Proc. Natl. Acad. Sci. USA Vol. 92, pp. 11721-11725, December 1995 Biochemistry Crystal structure of the complex of a catalytic antibody Fab fragment with a transition state analog: Structural similarities in esterase-like catalytic antibodies (phosphonate hapten/ester hydrolysis/oxyanion hole/hapten-induced conformational change) JEAN-BAPTISTE CHARBONNIER*, ELIZABETH CARPENTER*t, BENOIT GIGANT*, BEIATRICE GOLINELLI-PIMPANEAU*, ZELIG ESHHARt, BERNARD S. GREEN§, AND MARCEL KNOSSOW*¶ *Laboratoire de Biologie Structurale, UMR 9920, Centre National de la Recherche Scientifique, Universite Paris Sud, BAt. 34, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France; tDepartment of Chemical Immunology, Weizmann Institute, Rehovot, Israel; and §Department of Pharmaceutical Chemistry, The Hebrew University School of Pharmacy, Jerusalem 91120, Israel Communicated by Don C. Wiley, Harvard University, Cambridge, MA, July 24, 1995 ABSTRACT The x-ray structure of the complex of a tween the TSA and the structure of the catalytic antibody catalytic antibody Fab fragment with a phosphonate transi- raised against it (10). To this end, we have determined the tion-state analog has been determined. The antibody structure of the Fab fragment of an esterase-like catalytic (CNJ206) catalyzes the hydrolysis ofp-nitrophenyl esters with antibody, CNJ206, with bound hapten; the conformation of the significant rate enhancement and substrate specificity. Com- combining site differs from the one previously found in parison of this structure with that of the uncomplexed Fab uncomplexed CNJ206 (11). fragment suggests hapten-induced conformational changes: Antibody CNJ206 catalyzes the hydrolysis ofp-nitrophenyl the shape of the combining site changes from a shallow groove ester (compound 1 in Fig. 1) wit-h significant rate enhancement in the uncomplexed Fab to a deep pocket where the hapten is (kcat/kuncat = 1600) and substrate specificity and displays buried. Three hydrogen-bond donors appear to stabilize the multiple cycles (12). CNJ206 has been obtained by immuni- charged phosphonate group of the hapten: two NH groups of zation with a keyhole limpet hemocyanin conjugate of phos- the heavy (H) chain complementarity-determining region 3 phonate hapten 2 in Fig. 1 and has been selected for its high (H3 CDR) polypeptide chain and the side-chain of histidine- affinity for the short TSA 3 in Fig. 1 (13). We report here the H35 in the H chain (His-H35) in the Hi CDR. The combining structure of CNJ206 Fab fragment complexed with 3.11 We site shows striking structural similarities to that of antibody describe the amino acids present in the combining site, analyze 17E8, which also has esterase activity. Both catalytic antibody their relationship with the inducing hapten, and discuss their ("abzyme") structures suggest that oxyanion stabilization possible role in catalysis; we then compare this structure to that plays a significant role in their rate acceleration. Additional of Fab 17E8, the only other catalytic antibody Fab with catalytic groups that improve efficiency are not necessarily esterase activity whose structure has been solved (14). induced by the eliciting hapten; these groups may occur because of the variability in the combining sites of different monoclonal antibodies that bind to the same hapten. MATERIALS AND METHODS Affinity and Kinetic Measurements. Km and kcat for hydro- The exquisite stereospecificity and high turnover number of lysis of 1 were determined as described (13). Affinity mea- enzymes make them high-profile targets for bioorganic chem- surements were performed on the CNJ206 Fab by an indirect ists, who aim both to understand and mimic these catalysts. ELISA procedure (15). Enzyme catalysis is understood to occur because enzymes Crystallization and Data Collection. The preparation of selectively bind the transition state for a step of a particular monoclonal antibody CNJ206, hapten 3, and the preparation reaction (1, 2). To create binding entities with a catalytic and purification of CNJ206 Fab have been reported (11). The potential, synthetic host molecules have been made (3), and hanging drop procedure was used to grow crystals by vapor enzymes have been altered to change their specificity without diffusion; they were obtained in drops consisting of 2 ,tl of the destroying their catalytic activity (4, 5). Another approach has protein solution and 2 Al of a precipitating solution [8% been to use the remarkable capacity of the immune system, (wt/vol) polyethylene glycol 10,000/17% (vol/vol) glycerol/ which generates antibodies possessing high binding affinity 0.5 mM ZnSO4/1 mM hapten 3 in 0.1 M Tris-HCl, pH and structural specificity towards virtually any molecule, to 8.0/0.05% NaN3]. The initial protein solution was concen- produce catalytic antibodies ("abzymes") (6). trated at 10 mg/ml; the