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   containing a positive charge have provided antibody catalysts for
   important reactions such as acyl transfer ([41]7), elimination ([42]9,
   [43]10), and phosphodiester hydrolysis ([44]11). However, in the
   absence of structural data, it is not possible to establish
   unambiguously the nature and identity of the catalytic residue that has
   been induced and the relationship between the location of the haptenic
   charge and the position of the catalytic residue in the antibody
   combining site.
   [45]Figure 1
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   Figure 1

   Scheme of the reaction catalyzed by antibody 4B2. 4B2 catalyzes allylic
   rearrangement of α-cyclopent-1-en-1-yl-p-acetamidophenone 2 to
   α-cyclopentylidien-p-acetamidophenone 4 via the enediol intermediate 3,
   2-[4-(1-carboxy)propylamidobenzylamino]-3,4,5,6-tetrahydropyridinium.
   1a is the hapten used to generate 4B2. The structure that was
   determined is that of the complex of 4B2 with
   2-(4-aminobenzylamino)-3,4,5,6-tetrahydropyridinium 1b. Antibody 5C8
   was generated against hapten 5a, and its x-ray structure was determined
   in the presence of inhibitor 5b ([49]12).

   Indeed, in the few cases in which the use of a hapten containing a
   positively charged moiety successfully induced catalytic antibodies and
   in which the structure of the haptenÐantibody complex was determined,
   there was no negatively charged residue in the active site directly
   facing the positive charge, but stabilization of the haptenic charge
   was mediated mostly by cationй interactions ([50]12Ð[51]14). Herein,
   we report the structure, at 1.87- resolution, of the complex of an
   antibody catalyzing an allylic rearrangement with its cationic hapten.
   We provide direct evidence for an ionic pair interaction between the
   amidinium function of the hapten and a combining site carboxylate,
   which allows the precise positioning of this group, and show that this
   carboxylate is the general base responsible for catalysis.
   [52]Previous Section[53]Next Section

 ¤2¤ Materials and Methods ¤2¤

 ¤3¤ Fab Preparation, Purification, and Crystallization. ¤3¤

   The 4B2 antibody was purified from the ascitic fluid as described
   ([54]15). The Fab was generated by papain digestion of the antibody
   under standard conditions (30 mM Tris, pH 7.4/138 mM NaCl/1.25 mM
   EDTA/1.5 mM 2-mercaptoethanol) by using a 3% (wt/wt) papain-to-antibody
   ratio and a 9-h digestion time. Undigested IgG and Fc fragment were
   removed by DEAE anion exchange chromatography and gel filtration on a
   Sephacryl S100 HR column, and the Fab was purified further by ion
   exchange chromatography on a mono Q FPLC column by a NaCl gradient in
   20 mM ethanolamine buffer at pH 9.3.

   Crystals were grown at 4¡C by using the hanging-drop procedure in wells
   containing 1 ml of 16% (vol/vol) polyethylene glycol 4000, 3% (vol/vol)
   dioxan, 20% (vol/vol) glycerol, 0.2 M ammonium sulfate, 5 mM strontium
   chloride, and 20 mM sodium acetate (final pH 5). Drops consisting of a
   2-μl aliquot of a protein solution with hapten (0.25 mM hapten and 11.6
   mg of Fab per ml in 0.15 M NaCl/0.05% NaN[3]) were mixed with 2 μl of
   the well solution. Despite the simultaneous growth of thin needles and
   polyhedral-shaped crystals, this procedure yielded, in some drops,
   monocrystals of dimensions up to 0.7 × 0.45 × 0.35 mm^3.

 ¤3¤ X-Ray Data Collection and Structure Determination. ¤3¤

   Diffraction data were recorded by using one crystal kept at 4¡C on the
   W32 station of the Laboratoire pour l'Utilisation du Rayonnement
   ElectromagnŽtique (Orsay, France) synchrotron with a MAR Image Plate
   system. Data were processed with denzo and scalepack ([55]16), and
   statistics are shown in Table [56]1. The structure of the 4B2 Fab
   (IgG1, κ) was solved by molecular replacement with the program amore
   ([57]17); the models used were the Fv domain of Fab D23 (PDB code
   [58]1yec) and the CL-CH1 dimer of Fab 36-71 (PDB code [59]6fab). The
   atomic model was refined by alternating cycles of model reconstruction
   with the program o ([60]18) and of refinement with cns ([61]19). The
   final refinement statistics are given in Table [62]1. Fig. [63]2 AÐC
   was drawn with the program o ([64]18).
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   Table 1

   Data collection and refinement statistics for Fab 4B2 complexed with 1b
   [67]Figure 2
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   Figure 2

   (A) Schematic view of the active site of the 4B2-1b complex. The ligand
   is in red; water molecules are indicated as red crosses; Glu L34 is
   indicated in green; the other polar residues are represented in blue.
   The Cαs of residues L32ÐL36, L89ÐL97, H35ÐH37, and H94ÐH99 in
   hypervariable loops L1, L3, H1, and H3 are shown in yellow. The
   aromatic residues (Trp H47, Phe L89, Tyr L96, Tyr L32, Trp H103, and
   Phe L98) that have hydrophobic interactions with the hapten have been
   represented, except Phe L98 and Trp H103 for reasons of clarity.
   Nitrogen Nδ1 of His H35 is involved in a conserved H bond with Nɛ1 of
   Trp H47. His H35 is therefore neutral but cannot play the role of a
   general base, because its protonated nitrogen Nɛ2 points toward the
   inside of the cavity. (B) Hydrogen bonding network established with
   catalytic residue Glu L34. Hydrogen bonds are shown as dashed lines. A
   Fobs-Fcalc electron density map calculated without the amidinium ligand
   is superimposed on the structure. The map is contoured at the level of
   two standard deviations. One of the oxygens of Glu L34 is hydrogen
   bonded to the protonated nitrogen Nɛ2 of His L36. This tautomeric form
   of His L36 is stabilized by an additional hydrogen bond between its Nδ1
   and the NHɛ1 of Trp H103. Residue His L36 is therefore neutral but
   cannot play the role of a general base, because its unprotonated
   nitrogen Nɛ1 points away from the substrate. (C) Comparison of the
   environment of the charge of the hapten in antibodies 5C8 (in blue) and
   4B2 (in yellow). 5C8 catalyzes the disfavored cyclization of an
   epoxyalcohol ([71]12). The α-carbons of residues of the combining site
   (L32ÐL38, L43ÐL50, L86ÐL93, L96, L98, H32ÐH39, H91ÐH95, and H102ÐH104)
   of 5C8 have been superimposed on those of 4B2 (the rms deviation is