1h8s

From Proteopedia

Revision as of 14:27, 5 November 2007

THREE-DIMENSIONAL STRUCTURE OF ANTI-AMPICILLIN SINGLE CHAIN FV FRAGMENT COMPLEXED WITH THE HAPTEN.

Overview

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 differ mainly in the side-chain conformation of Trp H109, (according to a new consensus nomenclature Kabat residue number H95) in, the extremely short (three residues) CDR H3 and the presence or absence of, a well-resolved molecule of 2-methyl-pentane-2,4-diol 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, wild-type 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 Escherichia coli, and for directed evolution towards high antibiotic resistance.

About this Structure

1H8S is a Single protein structure of sequence from Mouse e 010090 with SO4 and AIC as ligands. Structure known Active Site: AIC. Full crystallographic information is available from OCA.

Reference

Selection, characterization and x-ray structure of anti-ampicillin single-chain Fv fragments from phage-displayed murine antibody libraries., Burmester J, Spinelli S, Pugliese L, Krebber A, Honegger A, Jung S, Schimmele B, Cambillau C, Pluckthun A, J Mol Biol. 2001 Jun 8;309(3):671-85. PMID:11397088

Page seeded by OCA on Mon Nov 5 16:32:27 2007