Electronic Measurements of Single-Molecule Catalysis by cAMP-Dependent Protein Kinase A

Sims, Patrick C.; Moody, Issa S.; Choi, Yongki; Dong, Chengjun; Iftikhar, Mariam; Corso, Brad L.; Gül, O. Tolga; Collins, Philip G.; Weiss, Gregory A.

doi:10.1021/ja311604j

Cited by 67 publications

(70 citation statements)

References 31 publications

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“…The dynamic apo enzyme toggles between three major conformational states along the free energy reaction coordinate (open, intermediate, and closed, Figure 1B). ATP binding shifts the enzyme conformational ensemble from an open to an intermediate state, increasing substrate affinity through a K- type binding cooperativity (Sims et al, 2013; Taylor et al, 2005; Taylor et al, 2004). Binding of substrate further shifts the ensemble toward the enzyme’s closed state.…”

Section: Introductionmentioning

confidence: 99%

Uncoupling Catalytic and Binding Functions in the Cyclic AMP-Dependent Protein Kinase A

Kim

Walters

et al. 2016

Structure

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SUMMARY The canonical function of kinases is to transfer a phosphoryl group to substrates, initiating a signaling cascade; while their non-canonical role is to bind other kinases or substrates, acting as scaffolds, competitors, and signal integrators. Here, we show how to uncouple kinases dual function by tuning the binding cooperativity between nucleotide (or inhibitors) and substrate allosterically. We demonstrate this new concept for the C-subunit of protein kinase A (PKA-C). Using thermocalorimetry and NMR, we found a linear correlation between the degree of cooperativity and the population PKA-C’s closed state. The non-hydrolysable ATP analog (ATPγC) does not follow this correlation, suggesting that changing the chemical groups around the phosphoester bond can uncouple kinases dual function. Remarkably, this uncoupling was also found for two ATP-competitive inhibitors, H89 and balanol. Since the mechanism for allosteric cooperativity is not conserved in different kinases, these results may suggest new approaches for designing selective kinase inhibitors.

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Section: Introductionmentioning

confidence: 99%

Uncoupling Catalytic and Binding Functions in the Cyclic AMP-Dependent Protein Kinase A

Kim

Walters

et al. 2016

Structure

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“…However, single molecule electronic measurements of PKAc catalysis indicate that not every open-close conformational cycle results in the phosphorylation reaction and/or product release, which can partially explain the relatively low catalytic efficiency of the enzyme (20). These experiments also caution us on correlating bulk kinetic values, such as the k cat , with the time it takes each individual molecule to go through a particular conformational change or a chemical reaction.…”

mentioning

confidence: 99%

Phosphoryl Transfer Reaction Snapshots in Crystals

Gerlits

Tian

Das

et al. 2015

Journal of Biological Chemistry

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Background: PKAc (catalytic subunit) catalyzes phosphorylation of protein substrates thereby regulating a myriad of cellular processes. Results: X-ray structures of PKAc complexes along the phosphoryl transfer reaction have been obtained. Conclusion:The phosphotransfer follows a multistep mechanism, including conformational changes of the substrate and product groups, a loose transition state, and metal movement. Significance: Mechanistic knowledge about the phosphorylation by PKAc will contribute to understanding of the kinase function and regulation.

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“…After docking, the dynamic phospho-acceptor samples a variety of conformations, some in which the appropriate elements within the ppERK2 active site, ATP, and the phosphorylatable sidechain of Ets-1 (and surrounding regions including the Pro at the P+1 position) are appropriately aligned to enable chemistry (high-activity states), and others in which the relevant elements are not correctly aligned (low-activity states). Indeed, states of higher and lower intrinsic activity have been demonstrated in single-molecule studies of the phosphorylation of a peptide substrate by protein kinase A (PKA) (44). Thus, higher turnover is obtained in cases with a substantial population of high-activity states in the docked ensemble.…”

Section: Molecular Recognition Of Erk2 By Ets-1 Involves Noncanonicalmentioning

confidence: 99%

Local destabilization, rigid body, and fuzzy docking facilitate the phosphorylation of the transcription factor Ets-1 by the mitogen-activated protein kinase ERK2

Piserchio

Warthaka

Kaoud

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Mitogen-activated protein (MAP) kinase substrates are believed to require consensus docking motifs (D-site, F-site) to engage and facilitate efficient site-specific phosphorylation at specific serine/ threonine-proline sequences by their cognate kinases. In contrast to other MAP kinase substrates, the transcription factor Ets-1 has no canonical docking motifs, yet it is efficiently phosphorylated by the MAP kinase ERK2 at a consensus threonine site (T38). Using NMR methodology, we demonstrate that this phosphorylation is enabled by a unique bipartite mode of ERK2 engagement by Ets-1 and involves two suboptimal noncanonical docking interactions instead of a single canonical docking motif. The N terminus of Ets-1 interacts with a part of the ERK2 D-recruitment site that normally accommodates the hydrophobic sidechains of a canonical D-site, retaining a significant degree of disorder in its ERK2-bound state. In contrast, the C-terminal region of Ets-1, including its Pointed (PNT) domain, engages in a largely rigid body interaction with a section of the ERK2 F-recruitment site through a binding mode that deviates significantly from that of a canonical F-site. This latter interaction is notable for the destabilization of a flexible helix that bridges the phospho-acceptor site to the rigid PNT domain. These two spatially distinct, individually weak docking interactions facilitate the highly specific recognition of ERK2 by Ets-1, and enable the optimal localization of its dynamic phospho-acceptor T38 at the kinase active site to enable efficient phosphorylation.MAP kinase | transcription factor | proximity-mediated catalysis | solution NMR T he mitogen activated protein (MAP) kinase ERK2 (extracellular signal-regulated kinase 2) lies at the terminus of a three-tiered phosphorylation-based response to a wide range of extracellular cues that include cytokines, hormones, and growth factors (1-4). ERK2 phosphorylates numerous substrates in both the nucleus and the cytoplasm, including a variety of transcription factors, regulatory kinases, phosphatases, proteins of the nuclear pore complex, and cytoskeleton proteins, among others (3, 4). ERK2-mediated phosphorylation occurs on specific serine/threonine residues that immediately precede a proline; however, the (S/T)P motif alone does not provide sufficient substrate affinity or selectivity to distinguish it from other proline-directed kinases, such as CDK2 (5). To attain high specificity toward native substrates, ERK2 and other MAP kinases use one of two so-called "docking sites," the D-recruitment site (DRS) or the F-recruitment site (FRS) (SI Appendix, Fig.

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Electronic Measurements of Single-Molecule Catalysis by cAMP-Dependent Protein Kinase A

Cited by 67 publications

References 31 publications

Uncoupling Catalytic and Binding Functions in the Cyclic AMP-Dependent Protein Kinase A

Uncoupling Catalytic and Binding Functions in the Cyclic AMP-Dependent Protein Kinase A

Phosphoryl Transfer Reaction Snapshots in Crystals

Local destabilization, rigid body, and fuzzy docking facilitate the phosphorylation of the transcription factor Ets-1 by the mitogen-activated protein kinase ERK2

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