Communication between tandem cAMP binding domains in the regulatory subunit of protein kinase A-Ialpha as revealed by domain-silencing mutations.
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.
McNicholl
E Tyler
ET
Department of Chemistry and Chemical Biology and Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8S 4M1, Canada.
Das
Rahul
R
Department of Chemistry and Chemical Biology and Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8S 4M1, Canada.
SilDas
Soumita
S
Department of Chemistry and Chemical Biology and Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8S 4M1, Canada.
Taylor
Susan S
SS
Department of Chemistry and Biochemistry, Department of Pharmacology, and Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California 92093-0654.
Melacini
Giuseppe
G
Department of Chemistry and Chemical Biology and Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8S 4M1, Canada. Electronic address: melacin@mcmaster.ca.
D000595:Amino Acid Sequence; D001665:Binding Sites; D000242:Cyclic AMP / Q000378:metabolism*; D054743:Cyclic AMP-Dependent Protein Kinase Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020868:Gene Silencing; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D017386:Sequence Homology, Amino Acid
D000242:Cyclic AMP; D054743:Cyclic AMP-Dependent Protein Kinase Type I