title: Molecular mechanism of antibody-mediated activation of β-galactosidase.
abstract: Binding of a single-chain Fv antibody to Escherichia coli β-galactosidase (β-gal) is known to stabilize the enzyme and activate several inactive point mutants, historically called antibody-mediated enzyme formation mutants. To understand the nature of this activation, we have determined by electron cryo-microscopy the structure of the complex between β-gal and the antibody scFv13R4. Our structure localizes the scFv13R4 binding site to the crevice between domains 1 and 3 in each β-gal subunit. The mutations that scFv13R4 counteracts are located between the antibody binding site and the active site of β-gal, at one end of the TIM-barrel that forms domain 3 where the substrate lactose is hydrolyzed. The mode of binding suggests how scFv stabilizes both the active site of β-gal and the tetrameric state.
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Kutti R
authors.initials: KR
authors.lastname: Vinothkumar
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Greg
authors.initials: G
authors.lastname: McMullan
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK. Electronic address: rh15@mrc-lmb.cam.ac.uk.
authors.forename: Richard
authors.initials: R
authors.lastname: Henderson
mesh_terms: D020134:Catalytic Domain; D004789:Enzyme Activation; D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D007785:Lactose / Q000737:chemistry; D008958:Models, Molecular; D011485:Protein Binding; D017510:Protein Folding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D057127:Single-Chain Antibodies / Q000737:chemistry*; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D001616:beta-Galactosidase; D007785:Lactose