ABSTRACT
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.
Subject(s)
raf Kinases/chemistry , raf Kinases/metabolism , Allosteric Regulation , Amino Acid Motifs , Amino Acid Sequence , Animals , Cell Line , Dimerization , Enzyme Activation , Humans , Mice , Models, Molecular , Molecular Sequence Data , Mutation , Phosphorylation , Protein Conformation , Protein Kinases/chemistry , Protein Kinases/genetics , Protein Kinases/metabolism , Proto-Oncogene Proteins B-raf/chemistry , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins c-raf/chemistry , Proto-Oncogene Proteins c-raf/genetics , Proto-Oncogene Proteins c-raf/metabolism , Sequence Alignment , Tryptophan/metabolism , raf Kinases/geneticsABSTRACT
Topological analysis of protein residue networks (PRNs) is a common method that can help to understand the roles of individual residues. Here, we used protein kinase A as a study object and asked what already known functionally important residues can be detected by network analysis. Along several traditional approaches to weight edges in PRNs we used local spatial pattern (LSP) alignment that assigns high weights to edges only if CαCß vectors for the corresponding residues retain their mutual positions and orientation. Our results show that even short molecular dynamic simulations of 10 to 20 ns can give convergent values for betweenness and degree centralities calculated from the LSP-based PRNs. Using these centralities, we were able to clearly distinguish a group of residues that are highly conserved in protein kinases and play important functional and regulatory roles. In comparison, traditional methods based on cross-correlation and linear mutual information were much less efficient for this particular task. These results call for reevaluation of the current methods to generate PRNs.
Subject(s)
Cyclic AMP-Dependent Protein Kinases , Molecular Dynamics SimulationABSTRACT
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.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/chemistry , Cyclic AMP-Dependent Protein Kinases/metabolism , Macromolecular Substances/metabolism , Catalytic Domain , Crystallography, X-Ray , Cyclic AMP/metabolism , Macromolecular Substances/chemistry , Phosphorylation , Protein Isoforms , Protein Structure, Tertiary , Signal TransductionABSTRACT
Conventional protein kinase C (cPKC) isozymes tune the signaling output of cells, with loss-of-function somatic mutations associated with cancer and gain-of-function germline mutations identified in neurodegeneration. PKC with impaired autoinhibition is removed from the cell by quality-control mechanisms to prevent the accumulation of aberrantly active enzyme. Here, we examine how a highly conserved residue in the C1A domain of cPKC isozymes permits quality-control degradation when mutated to histidine in cancer (PKCß-R42H) and blocks down-regulation when mutated to proline in the neurodegenerative disease spinocerebellar ataxia (PKCγ-R41P). Using FRET-based biosensors, we determined that mutation of R42 to any residue, including lysine, resulted in reduced autoinhibition as indicated by higher basal activity and faster agonist-induced plasma membrane translocation. R42 is predicted to form a stabilizing salt bridge with E655 in the C-tail and mutation of E655, but not neighboring E657, also reduced autoinhibition. Western blot analysis revealed that whereas R42H had reduced stability, the R42P mutant was stable and insensitive to activator-induced ubiquitination and down-regulation, an effect previously observed by deletion of the entire C1A domain. Molecular dynamics (MD) simulations and analysis of stable regions of the domain using local spatial pattern (LSP) alignment suggested that P42 interacts with Q66 to impair mobility and conformation of one of the ligand-binding loops. Additional mutation of Q66 to the smaller asparagine (R42P/Q66N), to remove conformational constraints, restored degradation sensitivity. Our results unveil how disease-associated mutations of the same residue in the C1A domain can toggle between gain- or loss-of-function of PKC.
Subject(s)
Neoplasms , Neurodegenerative Diseases , Humans , Isoenzymes/metabolism , Neurodegenerative Diseases/genetics , Protein Kinase C/genetics , Protein Kinase C/metabolism , Mutation , Neoplasms/geneticsABSTRACT
Since publication of the crystal structure of protein kinase (PK)A three decades ago, a structural portrait of the conserved kinase core has been drawn. The next challenge is to elucidate structures of full-length kinases and to address the intrinsically disordered regions (IDRs) that typically flank the core as well as the small linear motifs (SLiMs) that are embedded within the IDRs. It is increasingly apparent that unstructured regions integrate the kinase catalytic chassis into multienzyme-based regulatory networks. The extracellular signal-regulated kinase-ribosomal S6 PK-phosphoinositide-dependent kinase (ERK-RSK-PDK) complex is an excellent example to demonstrate how IDRs and SLiMs govern communication between four different kinase catalytic cores to mediate activation and how in molecular terms these promote the formation of kinase heterodimers in a context dependent fashion.
