The structure of MSK1 reveals a novel autoinhibitory conformation for a dual kinase protein

Mitogen and stress-activated kinase-1 (MSK1) is a serine/threonine protein kinase that is activated by either p38 or p42ERK MAPKs in response to stress or mitogenic extracellular stimuli. MSK1 belongs to a family of protein kinases that contain two distinct kinase domains in one polypeptide chain. We report the 1.8 A crystal structure of the N-terminal kinase domain of MSK1. The crystal structure reveals a unique inactive conformation with the ATP binding site blocked by the nucleotide binding loop. This inactive conformation is stabilized by the formation of a new three-stranded beta sheet on the N lobe of the kinase domain. The three beta strands come from residues at the N terminus of the kinase domain, what would be the alphaB helix in the active conformation, and the activation loop. The new three-stranded beta sheet occupies a position equivalent to the N terminus of the alphaC helix in active protein kinases.     

Auto‐activation mechanism of the Mycobacterium tuberculosis PknB receptor Ser/Thr kinase

Many Ser/Thr protein kinases are activated by autophosphorylation, but the mechanism of this process has not been defined. We determined the crystal structure of a mutant of the Ser/Thr kinase domain (KD) of the mycobacterial sensor kinase PknB in complex with an ATP competitive inhibitor and discovered features consistent with an activation complex. The complex formed an asymmetric dimer, with the G helix and the ordered activation loop of one KD in contact with the G helix of the other. The activation loop of this putative ‘substrate’ KD was disordered, with the ends positioned at the entrance to the partner KD active site. Single amino‐acid substitutions in the G‐helix interface reduced activation‐loop phosphorylation, and multiple replacements abolished KD phosphorylation and kinase activation. Phosphorylation of an inactive mutant KD was reduced by G‐helix substitutions in both active and inactive KDs, as predicted by the idea that the asymmetric dimer mimics a trans‐autophosphorylation complex. These results support a model in which a structurally and functionally asymmetric, ‘front‐to‐front’ association mediates autophosphorylation of PknB and homologous kinases.     

Structural insights into the autoactivation mechanism of p21-activated protein kinase

p21-activated kinases (PAKs) play an important role in diverse cellular processes. Full activation of PAKs requires autophosphorylation of a critical threonine/serine located in the activation loop of the kinase domain. Here we report crystal structures of the phosphorylated and unphosphorylated PAK1 kinase domain. The phosphorylated PAK1 kinase domain has a conformation typical of all active protein kinases. Interestingly, the structure of the unphosphorylated PAK1 kinase domain reveals an unusual dimeric arrangement expected in an authentic enzyme-substrate complex, in which the activation loop of the putative "substrate" is projected into the active site of the "enzyme." The enzyme is bound to AMP-PNP and has an active conformation, whereas the substrate is empty and adopts an inactive conformation. Thus, the structure of the asymmetric homodimer mimics a trans-autophosphorylation complex, and suggests that unphosphorylated PAK1 could dynamically adopt both the active and inactive conformations in solution.     

Structure and inhibition of the human cell cycle checkpoint kinase, Wee1A kinase: an atypical tyrosine kinase with a key role in CDK1 regulation

Phosphorylation is critical to regulation of the eukaryotic cell cycle. Entry to mitosis is triggered by the cyclin-dependent kinase CDK1 (Cdc2), which is inactivated during the preceding S and G2 phases by phosphorylation of T14 and Y15. Two homologous kinases, Wee1, which phosphorylates Y15, and Myt1, which phosphorylates both T14 and Y15, mediate this inactivation. We have determined the crystal structure of the catalytic domain of human somatic Wee1 (Wee1A) complexed with an active-site inhibitor at 1.8 A resolution. Although Wee1A is functionally a tyrosine kinase, in sequence and structure it most closely resembles serine/threonine kinases such as Chk1 and cAMP kinases. The crystal structure shows that although the catalytic site closely resembles that of other protein kinases, the activation segment contains Wee1-specific features that maintain it in an active conformation and, together with a key substitution in its glycine-rich loop, help determine its substrate specificity.     

