Dynamic architecture of a protein kinase McClendon, Christopher L; Kornev, Alexandr P; Gilson, Michael K ...
Proceedings of the National Academy of Sciences - PNAS,
10/2014, Letnik:
111, Številka:
43
Journal Article
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Significance Protein kinases represent a critically important family of regulatory enzymes. Their activity can be altered by mutations and binding events distant from the active site. To understand ...the nature of these long-distance effects, we used microsecond-timescale molecular dynamic simulation to subdivide a prototypical kinase, protein kinase A, into contiguous communities that exhibit internally correlated motions. Surprisingly, most of these unconventional structural entities were centered around known protein kinase functions. We thus propose a new framework for analysis of protein kinase structure and function that differs from traditional representations based simply on sequence motifs and secondary structure elements. These results extend our view on the dynamic nature of protein kinases and open a door to understanding of allosteric signaling in these enzymes.
Protein kinases are dynamically regulated signaling proteins that act as switches in the cell by phosphorylating target proteins. To establish a framework for analyzing linkages between structure, function, dynamics, and allostery in protein kinases, we carried out multiple microsecond-scale molecular-dynamics simulations of protein kinase A (PKA), an exemplar active kinase. We identified residue–residue correlated motions based on the concept of mutual information and used the Girvan–Newman method to partition PKA into structurally contiguous “communities.” Most of these communities included 40–60 residues and were associated with a particular protein kinase function or a regulatory mechanism, and well-known motifs based on sequence and secondary structure were often split into different communities. The observed community maps were sensitive to the presence of different ligands and provide a new framework for interpreting long-distance allosteric coupling. Communication between different communities was also in agreement with the previously defined architecture of the protein kinase core based on the “hydrophobic spine” network. This finding gives us confidence in suggesting that community analyses can be used for other protein kinases and will provide an efficient tool for structural biologists. The communities also allow us to think about allosteric consequences of mutations that are linked to disease.
Although RAF kinases are critical for controlling cell growth, their mechanism of activation is incompletely understood. Recently, dimerization was shown to be important for activation. Here we show ...that the dimer is functionally asymmetric with one kinase functioning as an activator to stimulate activity of the partner, receiver kinase. The activator kinase did not require kinase activity but did require N-terminal phosphorylation that functioned allosterically to induce cis-autophosphorylation of the receiver kinase. Based on modeling of the hydrophobic spine assembly, we also engineered a constitutively active mutant that was independent of Ras, dimerization, and activation-loop phosphorylation. As N-terminal phosphorylation of BRAF is constitutive, BRAF initially functions to activate CRAF. N-terminal phosphorylation of CRAF was dependent on MEK, suggesting a feedback mechanism and explaining a key difference between BRAF and CRAF. Our work illuminates distinct steps in RAF activation that function to assemble the active conformation of the RAF kinase.
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•RAF dimers are asymmetric with one component allosterically activating the other•N-terminal phosphorylation but not kinase activity is required on the activator•Dimerization induces cis-autophosphorylation of the RAF activation loop•Mutations that stabilize the active conformation do not require dimerization
RAF kinase dimerization is important for its activation. Dimerization allosterically induces the active kinase conformation through the asymmetric phosphorylation of the activation loop on one molecule, which activates the second molecule by inducing its cis-autophosphorylation.
Protein kinases are dynamic molecular switches that have evolved to be only transiently activated. Kinase activity is embedded within a conserved kinase core, which is typically regulated by ...associated domains, linkers and interacting proteins. Moreover, protein kinases are often tethered to large macromolecular complexes to provide tighter spatiotemporal control. Thus, structural characterization of kinase domains alone is insufficient to explain protein kinase function and regulation in vivo. Recent progress in structural characterization of cyclic AMP-dependent protein kinase (PKA) exemplifies how our knowledge of kinase signalling has evolved by shifting the focus of structural studies from single kinase subunits to macromolecular complexes.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Dynamics-Driven Allostery in Protein Kinases Kornev, Alexandr P.; Taylor, Susan S.
Trends in biochemical sciences (Amsterdam. Regular ed.),
11/2015, Letnik:
40, Številka:
11
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Protein kinases have very dynamic structures and their functionality strongly depends on their dynamic state. Active kinases reveal a dynamic pattern with residues clustering into semirigid ...communities that move in μs–ms timescale. Previously detected hydrophobic spines serve as connectors between communities. Communities do not follow the traditional subdomain structure of the kinase core or its secondary structure elements. Instead they are organized around main functional units. Integration of the communities depends on the assembly of the hydrophobic spine and phosphorylation of the activation loop. Single mutations can significantly disrupt the dynamic infrastructure and thereby interfere with long-distance allosteric signaling that propagates throughout the whole molecule. Dynamics is proposed to be the underlying mechanism for allosteric regulation in protein kinases.
Protein kinase allostery is based on changes in dynamics of large ensembles of residues that emerge stochastically inside the protein.
Active kinases are in a particular dynamic mode that is characterized by an ATP-dependent μs–ms timescale that radiates across the molecule.
The protein kinase core consists of groups of residues (communities) that move as semi-rigid bodies and correlate with catalytic and regulatory functions. Non-conserved elements are integrated with the core communities and are essential for the dynamic regulation of the core.
