibody-combining site and makes numerous contacts with the antigen (Fig. 2). Its C-terminal region significantly influences the shape of the bottom of the binding pocket. The conformational flexibility of the H3 loop makes H3 particularly sensitive to its environment, and thus prediction of the H3 structure extremely difficult (Morea et al., 1998). The presence of a small residue at position H100c of CDR H3 allows the hapten to be deeply buried in a hydrophobic pocket constructed essentially from framework residues (Figs. 1A and 2A – C) (GolinelliPimpaneau, 2000). The p-nitrophenyl groups of the haptens occupy the bottom of the cleft. When a bulky residue (Phe, Met) occupies position H100c, the H3 loop has the region proximal to the framework in the common h-bulge, or kinked conformation at position H101. This bulged conformation is most often stabilized by an ionic interaction between the side chains of
Fig. 1. The atom-coloring scheme for the ligand is as follows: carbons in a color close to that of the antibody (Fig. 1) or yellow (Fig. 2), oxygen in red, nitrogen in blue, phosphorus in magenta, fluoride in violet, chloride in maroon. The structures are that of the Fab – hapten complexes except for 43C9 which is a complex of the Fab – product complex. Comparison of the combining site residues of hydrolytic antibodies. The active site residues are a different color for different Fabs in the same figure. The a carbons of residues of the framework of the Fabs (L1 – L25, L33 – L49, L53 – L90, L97 – L107, H1 – H25, H36 – H51, H57 – H93, H103 – H111) are superimposed. (A – C) are presented in the same orientation. (A) Comparison of the combining site residues of hydrolytic antibodies. Fabs 48G7 germline (yellow), CNJ206 (blue; hapten in ˚ (48G7 cyan), D23 (red; hapten in pink) have been superimposed on 17E8 (green). The root mean squared deviations from 17E8 are 1.31 A ˚ (CNJ206), 1.53 A ˚ (D23). The aryl substituent of the haptens maximizes hydrophobic contacts with the antibody at the germline), 1.42 A hydrophobic bottom of the cavity, which is made of conserved residues of the framework (Trp H103, Phe L98, Trp H47, Val/Ile H37). This leads to a common shape for the bottom of the combining sites. The more variable residues in position L34, L36, L89, H35 and L91 form the walls of the cavity. (B) Comparison of the combining sites of hydrolytic antibodies 48G7, 48G7 germline and 43C9. Fabs 48G7 (brown), 48G7 germline ˚ (48G7), 1.31 A ˚ (yellow), 43C9 (gray; product in light gray) have been superimposed on 17E8. The root mean squared deviations are 1.26 A ˚ (43C9). The p-nitroaryl ring of the hapten occupies very different locations in the combining sites of 48G7 germline (48G7 germline), 1.73 A and 48G7 mature, due essentially to a Ser L34-Gly somatic mutation. In 43C9, the two water molecules that are replaced by the phosphonamidate oxygen atoms of the docked hapten in the antibody – hapten model are indicated in cyan. (C) Comparison of the combining sites of hydrolytic antibodies 7C8, 6D9 and of the sulfide oxidase antibody 28B4. Fabs 7C8 (yellow, hapten in salmon), 6D9 (red, hapten in pink), have been superimposed on sulfide oxidase Fab 28B4 (blue, hapten in cyan) which also possesses a bulky residue at position H100c of ˚ (7C8), 1.51 A ˚ (6D9). These antibodies possess a regular bulged conformation of the CDR H3. The root mean squared deviations are 1.54 A CDR H3 C-terminus that results in the same location for residue H100c at the bottom of the binding pocket (Morea et al., 1998), thereby preventing the haptens contacting buried Trp H103. The shapes of the pockets are more diversified and the haptens are bound more shallowly than in the other esterolytic antibodies. The aromatic rings of the acyl side chain are oriented differently in 7C8 and 6D9: towards the exterior of the cavity in 7C8 and into the combining site, for 6D9. The two aromatic rings of 6D9 and the p-nitrophenyl ring of 7C8 are sandwiched at the mouth of the combining site between residues in position H95 and H50 while the phosphonates are highly exposed.
