title: Limited Proteolysis Combined with Stable Isotope Labeling Reveals Conformational Changes in Protein (Pseudo)kinases upon Binding Small Molecules.
abstract: 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.
authors: Department of Medical Protein Research, VIB , A. Baertsoenkaai 3, 9000 Ghent, Belgium.|Department of Biochemistry, Ghent University , A. Baertsoenkaai 3, 9000 Ghent, Belgium.
authors: Michela
authors: M
authors: Di Michele
authors: Department of Medical Protein Research, VIB , A. Baertsoenkaai 3, 9000 Ghent, Belgium.|Department of Biochemistry, Ghent University , A. Baertsoenkaai 3, 9000 Ghent, Belgium.
authors: Elisabeth
authors: E
authors: Stes
authors: Department of Medical Protein Research, VIB , A. Baertsoenkaai 3, 9000 Ghent, Belgium.|Department of Biochemistry, Ghent University , A. Baertsoenkaai 3, 9000 Ghent, Belgium.
authors: Elien
authors: E
authors: Vandermarliere
authors: Institut de Chimie Organique et Analytique (ICOA), UMR 7311 CNRS-Université d'Orléans , Pôle de chimie, Rue de Chartres, 45100 Orléans, France.
authors: Rohit
authors: R
authors: Arora
authors: Biomotif AB , P.O. Box 156, SE-182 12 Danderyd, Sweden.
authors: Juan
authors: J
authors: Astorga-Wells
authors: Department of Medical Protein Research, VIB , A. Baertsoenkaai 3, 9000 Ghent, Belgium.|Department of Biochemistry, Ghent University , A. Baertsoenkaai 3, 9000 Ghent, Belgium.
authors: Jonathan
authors: J
authors: Vandenbussche
authors: Oncology Discovery, Janssen Research and Development, A Division of Janssen Pharmaceutica NV , Turnhoutseweg 30, B-2340 Beerse, Belgium.
authors: Erika
authors: E
authors: van Heerde
authors: Department of Medical Biochemistry and Biophysics, Karolinska Institutet , Scheelelaberatoriet Scheeles väg 2, SE-171 77 Stockholm, Sweden.
authors: Roman
authors: R
authors: Zubarev
authors: Institut de Chimie Organique et Analytique (ICOA), UMR 7311 CNRS-Université d'Orléans , Pôle de chimie, Rue de Chartres, 45100 Orléans, France.
authors: Pascal
authors: P
authors: Bonnet
authors: Oncology Discovery, Janssen Research and Development, A Division of Janssen Pharmaceutica NV , Turnhoutseweg 30, B-2340 Beerse, Belgium.
authors: Joannes T M
authors: JT
authors: Linders
authors: Oncology Discovery, Janssen Research and Development, A Division of Janssen Pharmaceutica NV , Turnhoutseweg 30, B-2340 Beerse, Belgium.
authors: Edgar
authors: E
authors: Jacoby
authors: Oncology Discovery, Janssen Research and Development, A Division of Janssen Pharmaceutica NV , Turnhoutseweg 30, B-2340 Beerse, Belgium.
authors: Dirk
authors: D
authors: Brehmer
authors: Department of Medical Protein Research, VIB , A. Baertsoenkaai 3, 9000 Ghent, Belgium.|Department of Biochemistry, Ghent University , A. Baertsoenkaai 3, 9000 Ghent, Belgium.
authors: Lennart
authors: L
authors: Martens
authors: Department of Medical Protein Research, VIB , A. Baertsoenkaai 3, 9000 Ghent, Belgium.|Department of Biochemistry, Ghent University , A. Baertsoenkaai 3, 9000 Ghent, Belgium.
authors: Kris
authors: K
authors: Gevaert
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000737:chemistry / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D041961:Deuterium Exchange Measurement; D006801:Humans; D007211:Indoles / Q000737:chemistry / Q000494:pharmacology; D007553:Isotope Labeling / Q000379:methods*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D013058:Mass Spectrometry / Q000379:methods; D056004:Molecular Dynamics Simulation; D009154:Mutation; D009536:Niacinamide / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D010455:Peptides / Q000032:analysis; D010671:Phenylurea Compounds / Q000737:chemistry / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000077157:Sorafenib; D013449:Sulfonamides / Q000737:chemistry / Q000494:pharmacology; D014357:Trypsin / Q000737:chemistry; D000077484:Vemurafenib
chemical_list: D007211:Indoles; D010455:Peptides; D010671:Phenylurea Compounds; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D000077484:Vemurafenib; D000266:Adenylyl Imidodiphosphate; D009536:Niacinamide; D000255:Adenosine Triphosphate; D000077157:Sorafenib; D011494:Protein Kinases; C097199:KSR-1 protein kinase; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D014357:Trypsin
keywords: B-Raf; KSR1; conformational change; inhibitor; kinase; kinase switch mechanism; limited proteolysis; method development; protein structure; small molecule