Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces.
J Mol Biol
; 425(22): 4595-613, 2013 Nov 15.
Article
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| MEDLINE
| ID: mdl-23978698
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 21 proteinligand 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.
Palabras clave
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
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Colorantes de Rosanilina
/
Región Variable de Inmunoglobulina
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Cadenas Ligeras de Inmunoglobulina
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Multimerización de Proteína
Tipo de estudio:
Prognostic_studies
Idioma:
En
Revista:
J Mol Biol
Año:
2013
Tipo del documento:
Article