162 B. Golinelli-Pimpaneau / Journal of Immunological Methods 269 (2002) 157–171 Fig. 2. The binding surface of the hydrolytic antibodies and of oxidase antibody 28B4. The molecular surfaces of the combining sites, in pink for the light chain and light yellow for the heavy chain, were obtained using the program GRASP. They are shown in a similar orientation with residues H35, Trp H47, Trp H103 and the H3 loop (residue H92 – H103, shown as a worm) indicated in white. Residue H100c is cyan. For 28G7 and 28B4, the position of the hapten in the germline – hapten complex is orange. Antibodies 6D9, 7C8, 28B4, 48G7 and 48G7 germline possess a bulge at residue H101, which positions H100c at the same location in the combining site. (A) 17E8, (B) CNJ206, (C) 48G7, (D) D23, (E) 43C9. The p-nitrophenyl of the product of antibody 43C9 lies at the surface of the combining site compared to the same part of the haptens of the other hydrolytic antibodies. If one assumes that the p-nitrophenyl of the phosphonate hapten of 43C9 occupies the same position as the p-nitrophenyl of the product (this is the case in antibody D23), this indicates that the hapten of 43C9 is not much buried in the hydrophobic pocket. Phe in position H100c partially hinders contact with Trp H103 although Phe H100c of 43C9 is not superimposable to Phe H100c of antibodies with the bulged conformation of CDR H3. (F) 7C8, (G) 6D9, (H) 28b4.
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Fig. 2 (continued ).
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Table 1 Sequence comparison of the active site residues of the structurally characterized hydrolytic antibodies (A) Hydrolytic antibodies with the ‘canonical’-binding pocket and amidase antibody 43C9 Residue
Location
48G7 48G7ger
17E8 29G11
D23 D24 D25
CNJ206
43C9
L32 L34 L36 L49 L89 L91 L96 L98 H33 H35 H37 H47 H50 H93 H95 H96 H97 H100 H100a H100b H100c H101 H103
CDR1 CDR1 FR2 FR2 CDR3 CDR3 CDR3 FR4 CDR1 CDR1 FR2 FR2 FR2 FR3 CDR3 CDR3 CDR3 CDR3 CDR3 CDR3 CDR3 CDR3 FR4
Tyr Gly Ser Leu Tyr Leu Tyr Arg Phe Tyr His Val Trp Arg Ala Tyrb – – – – Tyr Gly Ile Trp
Tyr Gly Tyr His Leu Tyr Arg Phe Ala Val His Val Trp Tyr Lys Ser Gly Tyrb Tyr Ser Arg Ser Tyr Asn Val Asp Trp
Tyr Asn Asn Ser Leu His Tyr Tyr Val Gly Tyr Phe Trp His Val Leu Val Trp Arg Thr Thr Val Trp Gly Phe Asp Tyr Val Ala Val Met Met Leu Asp Trp
Tyr Ser Leu Tyr Leu Tyr Tyr Phe Gly His Val Trp Tyr Ala Gly Aspb Tyrb Gly Ser Arg Gly Ala Trp
Tyr Ala Tyr Tyr Gln Hisa Arg Phe Asn His Val Trp Met Val Tyr Gly Tyr Gly Asp Arg Phe Ser Trp
(B) Antibodies with a bulky residue at position H100c and a bulged conformation of CDR H3 Residue
Location
7C8
6D9
28B4 28B4ger
L27d L32 L34 L36 L49 L89 L91 L94 L96 L98 H33 H35 H37 H47 H50 H52 H53 H93 H95 H100a H100b H100c
CDR1 CDR1 CDR1 FR2 FR2 CDR3 CDR3 CDR3 CDR3 FR4 CDR1 CDR1 FR2 FR2 FR2 FR2 CDR2 FR3 CDR3 CDR3 CDR3 CDR3
Asn Lys His Tyr Lys His Thr Arg Leu Phe Tyr Asn Leu Trp Trp Tyr Gly Ala Tyra Glu Gly Phe
His Tyr Asp Phe Tyr Phe Gly Val Pro Phe Ala Ser Val Trp Ser Ser Gly Ala Val Trp Tyr Phe
His Tyr Glu Tyr Tyr Phe Gly Val Arg Phe Tyr Asn Ser Ala Val Trp Phe Arg Lys Asn Ala Trp Asp Tyr Ala Met
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Asp H101 and Arg or Lys at position H94 as well as by a hydrogen bond between the carbonyl oxygen of residue H100c and the side chain of Trp H103 (Morea et al., 1998). This positions the bulky H100c residue at the bottom of the combining site, which makes the binding site shallower (Figs. 1C and 2F – H). The hapten is bound high in the binding pocket and the antibodies adopt different cavity shapes. This was also observed for nonhydrolytic catalytic antibodies (Golinelli-Pimpaneau, 2000). However, in D23, the C-terminal region of CDR H3 is unusual in that it continues the antiparallel hsheet structure of the antibody framework despite the presence of a bulky Met at position H100c, Arg at position H94 and Asp