Crystal structure of the kinase domain of WNK1, a kinase that causes a hereditary form of hypertension

WNK kinases comprise a small group of unique serine/threonine protein kinases that have been genetically linked to pseudohypoaldosteronism type II, an autosomal dominant form of hypertension. Here we present the structure of the kinase domain of WNK1 at 1.8 A resolution, solved in a low activity conformation. A lysine residue (Lys-233) is found in the active site emanating from strand beta2 rather than strand beta3 as in other protein kinases. The activation loop adopts a unique well-folded inactive conformation. The conformations of the P+1 specificity pocket, the placement of the conserved active site threonine (Thr-386), and the exterior placement of helix C, contribute to the low activity state. By homology modeling, we identified two hydrophobic residues in the substrate-binding groove that contribute to substrate specificity. The structure of the WNK1 catalytic domain, with its unique active site, may help in the design of therapeutic reagents for the treatment of hypertension.     

Structure and dimerization of the kinase domain from yeast Snf1, a member of the Snf1/AMPK protein family

The Snf1/AMPK kinases are intracellular energy sensors, and the AMPK pathway has been implicated in a variety of metabolic human disorders. Here we report the crystal structure of the kinase domain from yeast Snf1, revealing a bilobe kinase fold with greatest homology to cyclin-dependant kinase-2. Unexpectedly, the crystal structure also reveals a novel homodimer that we show also forms in solution, as demonstrated by equilibrium sedimentation, and in yeast cells, as shown by coimmunoprecipitation of differentially tagged intact Snf1. A mapping of sequence conservation suggests that dimer formation is a conserved feature of the Snf1/AMPK kinases. The conformation of the conserved alphaC helix, and the burial of the activation segment and substrate binding site within the dimer, suggests that it represents an inactive form of the kinase. Taken together, these studies suggest another layer of kinase regulation within the Snf1/AMPK family, and an avenue for development of AMPK-specific activating compounds.     

Crystal structure of an inactive Akt2 kinase domain

Akt/PKB represents a subfamily of three isoforms from the AGC serine/threonine kinase family. Amplification of Akt activity has been implicated in diseases that involve inappropriate cell survival, including a number of human malignancies. The structure of an inactive and unliganded Akt2 kinase domain reveals several features that distinguish it from other kinases. Most of the alpha helix C is disordered. The activation loop in this structure adopts a conformation that appears to sterically hinder the binding of both ATP and peptide substrate. In addition, an intramolecular disulfide bond is observed between two cysteines in the activation loop. Residues within the linker region between the N- and C-terminal lobes also contribute to the inactive conformation by partially occupying the ATP binding site.     

Exploring some of the physico-chemical properties of the LAMMER protein kinase DOA of Drosophila

Members of the highly conserved LAMMER family of protein kinases have been described in all eukaryotes. LAMMER kinases possess markedly similar peptide motifs in their kinase catalytic subdomains that are responsible for phosphotransfer and substrate interaction, suggesting that family members serve similar functions in widely diverged species. This hypothesis is supported by their phosphorylation of SR and SR-related proteins in diverged species. Here we describe a 3-dimensional homology model of the catalytic domain of DOA, a representative LAMMER kinase, encoded by the Drosophila locus Darkener of apricot (Doa). Homology modeling of DOA based on a Sky1p template revealed a highly conserved structural framework within conserved core regions. These adopt typical kinase folding like that of other protein kinases. However, in contrast to Sky1p, some structural features, such as those in helix ?C suggest that the DOA kinase is not a constitutively active enzyme but requires activation. This may occur by phosphorylation within an activation loop that forms a broad turn and in which interactions between the side chains occur across the loop. The fold of the activation loop is stabilized through interactions with residues in the C-terminal tail, which is not part of the conserved kinase core and is variable among protein kinases. Immediately following the activation loop in the segment between the ?9 sheet and helix ?F is a P+1 loop. The electrostatic surface potential of the DOA substrate binding groove is largely negative, as it is in other known SR protein kinases, suggesting that DOA substrates must be basic. All differences between D. melanogaster and other Drosophila species are single amino acid changes situated in regions outside of any ?-helices or ?-sheets, and after modeling these had absolutely no visible effect on protein structure. The absence of evolved amino acid changes among 12 Drosophila species that would cause at least predictable changes in DOA structure indicate that evolution has already selected evolved mutations for having minimal effect on kinase structure.     

