Resonant mirror biosensor analysis of type Iα cAMP‐dependent protein kinase B domain—Cyclic nucleotide interactions

Abstract

A resonant mirror biosensor was used to study cyclic nucleotide-receptor interactions. In particular, a novel method was developed to determine inhibition constants~K i ! from initial rates of ligate association to immobilized ligand. This approach was applied to the comparison of cyclic nucleotide-binding properties of the wild-type isolated B domain of the cAMP-dependent protein kinase type Ia regulatory subunit and its Ala-334-Thr~A334T! variant that has altered cyclic nucleotide specificity. A cUMP-saturated form of the B domain was used for all measurements. Under the conditions used, cUMP did not affect the kinetics of B domain association to immobilized cAMP. Triton X-100 was required to stabilize the protein at nanomolar concentrations. The association and dissociation rate constants for wild-type and A334T B domains yielded equilibrium dissociation constants of 11 and 16 nM. Heterogeneity of ligate and immobilized ligand, mass transport effects, and other factors were evaluated for their influence on biosensordetermined kinetic constants. Biosensor-determined relative inhibition constants~K9 i ϭ K i cAMP 0K i analog ! for 16 cyclic nucleotide analogs correlated well with those determined by a @ 3 H#cAMP binding assay. Previously published K9 i values for the B domain in the intact regulatory subunit were similar to those of the isolated B domain. The K9 i values for the wild-type and A334T B domains were essentially unchanged except for dramatic enhancements in affinity of cGMP analogs for the A334T B domain. These observations validate the isolated B domain as a simple model system for studying cyclic nucleotide-receptor interactions.

Mutations of PKA cyclic nucleotide-binding domains reveal novel aspects of cyclic nucleotide selectivity

“…Previous studies on isolated CNB domains of PKA RIα showed that CNB-B is more selective for cAMP compared with CNB-A [41,44]. In agreement with former studies, the selectivity of the isolated CNB-B remains the same as for CNB-B in the full-length PKA RIα [42]. In contrast, the isolated CNB-A shows a reduced selectivity compared with CNB-A in the full-length protein.…”

“…However, comparing the relative affinities, we demonstrate for the first time that CNB-B has a higher selectivity for cAMP compared with CNB-A (Figure 3 and Table 1). In contrast, previous studies using full-length PKA RIα revealed that CNB-A is more selective for cAMP than CNB-B [14,18,42]. However, as cAMP binding to PKA RIα is highly cooperative, cyclic nucleotide binding to the full-length protein and the isolated CNB domains cannot be directly compared [43].…”

Interfering with the high-affinity interaction between wheat amylase trypsin inhibitor CM3 and toll-like receptor 4: in silico and biosensor-based studies

“…A working volume of 80 μL was used throughout, and the temperature was set at 37 °C. The possible influence of mass transport on the determination of kinetic parameters 39 was considered and reduced by setting the stirrer rate to 95%.…”

Molecular Determinants and Signaling Effects of PKA RIα Phase Separation

“…Binding of cAMP to RIα is mediated by a pair of cooperative CNB domains that exhibit distinct affinities for cAMP 11,38 . The CNB-A domain shows a 50-fold higher EC 50 for cAMP compared with the CNB-B domain, and cAMP is thought to first bind to CNB-B, resulting in increased affinity of CNB-A for cAMP 39,40 . While our prior truncation study suggested that RIα LLPS requires the CNB domains 21 , we sought to test the individual contribution of cAMP binding to each CNB domain to RIα LLPS.…”