Abstract
Protein kinase A (PKA) is the main receptor for the universal cAMP second messenger. PKA is a tetramer with two catalytic (C) and two regulatory (R) subunits, each including two tandem cAMP binding domains, i.e. CBD-A and -B. Structural investigations of RIalpha have revealed that although CBD-A plays a pivotal role in the cAMP-dependent inhibition of C, the main function of CBD-B is to regulate the access of cAMP to site A. To further understand the mechanism underlying the cross-talk between CBD-A and -B, we report here the NMR investigation of a construct of R, RIalpha-(119-379), which unlike previous fragments characterized by NMR, spans in full both CBDs. Our NMR studies were also extended to two mutants, R209K and the corresponding R333K, which severely reduce the affinity of cAMP for CBD-A and -B, respectively. The comparative NMR analysis of wild-type RIalpha-(119-379) and of the two domain silencing mutations has led to the definition at an unprecedented level of detail of both intra- and interdomain allosteric networks, revealing several striking differences between the two CBDs. First, the two domains, although homologous in sequence and structure, exhibit remarkably different responses to the R/K mutations especially at the beta2-3 allosteric "hot spot." Second, although the two CBDs are reciprocally coupled at the level of local unfolding of the hinge, the A-to-B and B-to-A pathways are dramatically asymmetrical at the level of global unfolding. Such an asymmetric interdomain cross-talk ensures efficiency and robustness in both the activation and de-activation of PKA.
Highlights
This paper is dedicated to the memory of Captain George A
Class I consists of residues that are highly protected (PF Ͼ 6.5) and rely mainly on transient global unfolding H/D exchange pathways, i.e. the solvent protection of class I residues is not modulated by local unfolding
Our data reveal that the tandem CBD disposition and the transdomain lid of CBD-A, embedded within the N3A motif of CBD-B, ensure that Cyclic adenosine monophosphate (cAMP) binding to either phosphate binding cassette (PBC) affects the hinge regions of both domains, resulting in a fully bidirectional interdomain coupling at the level of local unfolding
Summary
This paper is dedicated to the memory of Captain George A. Mapping the Effects of the R333K Mutation on RI␣-(119 – 379) by Chemical Shifts— to the R209K mutant, the secondary chemical shifts measured for R333K in the presence of 2 mM excess cAMP do not exhibit any appreciable differences from those of the wild-type construct (supplemental Fig. S1, a and c), indicating that the overall structure is not significantly perturbed by the R333K mutation.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have