Dysfunctional Conformational Dynamics of Protein Kinase a from R14 Deletion of Phospholamban

Protein Kinase A (PKA) is a ubiquitous signaling enzyme responsible that mediates a myriad of cellular signaling events. A target of PKA during regulation of muscle contraction is Phospholamban (PLN). PLN phosphorylation reverses its inhibitory effect on the sarcoplasmic reticulum Ca2+-ATPase (SERCA), increasing SERCA's affinity for Ca2+ ions and augmenting diastole. A deletion at the P-2 site of PLN recognition sequence (R14del) found in humans reduces PLN phosphorylation levels, leading to dilated cardiomyopathy. In this study we utilize solution state NMR, isothermal titration calorimetry, and MD simulations to elucidate the molecular mechanism of aberrant substrate recognition. Here, we show that this single substrate deletion affects the structural conformation and dynamics of the PKA/PLN complex, causing dysfunctional conformational fluctuations throughout the enzyme, disrupting binding and reducing enzymatic efficiency. These results unveil a crucial role of kinase substrates for the formation of the catalytically-competent conformation and intramolecular allosteric signaling. These findings illuminate a molecular mechanism for the pathophysiology of R14del and aberrant Ca2+ signaling, underscoring the importance of a well-tuned structural and dynamic interplay between enzyme and substrate to achieve physiological phosphorylation levels for normal cardiac function.