A biosensor of SRC family kinase conformation by exposable tetracysteine useful for cell-based screening.
We developed a new approach to distinguish distinct protein conformations in live cells. The method, exposable tetracysteine (XTC), involved placing an engineered tetracysteine motif into a target protein that has conditional access to biarsenical dye binding by conformational state. XTC was used to distinguish open and closed regulatory conformations of Src family kinases. Substituting just four residues with cysteines in the conserved SH2 domain of three Src-family kinases (c-Src, Lck, Lyn) enabled open and closed conformations to be monitored on the basis of binding differences to biarsenical dyes FlAsH or ReAsH. Fusion of the kinases with a fluorescent protein tracked the kinase presence, and the XTC approach enabled simultaneous assessment of regulatory state. The c-Src XTC biosensor was applied in a boutique screen of kinase inhibitors, which revealed six compounds to induce conformational closure. The XTC approach demonstrates new potential for assays targeting conformational changes in key proteins in disease and biology.
Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute and §Department of Medical Biology, University of Melbourne , Melbourne, Victoria 3010, Australia.
Sevgi
S
Irtegun
Rebecca
R
Wood
Kurt
K
Lackovic
Jörg
J
Schweiggert
Yasmin M
YM
Ramdzan
David C S
DC
Huang
Terrence D
TD
Mulhern
Danny M
DM
Hatters
D000595:Amino Acid Sequence; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D004396:Coloring Agents / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D018909:src Homology Domains / Q000187:drug effects; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
D004396:Coloring Agents; D047428:Protein Kinase Inhibitors; D019061:src-Family Kinases; D003545:Cysteine