Signaling through dynamic linkers as revealed by PKA

Abstract

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 freeenergy 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.R egulation of signaling systems often relies on multidomain proteins, which function as conformational switches controlled by allosteric effectors (1-13). The structural and dynamic changes experienced by folded domains during effector-dependent allosteric transitions have been extensively investigated (1-21). However, the current understanding of the allosteric role of partially unstructured linkers is at best scant. Although the role of covalent linkage in colocalization of protein domains is well known, it has recently been hypothesized that linkers, although generally quite flexible, have evolved to serve not simply as passive covalent threads connecting one domain to the next (i.e., "beadson-a-string" model), but also as active components of functionally relevant allosteric networks (22)(23)(24)(25). To test this hypothesis, here we have investigated the allosteric role of a critical linker in the regulatory subunit (R) of protein kinase A (PKA). This linker bridges the inhibitory site (IS) and a critical cAMP-binding domain (CBD) of R [i.e., RIα (119-244) or CBD domain A (CBD-A), ref. 26, Fig. 1A]. The R construct spanning the IS, the linker, and CBD-A [i.e., RIα (91-244) or R A in short, Fig. 1A] exhibits affinities for the catalytic subunit of PKA (C) and for cAMP as well as structures that are similar...

A Mechanism for the Auto-inhibition of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) Channel Opening and Its Relief by cAMP

“…However, it is likely that differences may still exist between the CBDs of PKA and HCN. For instance, for one of the PKA CBDs, the apo pre-equilibrium does not appear to be as highly skewed toward the inactive state as in the CBD of the apo-HCN IR (35), explaining why the regulatory subunit of PKA, unlike HCN (11), binds cAMP with a low approximate nanomolar affinity. The differences in binding and allosteric properties among different eukaryotic CBDs will facilitate the selective targeting of CBDs as required for therapeutic purposes (77)(78)(79)(80).…”

“…HN-NOE Measurements-HN-NOE data were acquired as reported previously (35,36) and processed without linear prediction. A 10-s recycle delay was used to include a 5-s proton saturation period.…”

“…H/H Exchange-Amide proton H/H exchange spectra were acquired as described previously (32,35) N] HCN4(579 -707). The protein was expressed in deuterated M9 media, and 1 h prior to induction, 100 mg of methyl-13 C-␣-ketoisovaleric acid and 100 mg of methyl 13 C-␣-ketobutyric acid were added to the media.…”

“…Spin Labeling and Paramagnetic Relaxation Enhancement (PRE) Analysis-The spin label 1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl-methanethiosulfonate (MTSL) was covalently attached to the free cysteine residue in HCN4(579 -707). The reducing reagent was exchanged using a PD10 column before the addition of MTSL (35). The reduced HCN4(579 -707) was incubated with a 3-fold molar excess of MTSL for 3 h at room temperature.…”

“…Ninety six scans were recorded with a recycle delay of 1.50 s. After recording the data for the paramagnetic state, 5 mM DTT was added to obtain the diamagnetic control. The diamagnetic sample was incubated at room temperature for 1 h, after which we repeated the same experiments carried out for the paramag-netic sample (35). HSQC intensity ratios were then calculated as I ox /I red , where I ox is the intensity of signal with MTSL, and I red is the intensity in the reduced sample with DTT.…”