Assembly of the active kinase, as defined by the integrated communities, is mediated by the assembly of the regulatory spine and is often facilitated by activation loop phosphorylation.
Dynamics mediate the classic connection between a protein structure and its function even when major conformational changes are not apparent.
Eukayotic protein kinases evolved as a family of highly dynamic molecules with strictly organized internal architecture. A single hydrophobic F-helix serves as a central scaffold for assembly of the ...entire molecule. Two non-consecutive hydrophobic structures termed “spines” anchor all the elements important for catalysis to the F-helix. They make firm, but flexible, connections within the molecule, providing a high level of internal dynamics of the protein kinase. During the course of evolution, protein kinases developed a universal regulatory mechanism associated with a large activation segment that can be dynamically folded and unfolded in the course of cell functioning. Protein kinases thus represent a unique, highly dynamic, and precisely regulated set of switches that control most biological events in eukaryotic cells.
The fidelity of intracellular signaling hinges on the organization of dynamic activity architectures. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G ...protein-coupled receptors achieve specificity despite converging on a ubiquitous messenger, cyclic adenosine monophosphate (cAMP). However, the mechanisms responsible for spatially constraining this diffusible messenger remain elusive. Here, we reveal that the type I regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS) as a function of cAMP signaling to form biomolecular condensates enriched in cAMP and PKA activity, critical for effective cAMP compartmentation. We further show that a PKA fusion oncoprotein associated with an atypical liver cancer potently blocks RIα LLPS and induces aberrant cAMP signaling. Loss of RIα LLPS in normal cells increases cell proliferation and induces cell transformation. Our work reveals LLPS as a principal organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture.
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•PKA RIα undergoes liquid-liquid phase separation in response to cAMP dynamics•RIα condensates dynamically sequester cAMP and retain high PKA activity•RIα phase separation is necessary for effective cAMP compartmentation•A PKA oncoprotein disrupts RIα bodies, leading to aberrant signaling and growth
cAMP-responsive condensate formation by PKA’s RIα subunit controls local signaling, and disruption of phase separation in this context contributes to tumorigenesis.
Eukaryotic protein kinases (EPKs) regulate numerous signaling processes by phosphorylating targeted substrates through the highly conserved catalytic domain. Our previous computational studies ...proposed a model stating that a properly assembled nonlinear motif termed the Regulatory (R) spine is essential for catalytic activity of EPKs. Here we define the required intramolecular interactions and biochemical properties of the R-spine and the newly identified "Shell" that surrounds the R-spine using site-directed mutagenesis and various in vitro phosphoryl transfer assays using cyclic AMP-dependent protein kinase as a representative of the entire kinome. Analysis of the 172 available Apo EPK structures in the protein data bank (PDB) revealed four unique structural conformations of the R-spine that correspond with catalytic inactivation of various EPKs. Elucidating the molecular entities required for the catalytic activation of EPKs and the identification of these inactive conformations opens new avenues for the design of efficient therapeutic EPK inhibitors.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
This randomized trial showed no effect of early adenotonsillectomy, as compared with watchful waiting, on the primary outcome of attention and executive functioning in children with obstructive sleep ...apnea. Many secondary outcomes favored early surgery.
The childhood obstructive sleep apnea syndrome is associated with numerous adverse health outcomes, including cognitive and behavioral deficits.
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The most commonly identified risk factor for the childhood obstructive sleep apnea syndrome is adenotonsillar hypertrophy. Thus, the primary treatment is adenotonsillectomy, which accounts for more than 500,000 procedures annually in the United States alone.
2
Nevertheless, there has been no controlled study evaluating the benefits and risks of adenotonsillectomy, as compared with watchful waiting, for the management of the obstructive sleep apnea syndrome.
The Childhood Adenotonsillectomy Trial (CHAT) was designed to evaluate the efficacy of early adenotonsillectomy versus watchful waiting with supportive . . .
The 2 major molecular switches in biology, kinases and GTPases, are both contained in the Parkinson disease-related leucine-rich repeat kinase 2 (LRRK2). Using hydrogen-deuterium exchange mass ...spectrometry (HDX-MS) and molecular dynamics (MD) simulations, we generated a comprehensive dynamic allosteric portrait of the C-terminal domains of LRRK2 (LRRK2RCKW). We identified 2 helices that shield the kinase domain and regulate LRRK2 conformation and function. One helix in COR-B (COR-B Helix) tethers the COR-B domain to the αC helix of the kinase domain and faces its activation loop, while the C-terminal helix (Ct-Helix) extends from the WD40 domain and interacts with both kinase lobes. The Ct-Helix and the N-terminus of the COR-B Helix create a "cap" that regulates the N-lobe of the kinase domain. Our analyses reveal allosteric sites for pharmacological intervention and confirm the kinase domain as the central hub for conformational control.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Protein kinases constitute a large protein family of important regulators in all eukaryotic cells. All of the protein kinases have a similar bilobal fold, and their key structural features have been ...well studied. However, the recent discovery of non-contiguous hydrophobic ensembles inside the protein kinase core shed new light on the internal organization of these molecules. Two hydrophobic “spines” traverse both lobes of the protein kinase molecule, providing a firm but flexible connection between its key elements. The spine model introduces a useful framework for analysis of intramolecular communications, molecular dynamics, and drug design.
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