Novel isoform-specific interfaces revealed by PKA RIIbeta holoenzyme structures

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• cAMP-dependent protein kinase catalytic (C) subunit activity is regulated by RI and RII subunits.
• RII subunits uniquely function as both inhibitors and substrates of the C subunit.
• RIIbeta holoenzyme is a therapeutic target due to disease phenotypes in knockout mice.

Purpose of the Study:

• To understand RIIbeta's dual role as inhibitor and substrate.
• To elucidate the structural function of the linker region in RIIbeta holoenzyme architecture.
• To engineer RIIbeta constructs for structural and binding studies.

Main Methods:

• Engineering of RIIbeta constructs (RIIbeta(108-268) and RIIbeta(102-265)).
• Co-crystallization and X-ray crystallography of RIIbeta holoenzyme complexes.
• Binding affinity measurements using AMP-PNP (a non-hydrolyzable ATP analog).

Main Results:

• RIIbeta(108-268) showed poor binding to C subunit without nucleotide but high affinity (11 nM) with AMP-PNP/Mn(2+).
• The RIIbeta(108-268) holoenzyme structure revealed an enzyme:substrate complex in a closed conformation.
• Additional linker residues in RIIbeta(102-265) significantly increased affinity (1.6 nM) and docked onto the C subunit's large lobe surface.
• The linker's novel orientation suggests a role in RIIbeta holoenzyme organization.

Conclusions:

• AMP-PNP/Mn(2+) enhances binding affinity, a strategy applicable to other protein kinase substrates.
• The RIIbeta linker region plays a critical structural role in holoenzyme organization.
• Understanding RIIbeta structure-function provides a basis for developing isoform-specific modulators.