Allosteric communication between cAMP binding sites in the RI subunit of protein kinase A revealed by NMR.
The activation of protein kinase A involves the synergistic binding of cAMP to two cAMP binding sites on the inhibitory R subunit, causing release of the C subunit, which subsequently can carry out catalysis. We used NMR to structurally characterize in solution the RIalpha-(98-381) subunit, a construct comprising both cyclic nucleotide binding (CNB) domains, in the presence and absence of cAMP, and map the effects of cAMP binding at single residue resolution. Several conformationally disordered regions in free RIalpha become structured upon cAMP binding, including the interdomain alphaC:A and alphaC':A helices that connect CNB domains A and B and are primary recognition sites for the C subunit. NMR titration experiments with cAMP, B site-selective 2-Cl-8-hexylamino-cAMP, and A site-selective N(6)-monobutyryl-cAMP revealed that cyclic nucleotide binding to either the B or A site affected the interdomain helices. The NMR resonances of this interdomain region exhibited chemical shift changes upon ligand binding to a single site, either site B or A, with additional changes occurring upon binding to both sites. Such distinct, stepwise conformational changes in this region reflect the synergistic interplay between the two sites and may underlie the positive cooperativity of cAMP activation of the kinase. Furthermore, nucleotide binding to the A site also affected residues within the B domain. The present NMR study provides the first structural evidence of unidirectional allosteric communication between the sites. Trp(262), which lines the CNB A site but resides in the sequence of domain B, is an important structural determinant for intersite communication.
Byeon
In-Ja L
IJ
Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260, USA.
Dao
Khanh K
KK
Jung
Jinwon
J
Keen
Jeffrey
J
Leiros
Ingar
I
Døskeland
Stein O
SO
Martinez
Aurora
A
Gronenborn
Angela M
AM
D000494:Allosteric Regulation / Q000502:physiology; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D000242:Cyclic AMP