Phosphorylation Reaction in cAPK Protein Kinase-Free Energy Quantum Mechanical/Molecular Mechanics Simulations

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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“…2). These values are similar to those predicted by theoretical calculations for the loose transition state in the S N 2 phosphoryl transfer (46).…”

Section: Discussionsupporting

confidence: 89%

Phosphoryl Transfer Reaction Snapshots in Crystals

Gerlits

Tian

Das

et al. 2015

Journal of Biological Chemistry

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“…Experimental and theoretical studies have demonstrated that, in phosphoryl transfer by kinases and polymerases, nucleophilic attack on the target phosphate proceeds through a conformation that resembles a trigonal-bipyramidal transition state (31) and that phosphoryl transfer can occur through either an associative or a dissociative mechanism (31)(32)(33). A conserved aspartate (D813 in EGFR, D166 in protein kinase A) is proposed to function as a base acceptor for proton transfer from the hydroxyl group of the substrate, as depicted by the red arrows in Fig.…”

Section: Quantum Mechanics/molecular Mechanics (Qm/mm) Simulation Ofmentioning

confidence: 99%

ErbB3/HER3 intracellular domain is competent to bind ATP and catalyze autophosphorylation

Shi

Telesco

Liu

et al. 2010

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

409

421

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ErbB3/HER3 is one of four members of the human epidermal growth factor receptor (EGFR/HER) or ErbB receptor tyrosine kinase family. ErbB3 binds neuregulins via its extracellular region and signals primarily by heterodimerizing with ErbB2/HER2/Neu. A recently appreciated role for ErbB3 in resistance of tumor cells to EGFR/ErbB2-targeted therapeutics has made it a focus of attention. However, efforts to inactivate ErbB3 therapeutically in parallel with other ErbB receptors are challenging because its intracellular kinase domain is thought to be an inactive pseudokinase that lacks several key conserved (and catalytically important) residuesincluding the catalytic base aspartate. We report here that, despite these sequence alterations, ErbB3 retains sufficient kinase activity to robustly trans-autophosphorylate its intracellular regionalthough it is substantially less active than EGFR and does not phosphorylate exogenous peptides. The ErbB3 kinase domain binds ATP with a K d of approximately 1.1 μM. We describe a crystal structure of ErbB3 kinase bound to an ATP analogue, which resembles the inactive EGFR and ErbB4 kinase domains (but with a shortened αC-helix). Whereas mutations that destabilize this configuration activate EGFR and ErbB4 (and promote EGFR-dependent lung cancers), a similar mutation conversely inactivates ErbB3. Using quantum mechanics/molecular mechanics simulations, we delineate a reaction pathway for ErbB3-catalyzed phosphoryl transfer that does not require the conserved catalytic base and can be catalyzed by the "inactive-like" configuration observed crystallographically. These findings suggest that ErbB3 kinase activity within receptor dimers may be crucial for signaling and could represent an important therapeutic target.dimerization | kinase inhibitor | catalytic mechanism | activation loop R eceptor tyrosine kinases (RTKs) from the EGF receptor (EGFR) or ErbB/HER family play important roles in animal development and disease (1) and are the targets of several important therapeutic agents used clinically to treat cancer. Each receptor contains a large extracellular ligand-binding region (targeted by therapeutic antibodies), a single transmembrane helix, and an intracellular tyrosine kinase domain (TKD) that is flanked by juxtamembrane and C-terminal regulatory regions and is targeted by specific small-molecule kinase inhibitors (1, 2). Ligand binding to the extracellular region promotes homo-or heterodimerization of ErbB receptors, leading to allosteric activation of their intracellular kinase domains through the formation of asymmetric dimers (3-5). Within an activated dimer, the C-terminal regulatory tail is trans-autophosphorylated on tyrosines and recruits downstream signaling molecules that contain phosphotyrosine-binding Src homology-2 (SH2) domains.ErbB3/HER3 is unique among the mammalian ErbB receptors in being generally considered as kinase-inactive (6). When first cloned (7,8), amino acid substitutions were noted at two particular sites that are conserved in other known kinases (9). ...

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“…5) with the subsequent binding of second inhibitory metal ion (13). The binding of the second metal ion promotes its action as a Lewis base that must work on the β-γ bond of the ATP molecule to aid the removal of the terminal γ-PO 4 (31). Efficient binding of the two metal ions at the active site by residues N171 and D184 forms the basis of an efficient phosphotransfer process.…”

Section: Y204a Mutation Abolishes the Synergy Between Nucleotide Andmentioning

confidence: 99%

Mutation of a kinase allosteric node uncouples dynamics linked to phosphotransfer

Ahuja

Kornev

McClendon

et al. 2017

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

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The expertise of protein kinases lies in their dynamic structure, wherein they are able to modulate cellular signaling by their phosphotransferase activity. Only a few hundreds of protein kinases regulate key processes in human cells, and protein kinases play a pivotal role in health and disease. The present study dwells on understanding the working of the protein kinase-molecular switch as an allosteric network of “communities” composed of congruently dynamic residues that make up the protein kinase core. Girvan–Newman algorithm-based community maps of the kinase domain of cAMP-dependent protein kinase A allow for a molecular explanation for the role of protein conformational entropy in its catalytic cycle. The community map of a mutant, Y204A, is analyzed vis-à-vis the wild-type protein to study the perturbations in its dynamic profile such that it interferes with transfer of the γ-phosphate to a protein substrate. Conventional biochemical measurements are used to ascertain the effect of these dynamic perturbations on the kinetic profiles of both proteins. These studies pave the way for understanding how mutations far from the kinase active site can alter its dynamic properties and catalytic function even when major structural perturbations are not obvious from static crystal structures.

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Phosphorylation Reaction in cAPK Protein Kinase-Free Energy Quantum Mechanical/Molecular Mechanics Simulations

Cited by 111 publications

References 58 publications

Phosphoryl Transfer Reaction Snapshots in Crystals

Phosphoryl Transfer Reaction Snapshots in Crystals

ErbB3/HER3 intracellular domain is competent to bind ATP and catalyze autophosphorylation

Mutation of a kinase allosteric node uncouples dynamics linked to phosphotransfer

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