Subject(s)
Intrinsically Disordered Proteins/chemistry , Protein Kinases/chemistry , Protein Kinases/metabolism , Humans , Intrinsically Disordered Proteins/metabolism , Models, Molecular , Protein DomainsABSTRACT
Allosteric regulation of proteins continues to be an engaging research topic for the scientific community. Models describing allosteric communication have evolved from focusing on conformation-based descriptors of protein structural changes to appreciating the role of internal protein dynamics as a mediator of allostery. Here, we explain a "violin model" for allostery as a contemporary method for approaching the Cooper-Dryden model based on redistribution of protein thermal fluctuations. Based on graph theory, the violin model makes use of community network analysis to functionally cluster correlated protein motions obtained from molecular dynamics simulations. This Review provides the theory and workflow of the methodology and explains the application of violin model to unravel the workings of protein kinase A.
Subject(s)
Community Networks , Molecular Dynamics Simulation , Humans , Allosteric Regulation , MotionABSTRACT
It is difficult to imagine where the signaling community would be today without the Protein Data Bank. This visionary resource, established in the 1970s, has been an essential partner for sharing information between academics and industry for over 3 decades. We describe here the history of our journey with the protein kinases using cAMP-dependent protein kinase as a prototype. We summarize what we have learned since the first structure, published in 1991, why our journey is still ongoing, and why it has been essential to share our structural information. For regulation of kinase activity, we focus on the cAMP-binding protein kinase regulatory subunits. By exploring full-length macromolecular complexes, we discovered not only allostery but also an essential motif originally attributed to crystal packing. Massive genomic data on disease mutations allows us to now revisit crystal packing as a treasure chest of possible protein:protein interfaces where the biological significance and disease relevance can be validated. It provides a new window into exploring dynamic intrinsically disordered regions that previously were deleted, ignored, or attributed to crystal packing. Merging of crystallography with cryo-electron microscopy, cryo-electron tomography, NMR, and millisecond molecular dynamics simulations is opening a new world for the signaling community where those structure coordinates, deposited in the Protein Data Bank, are just a starting point!
Subject(s)
Cyclic AMP-Dependent Protein Kinases/chemistry , Cyclic AMP-Dependent Protein Kinases/history , Animals , Cryoelectron Microscopy , History, 20th Century , History, 21st Century , Humans , Molecular Dynamics Simulation , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Quaternary , Structure-Activity RelationshipABSTRACT
A dense interplay between structure and dynamics underlies the working of proteins, especially enzymes. Protein kinases are molecular switches that are optimized for their regulation rather than catalytic turnover rates. Using long-simulations dynamic allostery analysis, this study describes an exploration of the dynamic kinase:peptide complex. We have used protein kinase A (PKA) as a model system as a generic prototype of the protein kinase superfamily of signaling enzymes. Our results explain the role of dynamic coupling of active-site residues that must work in coherence to provide for a successful activation or inhibition response from the kinase. Amino acid networks-based community analysis allows us to ponder the conformational entropy of the kinase:nucleotide:peptide ternary complex. We use a combination of 7 peptides that include 3 types of PKA-binding partners: Substrates, products, and inhibitors. The substrate peptides provide for dynamic insights into the enzyme:substrate complex, while the product phospho-peptide allows for accessing modes of enzyme:product release. Mapping of allosteric communities onto the PKA structure allows us to locate the more unvarying and flexible dynamic regions of the kinase. These distributions, when correlated with the structural elements of the kinase core, allow for a detailed exploration of key dynamics-based signatures that could affect peptide recognition and binding at the kinase active site. These studies provide a unique dynamic allostery-based perspective to kinase:peptide complexes that have previously been explored only in a structural or thermodynamic context.
