Signaling through dynamic linkers as revealed by PKA.
Protein kinase A (PKA) is a prototype of multidomain signaling proteins functioning as allosteric conformational switches. Allosteric transitions have been the subject of extensive structural and dynamic investigations focusing mainly on folded domains. However, the current understanding of the allosteric role of partially unstructured linkers flanking globular domains is limited. Here, we show that a dynamic linker in the regulatory subunit (R) of PKA serves not only as a passive covalent thread, but also as an active allosteric element that controls activation of the kinase subunit (C) by tuning the inhibitory preequilibrium of a minimally populated intermediate (apo R). Apo R samples both C-binding competent (inactive) and incompetent (active) conformations within a nearly degenerate free-energy landscape and such degeneracy maximally amplifies the response to weak (∼2RT), but conformation-selective interactions elicited by the linker. Specifically, the R linker that in the R:C complex docks in the active site of C in apo R preferentially interacts with the C-binding incompetent state of the adjacent cAMP-binding domain (CBD). These unanticipated findings imply that the formation of the intermolecular R:C inhibitory interface occurs at the expense of destabilizing the intramolecular linker/CBD interactions in R. A direct implication of this model, which was not predictable solely based on protein structure, is that the disruption of a linker/CBD salt bridge in the R:C complex unexpectedly leads to increased affinity of R for C. The linker includes therefore sites of R:C complex frustration and frustration-relieving mutations enhance the kinase inhibitory potency of R without compromising its specificity.
Department of Chemistry and Chemical Biology, McMaster University, Hamilton, ON, Canada L8S 4M1.
Madoka
M
Akimoto
Rajeevan
R
Selvaratnam
E Tyler
ET
McNicholl
Geeta
G
Verma
Susan S
SS
Taylor
Giuseppe
G
Melacini
D000494:Allosteric Regulation; D000242:Cyclic AMP / Q000378:metabolism; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D015398:Signal Transduction
D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D000242:Cyclic AMP
NMR; allostery; cAMP; dynamics; intrinsically disordered proteins