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Updated: Apr 5, 2026
Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
Published on: June 30, 2019
Limited Proteolysis Combined with Stable Isotope Labeling Reveals Conformational Changes in Protein (Pseudo)kinases
Michela Di Michele1,2, Elisabeth Stes1,2, Elien Vandermarliere1,2
1Department of Medical Protein Research, VIB , A. Baertsoenkaai 3, 9000 Ghent, Belgium.
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.
Insights
Small molecule kinase inhibitors can paradoxically promote cancer by stabilizing active protein conformations. This study reveals how B-Raf inhibitors stabilize its structure, activating signaling pathways and increasing cell survival, potentially explaining treatment failure.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Small molecule inhibitors are crucial in cancer therapy, but their mechanisms can be complex.
- Protein kinases, like B-Raf, are frequently dysregulated in cancer.
- Conformational changes induced by inhibitors can impact kinase activity and cellular outcomes.
Purpose of the Study:
- To investigate the conformational effects of small molecule binding on B-Raf kinase.
- To understand how these conformational changes influence kinase activity and cellular signaling.
- To develop and apply a novel method for monitoring protein conformational dynamics.
Main Methods:
- Limited proteolysis coupled with stable isotope labeling mass spectrometry (MS) to detect conformational changes.
- Hydrogen/deuterium exchange MS and molecular dynamics simulations for structural validation.
- Cellular assays assessing cell viability and phosphorylation profiles.
Main Results:
- ATP binding stabilized the structure of both wild-type (WT) and V600E mutated B-Raf.
- B-Raf inhibitors (vemurafenib, sorafenib) stabilized the kinase domain but differentially affected the Ras-binding domain.
- Inhibitor-induced B-Raf stabilization activated MAPK signaling and enhanced cell survival.
- The method was also applied to monitor conformational changes in KSR1 upon nucleotide binding.
Conclusions:
- Small molecule inhibitors can paradoxically promote tumorigenesis by stabilizing active kinase conformations.
- Inhibitor-induced stabilization of B-Raf leads to MAPK pathway activation and increased cell survival, contributing to treatment failure.
- The developed proteolysis-MS method is effective for studying kinase conformational dynamics and drug interactions.
