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 (FAP) is a VL domain that binds malachite green dye (MG) 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 (CDRs) 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 1,000-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.

Breaking the color barrier – a multi-selective antibody reporter offers innovative strategies of fluorescence detection

“…Incidentally, the lower observed signal may be attributed to the protein-binding pocket encapsulating and shielding the fluorogen from background activation when free in solution. A similar observation was previously identified when measuring the photoinsulating properties of scFv scaffolds and cognate fluorogens (Saurabh et al, 2015;Szent-Gyorgyi et al, 2013). Thus, here, we find that HL1.0.1-TO1 lacks the ability to stabilize a twisted conformation into a planar geometry for fluorescence activation.…”

Local retention of antibodies in vivo with an injectable film embedded with a fluorogen-activating protein

“…Compared to conventional fluorescent reporters, MG-dL5 has enhanced photo-stability. The FAP protects the fluorogen from oxidative damage as crystallographic studies show that a single MG molecule is flanked by two L5 domains [35]. This feature was exploited here to monitor the stability of the IgG binding sites, as pAG MG would escape from the film matrix to become non-fluorescent.…”

“…Relative to free MG, thefluorescenceofdL5-boundMGisenhanced by up to 20,000 folds, due to constraining of the rotatable bonds in MG by two L5 proteins. Crystallographic studies have shown that MG mediates dimerization of two L5 proteins to form a fluorescent ternary complex [35]. We hypothesized that the system of MG-conjugated pAG (pAG MG ), dL5_EAK and EAK16-II, when deployed together (called dL5 film), serves as a self-illuminating, injectable formulation for local deposition of IgG.…”

“…We hypothesized that the system of MG-conjugated pAG (pAG MG ), dL5_EAK and EAK16-II, when deployed together (called dL5 film), serves as a self-illuminating, injectable formulation for local deposition of IgG. The tight binding of dL5 to MG ensures a stable intermediate [35]. Release of IgG from pAG MG is driven by the diffusion of free IgG into tissues surrounding the injection site.…”

Fluoromodule-based reporter/probes designed for in vivo fluorescence imaging

“…The light chain of HL9-MG was previously found sufficient for fluorogen activation (33). Studies of similar light chain-only FAPs have shown that the assembled fluoromodule contains 1 fluorogen molecule bound between 2 light chains and that tethering of 2 light chains by a flexible linker increases binding affinity (28,34). A gene encoding a peptide-linked dimer of light chains from HL9-MG was generated, randomly mutagenized, and screened using a yeast expression system and cell sorting in hopes of finding clones with improved binding and fluorescence activation of a membrane-permeant MG derivative, MG-ester.…”