Subject(s)
Adenosine Triphosphate/chemistry , Cyclic AMP-Dependent Protein Kinases/chemistry , Enzyme Inhibitors/chemistry , Magnesium/chemistry , Peptides/chemistry , Adenosine Triphosphate/metabolism , Allosteric Regulation , Allosteric Site , Amino Acid Sequence , Catalytic Domain , Cyclic AMP-Dependent Protein Kinases/metabolism , Enzyme Inhibitors/metabolism , Kinetics , Magnesium/metabolism , Molecular Dynamics Simulation , Peptides/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Structure, Tertiary , Substrate Specificity , ThermodynamicsABSTRACT
Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein, and LRRK2 mutants are recognized risk factors for Parkinson's disease (PD). Although the precise mechanisms that control LRRK2 regulation and function are unclear, the importance of the kinase domain is strongly implicated, since 2 of the 5 most common familial LRRK2 mutations (G2019S and I2020T) are localized to the conserved DFGψ motif in the kinase core, and kinase inhibitors are under development. Combining the concept of regulatory (R) and catalytic (C) spines with kinetic and cell-based assays, we discovered a major regulatory mechanism embedded within the kinase domain and show that the DFG motif serves as a conformational switch that drives LRRK2 activation. LRRK2 is quite unusual in that the highly conserved Phe in the DFGψ motif, which is 1 of the 4 R-spine residues, is replaced with tyrosine (DY2018GI). A Y2018F mutation creates a hyperactive phenotype similar to the familial mutation G2019S. The hydroxyl moiety of Y2018 thus serves as a "brake" that stabilizes an inactive conformation; simply removing it destroys a key hydrogen-bonding node. Y2018F, like the pathogenic mutant I2020T, spontaneously forms LRRK2-decorated microtubules in cells, while the wild type and G2019S require kinase inhibitors to form filaments. We also explored 3 different mechanisms that create kinase-dead pseudokinases, including D2017A, which further emphasizes the highly synergistic role of key hydrophobic and hydrophilic/charged residues in the assembly of active LRRK2. We thus hypothesize that LRRK2 harbors a classical protein kinase switch mechanism that drives the dynamic activation of full-length LRRK2.
Subject(s)
Catalytic Domain , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/chemistry , Molecular Dynamics Simulation , HEK293 Cells , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Mutation, MissenseABSTRACT
MUSK encodes the muscle-specific receptor tyrosine kinase (MuSK), a key component of the agrin-LRP4-MuSK-DOK7 signaling pathway, which is essential for the formation and maintenance of highly specialized synapses between motor neurons and muscle fibers. We report a patient with severe early-onset congenital myasthenic syndrome and two novel missense mutations in MUSK (p.C317R and p.A617V). Functional studies show that MUSK p.C317R, located at the frizzled-like cysteine-rich domain of MuSK, disrupts an integral part of MuSK architecture resulting in ablated MuSK phosphorylation and acetylcholine receptor (AChR) cluster formation. MUSK p.A617V, located at the kinase domain of MuSK, enhances MuSK phosphorylation resulting in anomalous AChR cluster formation. The identification and evidence for pathogenicity of MUSK mutations supported the initiation of treatment with ß2-adrenergic agonists with a dramatic improvement of muscle strength in the patient. This work suggests uncharacterized mechanisms in which control of the precise level of MuSK phosphorylation is crucial in governing synaptic structure.
Subject(s)
Mutation , Myasthenic Syndromes, Congenital/diagnosis , Myasthenic Syndromes, Congenital/genetics , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Cholinergic/genetics , Synapses/genetics , Adrenergic beta-2 Receptor Agonists/pharmacology , Adrenergic beta-2 Receptor Agonists/therapeutic use , Alleles , Amino Acid Substitution , Animals , CRISPR-Cas Systems , Cell Line , DNA Mutational Analysis , Female , Gene Targeting , Humans , Mice , Models, Molecular , Molecular Conformation , Muscle Proteins/metabolism , Myasthenic Syndromes, Congenital/drug therapy , Myasthenic Syndromes, Congenital/metabolism , Pedigree , Phosphorylation , Receptor Protein-Tyrosine Kinases/chemistry , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Cholinergic/chemistry , Receptors, Cholinergic/metabolism , Structure-Activity Relationship , Synapses/metabolismABSTRACT
Protein kinase C (PKC) family members are multi-domain proteins whose function is exquisitely tuned by interdomain interactions that control the spatiotemporal dynamics of their signaling. Despite extensive mechanistic studies on this family of enzymes, no structure of a full-length enzyme that includes all domains has been solved. Here, we take into account the biochemical mechanisms that control autoinhibition, the properties of each individual domain, and previous structural studies to propose a unifying model for the general architecture of PKC family members. This model shows how the C2 domains of conventional and novel PKC isozymes, which have different topologies and different positions in the primary structure, can occupy the same position in the tertiary structure of the kinase. This common architecture of conventional and novel PKC isozymes provides a framework for understanding how disease-associated mutations impair PKC function.