at position H101. The occurrence of the unpredictable extended conformation of the H3 loop instead of the bulged one prevents Met H100c occupying the bottom of the binding pocket and allows the formation of the deep ‘canonical’binding pocket (Figs. 1A and 2D). The structural convergence observed towards the ‘canonical’-binding mode may be linked to the ligand-induced conformational change of D23 (see above) (Lindner et al., 1999). Crystallographic studies are necessary to find out if the nonactive conformer possesses the bulged conformation of the H3 loop. The 48G7 germline and the mature antibody have the same conformation as CDR H3. The bulged conformation positions Gly H100c at the bottom of the binding pocket. Nevertheless, the small size of this residue allows the p-nitrophenyl group of the germline precursor of 48G7 to lie in the ‘canonical’ pocket, whereas in mature 48G7 it is rotated to occupy the bottom of a hydrophobic cleft formed between CDRs L1 and H3 (Fig. 2C). 2.4. Why is 43C9 the only hydrolytic antibody that is also able to hydrolyze an aromatic amide? Residues that play the role of a nucleophile or a base in catalysis have rarely been found in antibody-
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combining sites as a result of the chemical diversity of the immune response. An exception is antibody 43C9 which catalyses the hydrolysis not only of esters but also of aromatic amides with an outstanding acceleration of 2.5 105. The combining site of 43C9 possesses a histidine residue in position L91 that presumably plays the role of a nucleophile. Substitution of HisL91 by Gln produced a mutant with no catalytic activity, but its affinity for ligands was nearly the same as that of the wild type (Stewart et al., 1994). This catalytic residue appeared fortuitously during the immunization process rather than the result of an elaborate strategy. A model of the antibody complexed to the transition-state analog has been built from the X-ray structure of the antibody complexed to the p-nitrophenol product (Thayer et al., 1999), assuming that the p-nitrophenyl ring occupies the same position in both cases. This hypothesis was demonstrated in the case of antibody D23 for which X-ray structures of both the antibody – hapten complex and the antibody– product complex have been determined (Gigant et al., 1997). The model of 43C9 complexed to the transition-state analog indicates that the oxyanion would be stabilized by H-bonds with His H35 and Arg L96 and that the nucleophilic attack of His L91 would be assisted by tyrosines L36 and H95 through a hydrogen bonding network. No residue would play the role of a general acid that protonates the leaving group. The hydrolysis of the acylenzyme intermediate by a hydroxide ion would limit the rate of the reaction. The crystal structure of antibody 43C9 complexed to the product has been compared to that of the other hydrolytic antibodies (Thayer et al., 1999; Tantillo and Houk, 2001). It shows that, instead of binding in the specific ‘canonical’ hydrophobic pocket, the pnitrophenol lies flat at the mouth of the combining site pocket between CDR H3 and L1 (Figs. 1B and 2E) (Thayer et al., 1999). The absence of a distinct deep pnitrophenol-binding pocket is correlated with the presence of Phe in position H100c of CDR H3 although
Notes to Table 1: The three residues preceding H101 are denoted H100a, H100b, H100c and the H3 supervariable loop was aligned according to Morea et al. (1998). This table can be compared to Table 2 of Golinelli-Pimpaneau (2000) which concerns nonhydrolytic antibodies. Common residues forming the combining site are indicated in bold in the residue column. Residues that hydrogen bind to the phosphonate oxygens and are capable of stabilizing the oxyanion intermediate in ester hydrolysis are indicated in bold. a Nucleophile. b H-bond occurring through the main chain NH group.
