Limited Proteolysis Combined with Stable Isotope Labeling Reveals Conformational Changes in Protein (Pseudo)kinases upon Binding Small Molecules.
Likely due to conformational rearrangements, small molecule inhibitors may stabilize the active conformation of protein kinases and paradoxically promote tumorigenesis. We combined limited proteolysis with stable isotope labeling MS to monitor protein conformational changes upon binding of small molecules. Applying this method to the human serine/threonine kinase B-Raf, frequently mutated in cancer, we found that binding of ATP or its nonhydrolyzable analogue AMP-PNP, but not ADP, stabilized the structure of both B-Raf(WT) and B-Raf(V600E). The ATP-competitive type I B-Raf inhibitor vemurafenib and the type II inhibitor sorafenib stabilized the kinase domain (KD) but had distinct effects on the Ras-binding domain. Stabilization of the B-Raf(WT) KD was confirmed by hydrogen/deuterium exchange MS and molecular dynamics simulations. Our results are further supported by cellular assays in which we assessed cell viability and phosphorylation profiles in cells expressing B-Raf(WT) or B-Raf(V600E) in response to vemurafenib or sorafenib. Our data indicate that an overall stabilization of the B-Raf structure by specific inhibitors activates MAPK signaling and increases cell survival, helping to explain clinical treatment failure. We also applied our method to monitor conformational changes upon nucleotide binding of the pseudokinase KSR1, which holds high potential for inhibition in human diseases.
['Di Michele|Michela|M|', 'Stes|Elisabeth|E|', 'Vandermarliere|Elien|E|', 'Arora|Rohit|R|', 'Astorga-Wells|Juan|J|', 'Vandenbussche|Jonathan|J|', 'van Heerde|Erika|E|', 'Zubarev|Roman|R|', 'Bonnet|Pascal|P|', 'Linders|Joannes T M|JT|', 'Jacoby|Edgar|E|', 'Brehmer|Dirk|D|', 'Martens|Lennart|L|', 'Gevaert|Kris|K|']
D000255:Adenosine Triphosphate D000266:Adenylyl Imidodiphosphate D045744:Cell Line, Tumor D002470:Cell Survival D041961:Deuterium Exchange Measurement D006801:Humans D007211:Indoles D007553:Isotope Labeling D020935:MAP Kinase Signaling System D013058:Mass Spectrometry D056004:Molecular Dynamics Simulation D009154:Mutation D009536:Niacinamide D010455:Peptides D010671:Phenylurea Compounds D010766:Phosphorylation D011485:Protein Binding D047428:Protein Kinase Inhibitors D011494:Protein Kinases D017433:Protein Structure, Secondary D017434:Protein Structure, Tertiary D059748:Proteolysis D040901:Proteomics D048493:Proto-Oncogene Proteins B-raf D000077157:Sorafenib D013449:Sulfonamides D014357:Trypsin D000077484:Vemurafenib
limited proteolysis conformational change small molecule inhibitor kinase B-Raf KSR1 kinase switch mechanism method development protein structure