Sequence determinants of a specific inactive protein kinase conformation

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Protein kinases are crucial enzymes, but few adopt specific inactive conformations like the DFG-out state.
• Understanding the sequence basis for adopting these inactive states is vital given high homology among kinases.

Purpose of the Study:

• To identify sequence-level determinants that confer sensitivity to inhibitors stabilizing the DFG-out inactive conformation in protein kinases.
• To explore the generalizability of these determinants across different kinase families.

Main Methods:

• Sequence analysis to identify conserved residue positions correlating with DFG-out conformation sensitivity.
• Structural and dynamic characterization of engineered kinase mutants.
• Inhibition assays using DFG-out stabilizing ligands on wild-type and mutant kinases.

Main Results:

• Two specific residue positions were identified that sensitize mitogen-activated protein kinases (MAPKs) to DFG-out stabilizing inhibitors.
• A non-MAPK family kinase (apoptosis signal-regulating kinase 1) was successfully sensitized to DFG-out ligands by modifying these same residue positions.
• Characterization of an inhibitor-sensitive MAPK mutant provided insights into structural and dynamic changes.

Conclusions:

• Specific residue positions can be engineered to control kinase sensitivity to DFG-out stabilizing inhibitors.
• This strategy is applicable beyond the MAPK family, demonstrating broad potential.
• Targeting specific inactive conformations may facilitate research into non-catalytic kinase functions, including protein interactions and scaffolding.