Subject(s)
Protein Kinase C/chemistry , Protein Kinase C/metabolism , Animals , Humans , Isoenzymes , Kinetics , Protein Conformation , Protein Domains , Signal TransductionABSTRACT
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.
Subject(s)
Allosteric Regulation , Cyclic AMP-Dependent Protein Kinases/chemistry , Cyclic AMP-Dependent Protein Kinases/genetics , Mutation , Algorithms , Allosteric Site , Animals , Biocatalysis , Catalytic Domain , Crystallography, X-Ray , Cyclic AMP-Dependent Protein Kinases/metabolism , Entropy , Kinetics , Mice , Models, Molecular , Phosphorylation , Protein ConformationABSTRACT
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.
Subject(s)
Protein Kinases/metabolism , Allosteric Regulation , Hydrophobic and Hydrophilic Interactions , PhosphorylationABSTRACT
The intricacies of allosteric regulation of protein kinases continue to engage the research community. Allostery, or control from a distance, is seen as a fundamental biomolecular mechanism for proteins. From the traditional methods of conformational selection and induced fit, the field has grown to include the role of protein motions in defining a dynamics-based allosteric approach. Harnessing of these continuous motions in the protein to exert allosteric effects can be defined by a "violin" model that focuses on distributions of protein vibrations as opposed to concerted pathways. According to this model, binding of an allosteric modifier causes global redistribution of dynamics in the protein kinase domain that leads to changes in its catalytic properties. This model is consistent with the "entropy-driven allostery" mechanism proposed by Cooper and Dryden in 1984 and does not require, but does not exclude, any major structural changes. We provide an overview of practical implementation of the violin model and how it stands amidst the other known models of protein allostery. Protein kinases have been described as the biomolecules of interest. © 2019 IUBMB Life, 71(6):685-696, 2019.
Subject(s)
Allosteric Regulation/genetics , Protein Kinases/chemistry , Proteins/chemistry , Binding Sites/genetics , Entropy , Molecular Dynamics Simulation , Protein Binding/genetics , Protein Conformation , Protein Kinases/genetics , Proteins/genetics , Signal Transduction/geneticsABSTRACT
Eukaryotic protein kinases (EPKs) regulate almost every biological process and have evolved to be dynamic molecular switches; this is in stark contrast to metabolic enzymes, which have evolved to be efficient catalysts. In particular, the highly conserved active site of every EPK is dynamically and transiently assembled by a process that is highly regulated and unique for every protein kinase. We review here the essential features of the kinase core, focusing on the conserved motifs and residues that are embedded in every kinase. We explore, in particular, how the hydrophobic core architecture specifically drives the dynamic assembly of the regulatory spine and consequently the organization of the active site where the γ-phosphate of ATP is positioned by a convergence of conserved motifs including a conserved regulatory triad for transfer to a protein substrate. In conclusion, we show how the flanking N- and C-terminal tails often classified as intrinsically disordered regions, as well as flanking domains, contribute in a highly kinase-specific manner to the regulation of the conserved kinase core. Understanding this process as well as how one kinase activates another remains as two of the big challenges for the kinase signaling community. © 2019 IUBMB Life, 71(6):672-684, 2019.