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Phe H100c is not located at the bottom of the binding pocket but lies much higher than expected, as the Cterminal region of CDR H3 does not adopt the bulged conformation (Table 1, Fig. 2E). This variation from the ‘canonical’-binding mode seems to be linked to the structure of the hapten used to generate 43C9 which contains two different hydrophobic groups (Scheme 1). The use of well-defined, distinct combining sites to accommodate these two groups would position the cleavable bond precisely near His L91. This is not the case for the other hydrolytic antibodies where the aryl group is buried in the active site, whereas the second lateral chain is linear, flexible or short and is exposed to the solvent (Fig. 2). It has been proposed that the combination of a nucleophile and of a combining site that immobilizes the two ends of the substrate but does not bury deeply the p-nitrophenyl group of the hapten confers on 43C9 its capacity to hydrolyze amides (Thayer et al., 1999). 2.5. Use of a structurally different hapten in the 6D9 family leads to more diversified binding pockets Antibodies 6D9 and 7C8 were raised against a single chloramphenicol phosphonate which also contains two aromatic groups (Scheme 1). They are the more efficient antibodies of the family that catalyze the hydrolysis of the corresponding chloramphenicol ester. Their X-ray structures have shown that these antibodies do not bind the hapten in the commonly found hydrophobic pocket, as do the other hydrolytic antibodies (Kristensen et al., 1998; Gigant et al., 1999) (Figs. 1C and 2F,G). While the use of similar haptens containing both a phosphonate and a single aryl group restricts the shape of the combining site pocket as do other similar hydrophobic hapten structures (Fig. 1A) (Golinelli-Pimpaneau, 2000), the use of a hapten that contains two different hydrophobic groups expands the structural diversity of the immune response, as shown previously for 43C9. In contrast to the other esterolytic antibodies, in the 6D9 family, position H100c is occupied by a phenylalanine residue that prevents the hapten burying itself deep into the combining site (Table 1, Figs. 1C and 2F,G). In addition, the H35 residue that makes part of the combining site of the esterolytic antibodies described previously, is not the commonly encountered histidine but either a serine (6D9) or an aspar-
agine (7C8) (Table 1). These two sequence differences have also been found in 28B4, which has also been induced by a hapten containing both a phosphonate and a p-nitroaryl moiety (Scheme 1). Indeed, the hapten-binding modes of these three antibodies show a great similarity (Fig. 1C).
