Epitopes described in "Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces."

Article Authors:Chris Szent-Gyorgyi; Robyn L Stanfield; Susan Andreko; Alison Dempsey; Mushtaq Ahmed; Sarah Capek; Alan S Waggoner; Ian A Wilson; Marcel P Bruchez
Article Title:Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces.
Reference Detail
Reference ID:1026855
Abstract:We report that a symmetric small-molecule ligand mediates the assembly of antibody light chain variable domains (VLs) into a correspondent symmetric ternary complex with novel interfaces. The L5* fluorogen activating protein is a VL domain that binds malachite green (MG) dye to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with inclusive C2 symmetry, where MG is almost entirely encapsulated between an antiparallel arrangement of the two VL domains. Unliganded L5* VL domains crystallize as a similar antiparallel VL/VL homodimer. The complementarity-determining regions are spatially oriented to form novel VL/VL and VL/ligand interfaces that tightly constrain a propeller conformer of MG. Binding equilibrium analysis suggests highly cooperative assembly to form a very stable VL/MG/VL complex, such that MG behaves as a strong chemical inducer of dimerization. Fusion of two VL domains into a single protein tightens MG binding over 1000-fold to low picomolar affinity without altering the large binding enthalpy, suggesting that bonding interactions with ligand and restriction of domain movements make independent contributions to binding. Fluorescence activation of a symmetrical fluorogen provides a selection mechanism for the isolation and directed evolution of ternary complexes where unnatural symmetric binding interfaces are favored over canonical antibody interfaces. As exemplified by L5*, these self-reporting complexes may be useful as modulators of protein association or as high-affinity protein tags and capture reagents.
Affiliations:Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA 15213, USA. Electronic address: css@andrew.cmu.edu.
Reference Type:Literature
PubMed ID:23978698
Journal:J Mol Biol
Journal Volume:425
Article Pages:4595-613
Journal ISSN:0022-2836
Article Chemical List:gov.nih.nlm.ncbi.www.jaxb.impl.NameOfSubstanceImpl@1fc79550;gov.nih.nlm.ncbi.www.jaxb.impl.NameOfSubstanceImpl@681bc77c;gov.nih.nlm.ncbi.www.jaxb.impl.NameOfSubstanceImpl@77c5d349;gov.nih.nlm.ncbi.www.jaxb.impl.NameOfSubstanceImpl@5cb29b0d;gov.nih.nlm.ncbi.www.jaxb.impl.NameOfSubstanceImpl@51e1a206;gov.nih.nlm.ncbi.www.jaxb.impl.NameOfSubstanceImpl@345461e9;gov.nih.nlm.ncbi.www.jaxb.impl.NameOfSubstanceImpl@fe822d6
Article MeSH List:Amino Acid Sequence; Binding Sites; Complementarity Determining Regions(chemistry; metabolism); Crystallography, X-Ray; Immunoglobulin Heavy Chains(chemistry; metabolism); Immunoglobulin Light Chains(chemistry); Immunoglobulin Variable Region(chemistry; metabolism); Ligands; Models, Molecular; Molecular Sequence Data; Protein Binding; Protein Interaction Domains and Motifs; Protein Multimerization; Protein Structure, Tertiary; Rosaniline Dyes(chemistry; metabolism); Sequence Alignment; Thermodynamics
Curation Last Updated:2015-01-18 01:06:02