Subject(s)
Amino Acid Motifs/genetics , Eukaryota/genetics , Protein Kinases/genetics , Adenosine Triphosphate/genetics , Catalytic Domain/genetics , Conserved Sequence/genetics , Hydrophobic and Hydrophilic Interactions , Phosphates/metabolism , Protein Kinases/chemistry , Signal Transduction/genetics , Substrate SpecificityABSTRACT
Allostery is a fundamental regulatory mechanism in biology. Although generally accepted that it is a dynamics-driven process, the exact molecular mechanism of allosteric signal transmission is hotly debated. We argue that allostery is as a part of a bigger picture that also includes fractal-like properties of protein interior, hierarchical protein folding and entropy-driven molecular recognition. Although so far all these phenomena were studied separately, they stem from the same common root: self-organization of polypeptide chains and, thus, has to be studied collectively. This merge will allow the cross-referencing of a broad spectrum of multi-disciplinary data facilitating progress in all these fields.
Subject(s)
Entropy , Proteins/metabolism , Allosteric Regulation , Fractals , Protein FoldingABSTRACT
Close-range electrostatic interactions that form salt bridges are key components of protein stability. Here we investigate the role of these charged interactions in modulating the allosteric activation of protein kinase A (PKA) via computational and experimental mutational studies of a conserved basic patch located in the regulatory subunit's B/C helix. Molecular dynamics simulations evidenced the presence of an extended network of fluctuating salt bridges spanning the helix and connecting the two cAMP binding domains in its extremities. Distinct changes in the flexibility and conformational free energy landscape induced by the separate mutations of Arg239 and Arg241 suggested alteration of cAMP-induced allosteric activation and were verified through in vitro fluorescence polarization assays. These observations suggest a mechanical aspect to the allosteric transition of PKA, with Arg239 and Arg241 acting in competition to promote the transition between the two protein functional states. The simulations also provide a molecular explanation for the essential role of Arg241 in allowing cooperative activation, by evidencing the existence of a stable interdomain salt bridge with Asp267. Our integrated approach points to the role of salt bridges not only in protein stability but also in promoting conformational transition and function.
Subject(s)
Arginine/chemistry , Aspartic Acid/chemistry , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/chemistry , Cyclic AMP/chemistry , Allosteric Regulation , Allosteric Site , Amino Acid Sequence , Arginine/metabolism , Aspartic Acid/metabolism , Catalytic Domain , Cloning, Molecular , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/genetics , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/metabolism , Enzyme Activation , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Kinetics , Molecular Docking Simulation , Molecular Dynamics Simulation , Mutation , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Salts/chemistry , Sequence Alignment , Static Electricity , ThermodynamicsABSTRACT
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.
Subject(s)
Molecular Dynamics Simulation , Protein Kinases/chemistry , Adenosine Triphosphate/metabolism , Allosteric Regulation , Catalytic Domain , Ligands , Magnesium/metabolism , Mutagenesis , Reproducibility of Results , Templates, GeneticABSTRACT
Eukaryotic protein kinases have evolved to be highly regulated and dynamic molecular switches that are typically kept in an inactive state and then activated in response to extracellular signals. The hallmark signature of an active kinase is a hydrophobic spine called the regulatory (R) spine, which consists of four residues, two in the N-lobe and two in the C-lobe. RS1 is in the catalytic loop, RS2 is the Phe in the DFG motif, RS3 is at the C-terminus of the αC-Helix, and RS4 is at the beginning of ß4. Assembly of the R-spine is typically facilitated by phosphorylation of the Activation Loop. The assembled R-spine brings together all of the functional motifs that are essential for transferring the phosphate from ATP to a tethered protein substrate. This includes the G-Loop, which anchors the ATP, the catalytic loop, the DFG motif fused to the Activation Loop, and the αC-Helix. We focus here on the properties of the αC-Helix showing 1) how residues communicate with different parts of the molecule, 2) how it is recruited to the active site as a consequence of assembling of the R-spine, and 3) how it is regulated by linkers/motifs/proteins that lie outside the conserved kinase core. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.
Subject(s)
Protein Kinases/chemistry , Protein Structure, Secondary , Signal Transduction/genetics , Structure-Activity Relationship , Adenosine Triphosphate/chemistry , Amino Acid Motifs , Catalytic Domain , Eukaryota , Phosphorylation , Protein Kinases/metabolismABSTRACT
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.