Consequences of cAMP and catalytic-subunit binding on the flexibility of the A-kinase regulatory subunit

A combination of site-directed labeling and time-resolved fluorescence anis otropy was used to further elucidate the structure and underlying dynamic f eatures of the type I regulatory (R-alpha(I)) subunit of the cAMP-dependent protein kinase. Specifically, the consequences of cAMP and the catalytic ( C)-subunit binding on the backbone flexibility around seven sites of cystei ne substitution and fluorescein maleimide labeling (Thr(6)Cys, Leu(66)Cys, Ser(75)Cys, Ser(81)Cys, Ser(99)Cys, Ser(145)Cys, and Ser(373)Cys) in the R- alpha(I), subunit were assessed. Focusing on the fast rotational correlatio n time, the results indicate that most of the interdomain segment connectin g the dimerization/docking (D/D) and tandem cAMP-binding domains is probabl y weakly associated with the latter domain. Also, this segment becomes more tightly bound to the C subunit upon holoenzyme formation. The results also suggest that there is a short 'hinge' segment (around Leu(66)Cys) that cou ld allow the structured interdomain/cAMP-binding and D/D domains to pivot a bout each other. Finally, cAMP binding dramatically reduces the backbone fl exibility around only the two sites of cysteine substitution in the cAMP-bi nding domains, suggesting a selective structural stabilization caused by cA MP and a "tight" coupling of low-nanosecond fluctuations selectively within the tandem cAMP-binding domains.