3. Structural convergence or divergence inside a family of antibodies? Comparison of the structures of different antibodies elicited against a single hapten during a single immunization protocol (defined as belonging to a ‘family of antibodies’) has also given insight into the diversity of the immune response. 3.1. Diverse structural solutions in the 6D9 family Hydrolytic antibodies from the 6D9 family usually possess a high sequence identity and therefore probably a high structural homology (Miyashita et al., 1994; Fujii et al., 1995). However, antibody 7C8 differs from the other antibodies. It presents only 50% sequence identity to 6D9 (Table 1) and it uses a very different hapten-binding mode with the aromatic ring of the acyl side chain oriented towards the exterior of the cavity in 7C8 (Figs. 1C and 2F) while, for 6D9, it is oriented into the combining site (Fig. 2G). Moreover, the two phosphonate groups occupy very different positions at the surface of the antibody combining sites. The hapten used to generate 6D9 and 7C8 appears to bear multiple epitopes, which explains the fact that the germline precursors of 6D9 and 7C8 are very different. Consequently, the specificities, the catalytic residues, the rate-limiting steps and the catalytic mechanisms are different in the two antibodies. 3.2. Imprecise positioning of the catalytic residues in the 17E8 family The structures of antibodies 17E8 and 29G11 that have been induced to the same norleucine-phosphonate transition-state analog (Scheme 1) and that possess the highest level of catalytic activity towards the norleucine phenyl ester have been compared (Buchbinder et al., 1998). Despite their great sequence similarity (eight substitutions in the heavy chain
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variable region, including four in the hapten-combining site; Table 1), the same length of CDR H3, the presence of the same catalytic groups in the combining site and the similar location and orientation of their hapten, these antibodies have their catalytic ˚ . The groups positioned with differences of 1– 2 A different positioning of residues in the hapten-binding pocket comes from a very different association of the VH and VL domains in the two antibodies. The lack of subtle structure conservation, inside a family of antibodies, contrasts with natural enzymes that have evolved active sites with precise positioning of catalytic and substrate-binding residues. It has been proposed that the generally lower efficiencies of catalytic antibodies compared to enzymes may reflect a relatively inaccurate alignment of the bond-making and bond-breaking apparatus (Buchbinder et al., 1998). A more precise arrangement of the catalytic residues in antibody-combining sites is obtained by the ‘bait and switch’ and ‘reactive immunization’ strategies (see below). 3.3. A same hapten-binding mode despite different conformations of CDR H3 in the D23 family The structures of three antibodies D23, D24 and D25, which are the most efficient antibodies of the family to catalyze the hydrolysis of p-nitrobenzylesters, have been compared (Charbonnier et al., 1997b). These antibodies were identified by screening the entire hybridoma repertoire for catalysis (Tawfik et al., 1993). The amino acid sequences of the variable domains of D24 and D25 differ from D23 in 16 and 14 positions, respectively (Table 1). These differences come from somatic mutations from the same germline precursor (Kim et al., 1997). The sequence of CDR H3 of D24 possesses four differences and one insertion compared to D23 resulting from a difference in the recombination at the junction of the D and J segments. This leads to a very different conformation of this loop for the two antibodies. Although the antibodies have different conformations of the H3 loop, they bind their hapten in a similar mode with Tyr H100a and residue L34 hydrogen binding to one of the phosphonate oxygens and Trp H95 to the other. In the structure of D23 complexed to an amide inhibitor, the presence of a single hydrogen bond between the carbonyl oxygen of the substrate analog
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and Tyr H100a has identified Tyr H100a and residue L34 as the oxyanion hole (Gigant et al., 1997). As the H-bond to one of the phosphonate oxygens that occurs with Asn L34 in D23 is replaced by a water mediated H-bond with Ser L34 in D25, the catalytic motif that constitutes the oxyanion hole is similar.