Anatomy of a structural pathway for activation of the catalytic domain of Src kinase Hck

Src kinase activity is implicated in the regulation of downstream signal transduction pathways involved in cell growth processes. Crystallographic studies indicate that activation of Hematopoietic cell kinase (Hck), a member of the Src kinase family, is accompanied structurally by a large conformational change in two specific parts of its catalytic domain: the alpha-C helix and the activation loop. In the present study, molecular dynamics (MD) simulations are used to characterize the transformation pathway from the inactive to the active state. Four different conditions are considered: the presence or absence of Tyr416 phosphorylation in the activation loop, and the presence or absence of substrate ATP-2Mg(+2) in the active site. Effective free energy landscapes for local residues are determined using a combination of restrained MD simulations with a Root Mean Square Distance (RMSD) biasing potential to enforce the change followed by free MD simulations to allow relaxation from artificially enforced intermediates. A conceptual subdivision of the kinase catalytic domain into four moving parts: the flexible activation loop segment, the buried activation loop segment, the alpha-C helix, and the N-terminal end linker, leads to a concise hypothesis in which each of the moving parts are only required to be coupled to their nearest neighbor to ensure bidirectional allostery in the regulation of protein tyrosine kinases. Both Tyr416 phosphorylation and ATP-2Mg(+2) affect the local backbone torsional free energy landscapes accompanying the structural transition. When these two factors are present together, a metastable coordinated state of ATP-2Mg(+2) and the phosphorylated Tyr416 is observed that offers a possible explanation for the inhibition of protein kinase activity due to increase in Mg(+2) ion concentration.     

Crystal structures of IRAK-4 kinase in complex with inhibitors: a serine/threonine kinase with tyrosine as a gatekeeper

Interleukin-1 (IL-1) receptor-associated kinase-4 (IRAK-4) is a serine/threonine kinase that plays an essential role in signal transduction by Toll/IL-1 receptors (TIRs). Here, we report the crystal structures of the phosphorylated human IRAK-4 kinase domain in complex with a potent inhibitor and with staurosporine to 2.0 and 2.2 A, respectively. The structures reveal that IRAK-4 has a unique tyrosine gatekeeper residue that interacts with the conserved glutamate from helix alphaC. Consequently, helix alphaC is "pulled in" to maintain the active orientation, and the usual pre-existing hydrophobic back pocket of the ATP-binding site is abolished. The peptide substrate-binding site is more open when compared with other protein kinases due to a marked movement of helix alphaG. The pattern of phosphate ligand interactions in the activation loop bears a close resemblance to that of a tyrosine kinase. Our results provide insights into IRAK-4 function and the design of selective inhibitors.     

The crystal structure of a plant 3-ketoacyl-CoA thiolase reveals the potential for redox control of peroxisomal fatty acid beta-oxidation

Crystal structures of peroxisomal Arabidopsis thaliana 3-ketoacyl-CoA thiolase (AtKAT), an enzyme of fatty acid beta-oxidation, are reported. The subunit, a typical thiolase, is a combination of two similar alpha/beta domains capped with a loop domain. The comparison of AtKAT with the Saccharomyces cerevisiae homologue (ScKAT) structure reveals a different placement of subunits within the functional dimers and that a polypeptide segment forming an extended loop around the open catalytic pocket of ScKAT converts to alpha-helix in AtKAT, and occludes the active site. A disulfide is formed between Cys192, on this helix, and Cys138, a catalytic residue. Access to Cys138 is determined by the structure of this polypeptide segment. AtKAT represents an oxidized, previously unknown inactive form, whilst ScKAT is the reduced and active enzyme. A high level of sequence conservation is observed, including Cys192, in eukaryotic peroxisomal, but not mitochondrial or prokaryotic KAT sequences, for this labile loop/helix segment. This indicates that KAT activity in peroxisomes is influenced by a disulfide/dithiol change linking fatty acid beta-oxidation with redox regulation.     

Structure of the catalytic and ubiquitin-associated domains of the protein kinase MARK/Par-1

The Ser/Thr kinase MARK2 phosphorylates tau protein at sites that cause detachment from microtubules in Alzheimer neurofibrillary degeneration. Homologs of MARK2 include Par-1 in C. elegans and Drosophila, which generates embryonic polarity. We report the X-ray structure of the catalytic and ubiquitin-associated domains (UBA) of human MARK2. The activity was altered by mutations in the ATP binding site and/or activation loop. The catalytic domain shows the small and large lobes typical of kinases. The substrate cleft is in an inactive, open conformation in the inactivated and the wild-type structure. The UBA domain is attached via a taut linker to the large lobe of the kinase domain and leans against a hydrophobic patch on the small lobe. The UBA structure is unusual because the orientation of its third helix is inverted, relative to previous structures. Possible implications of the structure for the regulation of kinase activity are discussed.     

Crystal structure of Hck in complex with a Src family-selective tyrosine kinase inhibitor.

