5J74

Fluorogen activating protein AM2.2 in complex with TO1-2p

Summary for 5J74

• Entry DOI: 10.2210/pdb5j74/pdb
• Related: 5J75
• Descriptor: scFv AM2.2, PHOSPHATE ION, 1-(17-amino-5,8-dioxo-12,15-dioxa-4,9-diazaheptadecan-1-yl)-4-{[3-(3-sulfopropyl)-1,3-benzothiazol-3-ium-2-yl]methyl}quinolin-1-ium, ... (4 entities in total)
• Functional Keywords: fluorogen activating protein drug discovery biosensor protein trafficking, protein binding, immune system
• Biological source: Homo sapiens
• Total number of polymer chains: 2
• Total formula weight: 56081.26

Authors

Stanfield, R.L.,Wilson, I.A.,Wu, Y. (deposition date: 2016-04-05, release date: 2016-05-25, Last modification date: 2024-11-13)

Primary citation

Wu, Y.,Stauffer, S.R.,Stanfield, R.L.,Tapia, P.H.,Ursu, O.,Fisher, G.W.,Szent-Gyorgyi, C.,Evangelisti, A.,Waller, A.,Strouse, J.J.,Carter, M.B.,Bologa, C.,Gouveia, K.,Poslusney, M.,Waggoner, A.S.,Lindsley, C.W.,Jarvik, J.W.,Sklar, L.A.
Discovery of Small-Molecule Nonfluorescent Inhibitors of Fluorogen-Fluorogen Activating Protein Binding Pair.
J Biomol Screen, 21:74-87, 2016

PubMed Abstract: A new class of biosensors, fluorogen activating proteins (FAPs), has been successfully used to track receptor trafficking in live cells. Unlike the traditional fluorescent proteins (FPs), FAPs do not fluoresce unless bound to their specific small-molecule fluorogens, and thus FAP-based assays are highly sensitive. Application of the FAP-based assay for protein trafficking in high-throughput flow cytometry resulted in the discovery of a new class of compounds that interferes with the binding between fluorogens and FAP, thus blocking the fluorescence signal. These compounds are high-affinity, nonfluorescent analogs of fluorogens with little or no toxicity to the tested cells and no apparent interference with the normal function of FAP-tagged receptors. The most potent compound among these, N,4-dimethyl-N-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)benzenesulfonamide (ML342), has been investigated in detail. X-ray crystallographic analysis revealed that ML342 competes with the fluorogen, sulfonated thiazole orange coupled to diethylene glycol diamine (TO1-2p), for the same binding site on a FAP, AM2.2. Kinetic analysis shows that the FAP-fluorogen interaction is more complex than a homogeneous one-site binding process, with multiple conformational states of the fluorogen and/or the FAP, and possible dimerization of the FAP moiety involved in the process.

PubMed: 26442911
DOI: 10.1177/1087057115609145

Experimental method

X-RAY DIFFRACTION (2.7 Å)