Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces

J Mol Biol. 2013 Nov 15;425(22):4595-613. doi: 10.1016/j.jmb.2013.08.014. Epub 2013 Aug 23.


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.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Complementarity Determining Regions / chemistry
  • Complementarity Determining Regions / metabolism
  • Crystallography, X-Ray
  • Immunoglobulin Heavy Chains / chemistry
  • Immunoglobulin Heavy Chains / metabolism
  • Immunoglobulin Light Chains / chemistry*
  • Immunoglobulin Variable Region / chemistry*
  • Immunoglobulin Variable Region / metabolism
  • Ligands
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Multimerization*
  • Protein Structure, Tertiary
  • Rosaniline Dyes / chemistry*
  • Rosaniline Dyes / metabolism
  • Sequence Alignment
  • Thermodynamics


  • Complementarity Determining Regions
  • Immunoglobulin Heavy Chains
  • Immunoglobulin Light Chains
  • Immunoglobulin Variable Region
  • Ligands
  • Rosaniline Dyes
  • malachite green

Associated data

  • PDB/4K3G
  • PDB/4K3H