4. How does the mature antibody structurally differ from its germline precursor? 4.1. Conformational flexibility of the active site of the germline antibody During the process of affinity maturation, somatic mutations are introduced that increase the binding affinity of the antibody to the antigen. To determine the effects of affinity maturation on binding and catalysis, the structures of the hapten-bound and free germline precursor and affinity-matured antibodies have been determined for three catalytic antibodies: esterolytic antibody 48G7 (Wedemayer et al., 1997a), oxy Cope antibody AZ-28 (Mundorff et al., 2000) and sulfide oxidase antibody 28B4 (Yin et al., 2001). In all cases, the germline antibodies show significant structural changes upon hapten binding, in particular a significant rearrangement of CDR H3 that improves packing interactions with the hapten. In contrast, in the affinity-matured antibody, somatic mutations have led to a lock-and-key binding mode in which the antibody-combining site is pre-organized for hapten binding. It has been proposed that the conformational flexibility seen in the germline antibodies significantly enhances the binding potential of the primary antibody repertoire (Wedemayer et al., 1997a). In contrast, the structure of the unliganded germline precursor to Diels Alderase antibody 39-A11 is very similar to that of the 39-A11 hapten complex (Romesberg et al., 1998). Hence, neither somatic mutation nor ligand binding seems to result in substantial conformational changes in the active site for antibody 39-A11. This is not unexpected as the germline precursor differs from the mature antibody by only two somatic mutations. One of them, SerL91Val, increases the affinity of the antibody to the hapten by eliminating a cavity near the combining site, thereby improving the packing between the hapten and the antibody. Despite the small sequence differ-
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ences between the germline and mature antibodies, the germline antibody possesses a polyspecific-combining site, unlike the mature antibody. It was suggested that the selection of polyspecific germline genes could be a general mechanism for rapid generation of antibodies of moderate to high affinity for a broad range of antigens that could expand the binding potential associated with the structural diversity of the primary antibody repertoire (Romesberg et al., 1998). 4.2. Have the germline and mature antibodies the same hapten-binding mode? In antibody AZ-28, the hapten in the germline complex is bound in a conformation similar to that seen in the affinity-matured antibody complex. Five out of the six somatic mutations appear to increase the binding affinity of the mature antibody predominantly through secondary sphere hydrogen-bonding interactions while the SerL34-Asn mutation, localized in the combining site, reduced the catalytic rate by fixing the transition-state analog in an unfavorable conformation. Here, the decreased degree of flexibility in the CDR H3 in the mature antibody was unresponsive to catalysis. Mutations introduced into the germline antibody during the affinity maturation process may also lead to differences in the hapten-binding mode. In contrast to antibody AZ-28, in 48G7 and 28B4, the haptens are bound in different conformations in the germline and mature antibody (Wedemayer et al., 1997a; Yin et al., 2001) (Figs. 1B and 2C,H). Antibodies 48G7 and 28B4 have been induced against haptens containing both a p-nitrophenyl ring and a phosphonate part. The germline antibodies bind the p-nitrophenyl ring of haptens in an orientation significantly different from that seen in the affinity-matured antibodies, whereas the phosphonate moiety is bound in a similar mode. It is very surprising to see that for 48G7germline, the p-nitrophenyl part of hapten is fitted in the commonly found orientation in the hydrophobic pocket, while for the affinity-matured antibody the p-nitrophenyl ring occupies a different position in the combining site, lying flat, nearer to the mouth of the combining site (Wedemayer et al., 1997a) (Figs. 1B and 2C). A single somatic mutation near the combining site, Ser L34-Gly, is essentially responsible for this huge effect. The other eight somatically mutated residues do not directly
contact the bound hapten but likely play an important role in organizing the conformation of residues that make up the active site and in limiting flexibility in the germline antibody. In this way, an additional H-bond occurs in the mature antibody complex relative to the germline complex between residue Tyr H33 and one of the phosphonate oxygens. In 28B4, three out of the nine replacement mutations occur at the hapten-binding site (Table 1) but the new orientation of the bound hapten is caused mainly by one of the mutations, AspH95-Trp in CDR H3. The presence of this mutation is a prerequisite for other somatic mutations to enhance the binding affinity for the hapten like the AsnH53-Lys mutation that introduces two new hydrogen bonds between the antibody and the phosphonate moiety. Affinity maturation allows antibody 48G7 to avoid burying the hapten deep into the hydrophobic-combining site pocket thereby enhancing the catalytic activity. On the contrary, the p-nitrophenyl group of the hapten is buried further in mature 28B4 than in the germline antibody. As the phosphonate group of the haptens is bound in a similar fashion in the germline and affinitymatured antibodies, the catalytic residues are conserved but the H-bonds and the complementarity of the hapten to the antibody are optimized during the evolution of the germline antibody. It is likely that if the antibodies had been screened directly for catalytic efficiency, there would be more evolution of the catalytic residues. 4.3. How is the transition-state analog strategy translated into the antibodies? The increase of affinity for the transition-state analog that comes with the affinity maturation most often leads to an increase of the catalytic activity. This confirms that the major part of catalysis comes from the complementarity to the transition state. However, in a few cases, the catalytic constants and the hapten affinities do not agree with the transition-state theory. Compared to 17E8, the affinity of 29G11 for the transition-state analog is actually 19 times better while the activity is four times less. Moreover, the improvement of the affinity to the transition-state analog did not lead to mutants with a better catalytic efficiency (Baca et al., 1997).
