Abstract
BackgroundThe regulatory subunit (R) of cAMP-dependent protein kinase (PKA) is a modular flexible protein that responds with large conformational changes to the binding of the effector cAMP. Considering its highly dynamic nature, the protein is rather stable. We studied the thermal denaturation of full-length RIα and a truncated RIα(92-381) that contains the tandem cyclic nucleotide binding (CNB) domains A and B.Methodology/Principal FindingsAs revealed by circular dichroism (CD) and differential scanning calorimetry, both RIα proteins contain significant residual structure in the heat-denatured state. As evidenced by CD, the predominantly α-helical spectrum at 25°C with double negative peaks at 209 and 222 nm changes to a spectrum with a single negative peak at 212–216 nm, characteristic of β-structure. A similar α→β transition occurs at higher temperature in the presence of cAMP. Thioflavin T fluorescence and atomic force microscopy studies support the notion that the structural transition is associated with cross-β-intermolecular aggregation and formation of non-fibrillar oligomers.Conclusions/SignificanceThermal denaturation of RIα leads to partial loss of native packing with exposure of aggregation-prone motifs, such as the B' helices in the phosphate-binding cassettes of both CNB domains. The topology of the β-sandwiches in these domains favors inter-molecular β-aggregation, which is suppressed in the ligand-bound states of RIα under physiological conditions. Moreover, our results reveal that the CNB domains persist as structural cores through heat-denaturation.
Highlights
Adenosine cyclic 39,59-phosphate and guanosine cyclic 39,59-phosphate act as second messengers for many cellular processes [1]
The cyclic nucleotide binding (CNB) fold – classified as the double-stranded beta-helix fold in SCOP [2] 2 exhibits a b-sandwich topology and is present in several protein families, i.e. i) proteins with cAMP binding domains, such as the regulatory subunit (R) of cAMP-dependent protein kinase (PKA), Rap1 guanine exchange factor (Epac), cGMP-dependent protein kinase (PKG), cNMPgated ion channels and the gene activator protein (CAP) [3], and ii) proteins with cAMP-binding-like domains, such as the COsensing protein CooA [4] and the listeriolysin regulatory protein PrfA [5]
Comparison of the crystal structures of RIa(103– 376) with cAMP bound at the A and B sites [7] with that of RIa(91–379:R333K), complexed with C subunit [11], reveals large conformational changes that accompany the functional cycle of PKA
Summary
Adenosine cyclic 39,59-phosphate (cAMP) and guanosine cyclic 39,59-phosphate (cGMP) act as second messengers for many cellular processes [1]. The sugar phosphate moiety of cAMP interacts with the so-called phosphate binding cassette (PBC), consisting of two pairs of antiparallel strands, linked by a short, one-turn helix that is characteristic of the double-stranded beta-helix fold. This short helix – referred to as B’ in the CNB domain nomenclature (i.e. residues 200–205 in domain A (aB’:A) and 324–329 in domain B (aB’:B helices) for bovine RIa Comparison of the crystal structures of RIa(103– 376) with cAMP bound at the A and B sites [7] with that of RIa(91–379:R333K), complexed with C subunit [11], reveals large conformational changes that accompany the functional cycle of PKA. We studied the thermal denaturation of full-length RIa and a truncated RIa(92-381) that contains the tandem cyclic nucleotide binding (CNB) domains A and B
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