Volume 2, Issue 3, Pages 175-183 (March 1994) Crystal structure of a catalytic antibody Fab with esterase- like activity  B Golinelli-Pimpaneau, B Gigant, T Bizebard, J Navaza, P Saludjian, R Zemel, DS Tawfik, Z Eshhar, BS Green, M Knossow  Structure  Volume 2, Issue 3, Pages 175-183 (March 1994) DOI: 10.1016/S0969-2126(00)00019-8

Figure 1 Diagrams of the hydrolysis reaction catalyzed by CNJ206 and of the compounds used in this study. 1 is the substrate (a p- nitrophenyl ester); 2 is the transition state analog (TSA) hapten used to elicit CNJ206; 3 is a short TSA used to select catalytic antibodies; 4 and 5 were used in binding studies with CNJ206. Structure 1994 2, 175-183DOI: (10.1016/S0969-2126(00)00019-8)

Figure 2 Stereoview of the electron density map. The region of the combining site cavity is displayed in this 2F o–F cmap contoured at 1.0 standard deviation. Key residues lining the groove and discussed in the text are labeled. [The model used to calculate this map (and Figure 3, Figure 4 and Figure 5) was refined in a unit cell with a halved acell edge. The molecular model of CNJ206 Fab is essentially unaffected when all the reflections in the complete unit cell are included in the refinement (see Materials and methods).] Structure 1994 2, 175-183DOI: (10.1016/S0969-2126(00)00019-8)

Figure 3 Stereoview of the combining site. The groove, limited by the framework regions of the heavy chain (on the right) and of the light chain (on the left), is shown in this view looking down the pseudo two-fold axis of the Fv dimer. The groove is also limited by the hypervariable loop L3 (residues L91 to L96) and part of the hypervariable loop H3 (residues H99 to H101). The rest of hypervariable loop H3, of which only residues H102 to H104 are shown here, is located at the top of this view and lies outside the combining site groove. Hydrogen bonds are shown as dotted lines. The remaining complementarity determining loops, L1, L2, H1 and H2 lie outside the groove and are each indicated by one central residue; Ile L29, Ser L52, Phe H29 and Gly H54, respectively. Structure 1994 2, 175-183DOI: (10.1016/S0969-2126(00)00019-8)

Figure 4 Comparison of the combining site cavities in CNJ206 and NQ10/12.5. The heavy chains of the two Fabs have been superposed and the residues lining the combining site cavities are represented here. Atom color coding is used for CNJ206 (carbon, white; nitrogen, blue; oxygen, red), NQ10/12.5 is in blue and phenyloxazolone (the ligand for NQ10/12.5) is in red. Tyr H101 of CNJ206 is seen colliding with phenyloxazolone thereby preventing the formation of the type of deep cavity observed in NQ10/12.5. Structure 1994 2, 175-183DOI: (10.1016/S0969-2126(00)00019-8)

Figure 5 Model of the transition-state analog bound to CNJ206. (a) The same view as in Figure 2, illustrating a model of p-nitrophenyl methylphosphonate (compound 3 of Figure 1) bound to CNJ206. The inhibitor 3 was modeled using SYBYL (Molecular Modeling Software, Tripos Associates, St Louis, MO) and structural data [43] and adjusted into the binding site of CNJ206 using FRODO [44]. Atomic positions were then subjected to energy refinement with X-PLOR [41]. Atoms further than 9 å from the hapten were kept fixed, while soft harmonic constraints were applied to atoms in a shell between 7 and 9 å from the hapten. For residues within 7 å of the hapten, softer constraints were applied to main-chain atoms, while side chains were left unconstrained. Polar or charged residues lining the cavity are labeled. The intramolecular salt link between Arg L46 and Asp L55, which stabilizes the conformation of the arginine is shown. The orientation presented allows hydrogen bonds (dotted lines) to be made both to the nitro group and to the methyl phosphonate (atom colours as described for Figure 4). (b)A space-filling representation of the same model. The phosphorous atom is shown here in green with the phenyl ring and methyl group of compound 3 in yellow. In this orientation, compound 3 buries 242 å 2of surface, which is 71 % of its total accessible surface area (calculated using a 1.4 å radius probe). Structure 1994 2, 175-183DOI: (10.1016/S0969-2126(00)00019-8)