drop was equilibrated against a The majority of known catalytic antibodies have been pro- reservoir solution that consisted of the precipitating solution grammed to catalyze the hydrolysis of esters as well as other without TSA and to which 0.15 M NaCl was added. acyl transfer reactions. They have been raised against phos- Intensity data were collected on one crystal kept at 4°C on phonate haptens, transition state analogs (TSA) that model the the W32 station of the Laboratoire pour l'Utilisation du Rayon- structure of the putative tetrahedral transition states of these nement Electromagnetique (Orsay, France) synchrotron using reactions. The catalytic activity exhibited by these antibodies is a MAR Image Plate system. The crystal belongs to symmetry generally far below that of enzymes. The catalytic proficiency of these antibodies can be improved with phage display Abbreviations: H and L chains, heavy and light chains; C, constant; V, libraries (7) and development of effective screening method- variable; TSA, transition-state analog; CDR, complementarity- ologies (8, 9). For all monoclonal catalytic antibody ap- determining region; H1-H3 CDR, H chain CDRs 1-3. proaches, it is important to understand the relationship be- tPresent address: National Institute for Medical Research, The Ridge- way, Mill Hill, London NW7 1AA, United Kingdom. ITo whom reprint requests should be addressed. The publication costs of this article were defrayed in part by page charge 11 The atomic coordinates have been deposited in the Protein Data payment. This article must therefore be hereby marked "advertisement" in Bank, Chemistry Department, Brookhaven National Laboratory, accordance with 18 U.S.C. §1734 solely to indicate this fact. Upton, NY 11973 (reference IKNO). 11721 Downloaded by guest on October 3, 2021 11722 Biochemistry: Charbonnier et al. Proc. Natl. Acad. Sci. USA 92 (1995) 0 -0@ + HO -* OH.4T'--- + HO0-& 02N H3 2 CH3 1 0 02N J-( CH2)3 COOH FIG. 1. Diagrams of the hydrolysis cat- 23 alyzed by antibody CNJ206 and of the compounds used in this study. 1 is the substrate (p-nitrophenyl acetate); 2 and 3 0 are different analogs used in this study: 2 O2N-15S- CH3 is the TSA hapten used to elicit CNJ206, 02N 3 and 3 is a short TSA used to select this catalytic antibody. The structure pre- sented here is the complex between 3 CNJ206 and 3. of space group P1 with a = 74.1 A, b = 76.9 A, c = 88.3 A, a of compound 1 in Fig. 1 (Km = 80 ± 10 AM; kcat = 0.4 ± 0.05 = 93.80, ,3 = 93.90, and y = 115.60. Intensities were evaluated min-1) are very similar to values obtained previously with the with the program MOSFLM as modified for the image plate (16) Fab and thep-nitrophenyl ester ofN-glutaryl caproate (11, 12); and were processed with programs of the CCP4 suite (17), they were measured at the pH (pH 8) of the crystal growth. Kd giving an overall Rmerge of 11.2%. The data set used for for the p-nitrophenol product was measured at this pH and at structure analysis had 27,401 unique reflections in the 20- to pH 4, well below the pK of p-nitrophenol (pK = 7.15). The 3.2-A resolution range with 96% completion (89% in the 3.3- dissociation constant forp-nitrophenol at pH 4 (Kd = 30 ± 3 to 3.2-A shell; 41% of the reflections in that shell were larger AM) is much lower than at pH 8 (Kd > 3 mM). than 2 SD; Rmerge in that shell was 44%). These data were Conformational Changes in the Fab upon Hapten Binding. measured with a 1.8 redundancy. The structure of the uncomplexed CNJ206 Fab has been solved Structure Determination. The structure was solved by mo- lecular replacement with the program AMoRe (18) and data at 3.0-A resolution and reported (11). The conformation of the in the 10- to 4-A resolution range. The Fv and the constant (C) C domain dimer is essentially unchanged in the complex and domain CL-CH1 dimer of the unliganded CNJ206 Fab (11) uncomplexed structures (rms deviation of Ca positions: 0.4 A; were used separately as models for the corresponding parts of 190 atoms compared), but there is a significant rearrangement the complex. At the end of molecular replacement, the model of the Fv (rms deviation of Co positions: 1.0 A; 208 atoms consisting of three Fvs and three C domain dimers had an R compared). Since individual light (L) and heavy (H) chain factor of 0.32 for data in the 10- to 4-A range. The three Fabs variable (V) domains VL and VH [except the CDR3 of the H in the asymmetric unit are related by two pseudo-twofold chain (H3 CDR)] superimpose very well to the corresponding symmetry operations. domain in uncomplexed CNJ206 (rms deviation of C" posi- The model was refined by using the conjugate-gradient tions 0.36 A and 0.49 A, respectively; 105 atoms compared), the protocol in X-PLOR (19), and progress was judged by decrease observed rearrangement is due to a movement of one of the V of the free R factor (20).