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Burmester, J., Spinelli, S., Pugliese, L., Krebber, A., Honegger, A., Jung, S., Schimmele, B., Cambilleau, C. and Plückthun, A.
- Selection, characterization and X-ray structure of anti-ampicillin single chain Fv fragments from phage-displayed murine antibody libraries
J.Mol.Biol., 309 (2001) 671-685
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- Single-chain Fv (scFv) antibody libraries were constructed from mice immunized with an ampicillin-bovine serum albumin conjugate. Several antibodies with specificity for intact ampicillin were selected by phage display and characterized. The antibody scFv fragment aL2 binds to intact ampicillin and shows no detectable cross-reactivity with hydrolyzed ampicillin. We determined the X-ray structures of two crystal forms of w.t. aL2, which mainly differ in the side-chain conformation of Trp H109 according to a new consensus nomenclature (Honegger & Plückthun, J. Mol. Biol. 2000; Kabat residue number H95) in the extremely short (3 residues) CDR H3 and the presence or absence of a well-resolved molecule of 2-methyl-pentane-2,4-diol (MPD) in the bottom of the binding pocket. Attempts to co-crystallize aL2 with its antigen or to diffuse ampicillin into the wild-type aL2 crystals were unsuccessful, since crystal contacts obstruct the binding pocket. However, a mutant with two point mutations near the N-terminus (Gln H6 replaced by Glu and Ala H10 (Kabat H9) replaced by Gly) crystallized in a form compatible with antigen binding. Although the mutations affect the conformation of framework I, the conformations of the binding pocket of the uncomplexed w.t. aL2 and of the mutant complex were almost identical. The structure explains the specificity of the antibody for intact ampicillin and the degree of cross-reactivity of aL2 with a wide variety of ampicillin analogs. This antibody system will be very useful as a diagnostic reagent for antibiotics use and abuse, as a model for the effect of expression of antibiotic binding molecules in E. coli, and for directed evolution towards high antibiotic resistance.
- Jung, S., Spinelli, S., Schimmele, B., Honegger, A., Pugliese, L., Cambilleau, C. and Plückthun, A.
The importance of framework residues H6, H7 and H10 in antibody heavy chains: experimental evidence for a new structural subclassification of antibody VH domain
- J.Mol.Biol., 309 (2001) 701-716
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- The N-terminal segment (FR-H1) of the heavy chain (VH) of antibodies shows significant conformational variability correlating with the nature of the amino acids H6, H7 and H10 (Kabat H9). In this study we have established a causal relationship between the local sequence and the structure of this framework region and linked this relationship to important biophysical properties such as affinity, folding yield and stability. We have generated six mutants of the scFv fragment aL2, covering some of the most abundant amino acid combinations in positions H6, H7 and H10 (Kabat H9). For the aL2 wildtype (w.t.) with the sequence 6Q7P10A and for two of the mutants, the X-ray structures have been solveddetermined. The structure of the triple mutant aL2-6E7S10G shows the FR-H1 backbone conformations predicted for this amino acid combination, which is distinctly different from the structure of the w.t, thus proving supporting our hypothesis that these residues determine the conformation of this segment. The mutant aL2-6E7P10G represents a residue combination not occurring in natural antibody sequences. It shows a completely different, unique structure in the first b-strand of VH, not observed in natural Fv fragments and forms a novel type of diabody. Two VH domains of the mutant associate by swapping the first b-strand. Concentration dependent changes in Trp fluorescence indicate that this dimerization is not just an artefact of crystallization, but occurs also under normal experimental conditions. The mutations in amino acids H6, H7 and H10 (Kabat H9) influence the dimerization behavior of the scFv and its thermodynamic stability. All the observations reported here have practical implications for the cloning of Fv fragments with degenerate primers, as well as for the design of new antibodies by CDR grafting or synthetic libraries.
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