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The germline precursor to AZ-28 catalyzes the reaction with a 35-fold higher rate despite a 40-fold lower binding affinity for the transition-state analog because affinity maturation fixes the transition state in a catalytically unfavorable conformation (Mundorff et al., 2000). As the increase of affinity to the transitionstate analog does not always translate into higher efficiency and as the transition-state analog approach is not appropriate to program a chemically active residue in the combining site, other strategies have been thought to lead more active antibodies.
5. Strategies different from the transition-state analog approach lead to broad substrate tolerance The ‘bait and switch’ strategy uses a charged hapten to induce a complementary charged residue at a predetermined location with respect to the hapten. The crystallographic structure of an antibody, in which the general base carboxylate residue was induced by this strategy, has validated this approach (Golinelli-Pimpaneau et al., 2000). The ‘reactive immunization’ method aims at generating a residue that accelerates the reaction through covalent catalysis. It involves the formation of a stable complex between the hapten and the functional residue of the antibody, that can be easily monitored to allow the selection of the catalytic clones (Wagner et al., 1995; Barbas et al., 1997; Karlstrom et al., 2000). The appearance of the covalent bond during the immunization procedure hinders further evolution of the immune response. These two methods result in broad substrate tolerance and lead to antibodies analogous to the polyspecific germline antibodies. It remains to be studied if the broad substrate tolerance is linked to a high conformational flexibility.
6. Conclusion The broad diversity initially present in the primary repertoire is reduced to a small set of solutions when the immune system is challenged to bind haptens that contain an aryl group. Indeed, the Xray structures of antibodies selected for tight binding to arylphosphonate transition-state analogs have revealed that the majority share a common deep
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hydrophobic-binding pocket. Structural convergence is even evident in antibodies raised against unrelated hydrophobic haptens (Golinelli-Pimpaneau, 2000). There is a strong selective pressure to bind the hydrophobic portion of the hapten that could be more easily removed if the antibodies were selected directly for catalysis rather than for hapten binding (Tawfik et al., 1993). The transition-state analog approach generates an oxyanion hole constituted from H-bond donating residues but does not program a nucleophile or a general base that accelerates the reaction. Comparison of antibodies resulting from a single immunization has shown that the transition-state analog approach actually can lead not only to an imprecise arrangement of the catalytic residues inside the binding pocket but also to completely different locations of the combining site residues themselves. The inadequacy of the transition-state analog approach in programming a precisely positioned functional residue gives little hope for obtaining a sophisticated antibody that operates, for example, via a catalytic dyad by this strategy. The diversity of the primary immune response appears to be significantly enhanced when antibodies adopt more than one binding site conformation. The structural changes that occur on binding of hapten to the germline antibody are further optimized by affinity maturation which stabilizes one of the conformers and decreases the structural flexibility intrinsic to the germline antibody to enhance the antibody –hapten complementarity. Hence, the hapten binds the mature antibody in a ‘lock-and-key’ fashion. This contrasts to enzymatic catalysis where conformational flexibility is expected to improve catalytic activity. However, an example where conformational restriction of the antibody limits its activity has been provided. To mimic the polyspecificity of the germline antibodies and obtain more structurally diversified active sites, more diversified hapten structures should be used. References Baca, M., Scanlan, T.S., Stephenson, R.C., Wells, J.A., 1997. Phage display of a catalytic antibody to optimize affinity for transitionstate analog. Proc. Natl. Acad. Sci. U. S. A. 94, 10063. Barbas III, C.F., Heine, A., Zhong, G., Hoffman, T., Gramatikova,
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