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Updated: Sep 20, 2025
Assaying Protein Kinase Activity with Radiolabeled ATP
Published on: May 26, 2017
Catalytic activity in vitro of the human protein kinase ASK1 mutants: Experimental and molecular simulation study
Yayu Xie1, Pei Cao1, Yuan Qin1
1Institute of Marine Drugs, Guangxi University of Chinese Medicine, Nanning 530200, PR China.
Abstract:
Kinases have become an important class of targets for drug discovery since the milestone approval of imatinib in 2001. Although a great success has been achieved for targeting kinases with over 70 inhibitors approved by the FDA, it is inevitable that drug resistance would emerge during treatment. Thus, assessment of the kinase mutations is an essential issue for the development of the next generation inhibitors. Apoptosis signal-regulating kinase 1 (ASK1) is a crucial regulator of classical mitogen-activated protein kinase cascade that is being explored under several clinical trials as a promising target. Herein, we investigate the catalytic activity in vitro of ASK1 by constructing two mutants: M754T and H729L, from gatekeeper and αC-helix, respectively. Compared to wild type, the mutation of M754T and H729L results in a roughly 3-fold and 2-fold decrease in binding affinity experimentally. In addition, their binding modes with substrate are theoretically predicted and compared by molecular dynamics. Trajectory analyses of simulations indicate that the decrease of binding affinity should be attributed to the loss of H-bond interaction with gatekeeper methionine. Unexpectedly, the conformation of αC-helix in H729L mutant did not alter significantly during the simulations, although the putatively important H-bond with H729 is lost. These simulations showed the regulatory role of H729 in αC-helix is maintained by leucine residue through the interaction with non-polar residues around H729 site.
Insights
Investigating mutations in Apoptosis signal-regulating kinase 1 (ASK1) is crucial for developing new kinase inhibitors. Mutations M754T and H729L reduced binding affinity, offering insights into drug resistance mechanisms.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Kinase inhibitors represent a significant advancement in targeted therapy, with over 70 approved by the FDA.
- Drug resistance remains a major challenge, necessitating the study of kinase mutations for next-generation inhibitor development.
- Apoptosis signal-regulating kinase 1 (ASK1) is a key regulator in signaling pathways and a promising target in ongoing clinical trials.
Purpose of the Study:
- To investigate the impact of specific mutations on the catalytic activity and binding affinity of ASK1.
- To explore the structural and dynamic consequences of gatekeeper (M754T) and αC-helix (H729L) mutations in ASK1.
- To provide theoretical insights into the binding modes and interactions of ASK1 mutants with their substrates.
Main Methods:
- In vitro enzymatic assays to determine the catalytic activity of wild-type and mutant ASK1.
- Construction and characterization of ASK1 mutants M754T and H729L.
- Molecular dynamics simulations to predict and compare binding modes and analyze conformational changes.
Main Results:
- The M754T and H729L mutations resulted in a 3-fold and 2-fold decrease in binding affinity, respectively, compared to wild-type ASK1.
- Molecular dynamics simulations revealed that the reduced binding affinity in M754T is due to the loss of hydrogen bond interactions with the gatekeeper methionine.
- The H729L mutation did not significantly alter the αC-helix conformation, suggesting leucine maintains the helix's regulatory role through interactions with surrounding non-polar residues.
Conclusions:
- Gatekeeper and αC-helix mutations in ASK1 significantly impact kinase activity and binding affinity, contributing to drug resistance.
- The study elucidates the molecular basis for reduced binding affinity caused by specific ASK1 mutations.
- Findings highlight the complex interplay of residues in maintaining kinase structure and function, crucial for designing effective inhibitors.
