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: Michela Di Michele, Elisabeth Stes, Elien Vandermarliere, Rohit Arora, Juan Astorga-Wells, Jonathan Vandenbussche, Erika van Heerde, Roman Zubarev, Pascal Bonnet, Joannes T M Linders, Edgar Jacoby, Dirk Brehmer, Lennart Martens, Kris Gevaert
• Journal: Journal of proteome research (J Proteome Res) Vol. 14 Issue 10 Pg. 4179-93 (Oct 02 2015) ISSN: 1535-3907 [Electronic] United States
• PMID: 26293246 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Indoles
• Peptides
• Phenylurea Compounds
• Protein Kinase Inhibitors
• Sulfonamides
• Vemurafenib
• Adenylyl Imidodiphosphate
• Niacinamide
• Adenosine Triphosphate
• Sorafenib
• Protein Kinases
• KSR-1 protein kinase
• BRAF protein, human
• Proto-Oncogene Proteins B-raf
• Trypsin

Topics

• Adenosine Triphosphate (chemistry, metabolism)
• Adenylyl Imidodiphosphate (chemistry, metabolism)
• Cell Line, Tumor
• Cell Survival (drug effects)
• Deuterium Exchange Measurement
• Humans
• Indoles (chemistry, pharmacology)
• Isotope Labeling (methods)
• MAP Kinase Signaling System (drug effects)
• Mass Spectrometry (methods)
• Molecular Dynamics Simulation
• Mutation
• Niacinamide (analogs & derivatives, chemistry, pharmacology)
• Peptides (analysis)
• Phenylurea Compounds (chemistry, pharmacology)
• Phosphorylation (drug effects)
• Protein Binding
• Protein Kinase Inhibitors (chemistry, pharmacology)
• Protein Kinases (chemistry, genetics, metabolism)
• Protein Structure, Secondary
• Protein Structure, Tertiary
• Proteolysis
• Proteomics (instrumentation, methods)
• Proto-Oncogene Proteins B-raf (antagonists & inhibitors, chemistry, genetics, metabolism)
• Sorafenib
• Sulfonamides (chemistry, pharmacology)
• Trypsin (chemistry)
• Vemurafenib