Molecular analyses of the C-terminal CRAF variants associated with cardiomyopathy reveal their opposing impacts on the active conformation of the kinase domain

Abstract

The germline mutations in the C-terminus of CRAF kinase, particularly L603, and S612T/L613V, are associated with congenital heart disorders, for example, dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). The experimental data suggest that genetic alternation at position 603 impairs, while those at positions 612/613 enhance the CRAF kinase activity. However, the underlying mechanistic details by which these mutations increase or decrease kinase activity remain elusive. Therefore, we applied molecular dynamic simulation to investigate the impacts of these point mutations on the conformation of the CRAF kinase domain. The results revealed that the substitution of Leucine 603 for proline transits the kinase domain to a state that exhibits the molecular hallmarks of an inactive kinase, for example, a closed activation loop, ‘αC-helix out’ conformation and a distorted regulatory hydrophobic spine. However, two HCM-associated variants (S612T and L613V) show features of an active conformation, such as an open activation loop conformation, ‘αC-helix in’, the assembly of the hydrophobic spine, and more surface-exposed catalytic residues of phosphoryl transfer reaction. Overall, our study provides a mechanistic basis for the contradictory effects of the CRAF variants associated with HCM and DCM.

Communicated by Ramaswamy H. Sarma

Keywords:

• CRAF
• kinase domain
• MAPK pathway
• molecular dynamic simulation
• conformational changes
• catalytic activity
• mutation
• serine/threonine kinase
• cardiomyopathy
• RAF1
• RASopathy
• Noonan syndrome

Acknowledgments

We are grateful to Dr. Ehsan Amin from the Medical Faculty of the Heinrich-Heine University Düsseldorf for his helpful advice, and stimulating discussions. We also gratefully thank the Ferdowsi University of Mashhad for the support (Grant No. 1/52668).

Authors’ contributions

Saeideh Nakhaei-Rad: Conceptualization, Investigation, Writing - Original Draft, Visualization, Formal analysis. Mohammad R. Housaindokht: Conceptualization, Methodology, Resources, Funding acquisition, Project administration, Supervision. Fatemeh Janatifard: Methodology, Software, Validation, Formal analysis, Investigation. Radovan Dvorsky: Methodology, Writing - Review & Editing. Mohammad R. Ahmadian: Writing - Review & Editing.

Disclosure statement

There are no conflicts of interest to declare.

Additional information

Funding

We acknowledge the funding by Ferdowsi University of Mashhad (FUM; grant number 52668 to M.R.H) and the European Network on Noonan Syndrome and Related Disorders (NSEuroNet, grant number: 01GM1621B to M.R.A.). Dr. Fatemeh Janati-Fard was partially supported by a post-doctorate program of the vice president for research of FUM (No. 55155).