title: Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces.
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.
authors: Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA 15213, USA. Electronic address: css@andrew.cmu.edu.
authors: Chris
authors: C
authors: Szent-Gyorgyi
authors: Robyn L
authors: RL
authors: Stanfield
authors: Susan
authors: S
authors: Andreko
authors: Alison
authors: A
authors: Dempsey
authors: Mushtaq
authors: M
authors: Ahmed
authors: Sarah
authors: S
authors: Capek
authors: Alan S
authors: AS
authors: Waggoner
authors: Ian A
authors: IA
authors: Wilson
authors: Marcel P
authors: MP
authors: Bruchez
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000737:chemistry*; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D012394:Rosaniline Dyes / Q000737:chemistry* / Q000378:metabolism; D016415:Sequence Alignment; D013816:Thermodynamics
chemical_list: D022801:Complementarity Determining Regions; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D008024:Ligands; D012394:Rosaniline Dyes; C005095:malachite green
keywords: CDR; CID; FACS; FAP; HIV; MG; MW; PBS; SAS; chemical inducer of dimerization; complementarity-determining region; cooperative binding; directed evolution; fluorescence activated cell sorting; fluorogen activating protein; human immunodeficiency virus; malachite green; molecular weight; phosphate-buffered saline; quaternary structure; solvent-accessible surface; yeast surface display
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