Multi-state recognition pathway of the intrinsically disordered protein kinase inhibitor by protein kinase A

Abstract

In the nucleus, the spatiotemporal regulation of the catalytic subunit of cAMP-dependent protein kinase A (PKA-C) is orchestrated by an intrinsically disordered protein kinase inhibitor, PKI, which recruits the CRM1/RanGTP nuclear exporting complex. How the PKA-C/PKI complex assembles and recognizes CRM1/RanGTP is not well understood. Using NMR, SAXS, fluorescence, metadynamics, and Markov model analysis, we determined the multi-state recognition pathway for PKI. After a fast binding step in which PKA-C selects PKI’s most competent conformations, PKI folds upon binding through a slow conformational rearrangement within the enzyme’s binding pocket. The high-affinity and pseudo-substrate regions of PKI become more structured and the transient interactions with the kinase augment the helical content of the nuclear export sequence, which is then poised to recruit the CRM1/RanGTP complex for nuclear translocation. The multistate binding mechanism featured by PKA-C/PKI complex represents a paradigm on how disordered, ancillary proteins (or protein domains) are able to operate multiple functions such as inhibiting the kinase while recruiting other regulatory proteins for nuclear export.

Cell-type and subcellular compartment-specific APEX2 proximity labeling reveals activity-dependent nuclear proteome dynamics in the striatum

“…Other than transcription factors, Pkia was downregulated in the nucleus of dSPNs after hM3Dq activation (two-sided t -test, p = 0.0136). Pkia is a potent inhibitor of the nuclear protein kinase A (PKA) activity and is believed to facilitate the export of PKA from the nucleus to the cytoplasm by recruiting nuclear export machinery 38 . To what extent PKA signaling pathway is involved in the Gα q signaling-mediated activation of dSPNs remains to be elucidated in future studies; yet Gα q signaling can modulate PKA activity via muscarinic acetylcholine receptors in hippocampus 39 .…”

Structural Basis for Allosteric Control of the SERCA-Phospholamban Membrane Complex by Ca2+and cAMP-dependent Phosphorylation

“…3a,b). The interactions between the cytoplasmic regions are transient and highly dynamic, resembling the conformational ensembles of intrinsically disordered complexes 56 . For both complexes, we observe persistent interactions between PLN-Glu2 and SERCA-Lys365, PLN-Glu19, and SERCA-Lys328, and to a lesser extent PLN-Lys3 and SERCA-Asp399 and PLN-Tyr6 and SERCA-Asp557.…”

Structural basis for allosteric control of the SERCA-Phospholamban membrane complex by Ca2+ and phosphorylation

“…From the analysis of the structural ensembles of the two complexes, it emerges that the relief of inhibition (i.e., activation) occurs via a rearrangement of the intramembrane contacts between the TM region of pPLN and SERCA, with a reconfiguration of electrostatic interactions near the phosphorylation site and a disruption of packing at the protein-protein interface (Figure 3A, B). The interactions between the cytoplasmic regions are transient and highly dynamic, resembling the conformational ensembles of intrinsically disordered complexes 57 . For both complexes, we observe persistent interactions between PLN-Glu2 and SERCA-Lys365, PLN-Glu19, and SERCA-Lys328, and to a lesser extent PLN-Lys3 and SERCA-Asp399 and PLN-Tyr6 and SERCA-Asp557.…”