4K3H

Immunoglobulin lambda variable domain L5(L89S) fluorogen activationg protein in complex with malachite green

Summary for 4K3H

• Entry DOI: 10.2210/pdb4k3h/pdb
• Related: 4K3G
• Descriptor: Immunoglobulin lambda variable domain L5(L89S), 4-{bis[4-(dimethylamino)phenyl]methyl}phenol, GLYCEROL, ... (5 entities in total)
• Functional Keywords: immunoglobulin fold, fluorescensce, malachite green, o-mannosylation, thr27, immune system-inhibitor complex, immune system/inhibitor
• Biological source: Homo sapiens (human)
• Total number of polymer chains: 8
• Total formula weight: 103293.38

Authors

Stanfield, R.L.,Szent-Gyorgyi, C.,Wilson, I.A. (deposition date: 2013-04-10, release date: 2013-10-09, Last modification date: 2024-11-27)

Primary citation

Szent-Gyorgyi, C.,Stanfield, R.L.,Andreko, S.,Dempsey, A.,Ahmed, M.,Capek, S.,Waggoner, A.S.,Wilson, I.A.,Bruchez, M.P.
Malachite Green Mediates Homodimerization of Antibody VL Domains to Form a Fluorescent Ternary Complex with Singular Symmetric Interfaces.
J.Mol.Biol., 425:4595-4613, 2013

PubMed 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.

PubMed: 23978698
DOI: 10.1016/j.jmb.2013.08.014

Experimental method

X-RAY DIFFRACTION (2.45 Å)