The crystal structure of the autoinhibited form of Hck has been determined at 2.0 A resolution, in complex with a specific pyrazolo pyrimidine-type inhibitor, PP1. The activation segment, a key regulatory component of the catalytic domain, is unphosphorylated and is visualized in its entirety. Tyr-416, the site of activating autophosphorylation in the Src family kinases, is positioned such that access to the catalytic machinery is blocked. PP1 is bound at the ATP-binding site of the kinase, and a methylphenyl group on PP1 is inserted into an adjacent hydrophobic pocket. The enlargement of this pocket in autoinhibited Src kinases suggests a route toward the development of inhibitors that are specific for the inactive forms of these proteins.     

Crystal structures of the N-terminal kinase domain of human RSK1 bound to three different ligands: Implications for the design of RSK1 specific inhibitors

The p90 ribosomal S6 kinases (RSKs) also known as MAPKAP-Ks are serine/threonine protein kinases that are activated by ERK or PDK1 and act as downstream effectors of mitogen-activated protein kinase (MAPK). RSK1, a member of the RSK family, contains two distinct kinase domains in a single polypeptide chain, the regulatory C-terminal kinase domain (CTKD) and the catalytic N-terminal kinase domain (NTKD). Autophosphorylation of the CTKD leads to activation of the NTKD that subsequently phosphorylates downstream substrates. Here we report the crystal structures of the unactivated RSK1 NTKD bound to different ligands at 2.0 A resolution. The activation loop and helix alphaC, key regulatory elements of kinase function, are disordered. The DFG motif of the inactive RSK1 adopts an "active-like" conformation. The beta-PO(4) group in the AMP-PCP complex adopts a unique conformation that may contribute to inactivity of the enzyme. Structures of RSK1 ligand complexes offer insights into the design of novel anticancer agents and into the regulation of the catalytic activity of RSKs.     

A phosphoserine/threonine-binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation

The growth factor-activated AGC protein kinases RSK, S6K, PKB, MSK and SGK are activated by serine/threonine phosphorylation in the activation loop and in the hydrophobic motif, C-terminal to the kinase domain. In some of these kinases, phosphorylation of the hydrophobic motif creates a specific docking site that recruits and activates PDK1, which then phosphorylates the activation loop. Here, we discover a pocket in the kinase domain of PDK1 that recognizes the phosphoserine/phosphothreonine in the hydrophobic motif by identifying two oppositely positioned arginine and lysine residues that bind the phosphate. Moreover, we demonstrate that RSK2, S6K1, PKBalpha, MSK1 and SGK1 contain a similar phosphate-binding pocket, which they use for intramolecular interaction with their own phosphorylated hydrophobic motif. Molecular modelling and experimental data provide evidence for a common activation mechanism in which the phosphorylated hydrophobic motif and activation loop act on the alphaC-helix of the kinase structure to induce synergistic stimulation of catalytic activity. Sequence conservation suggests that this mechanism is a key feature in activation of >40 human AGC kinases.     

Structure of mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 suggests a bifunctional switch that couples kinase activation with nuclear export

MAPK-activated protein kinase 2 (MAPKAPK2), one of several kinases directly phosphorylated and activated by p38 MAPK, plays a central role in the inflammatory response. The activated MAPKAPK2 phosphorylates its nuclear targets CREB/ATF1, serum response factor, and E2A protein E47 and its cytoplasmic targets HSP25/27, LSP-1, 5-lipoxygenase, glycogen synthase, and tyrosine hydroxylase. The crystal structure of unphosphorylated MAPKAPK2, determined at 2.8 A resolution, includes the kinase domain and the C-terminal regulatory domain. Although the protein is inactive, the kinase domain adopts an active conformation with aspartate 366 mimicking the missing phosphorylated threonine 222 in the activation loop. The C-terminal regulatory domain forms a helix-turn-helix plus a long strand. Phosphorylation of threonine 334, which is located between the kinase domain and the C-terminal regulatory domain, may serve as a switch for MAPKAPK2 nuclear import and export. Phosphorylated MAPKAPK2 masks the nuclear localization signal at its C terminus by binding to p38. It unmasks the nuclear export signal, which is part of the second C-terminal helix packed along the surface of kinase domain C-lobe, and thereby carries p38 to the cytoplasm.     

Crystal structure of a C-terminal deletion mutant of human protein kinase CK2 catalytic subunit

Protein kinase CK2 (formerly called: casein kinase 2) is a heterotetrameric enzyme composed of two separate catalytic chains (CK2alpha) and a stable dimer of two non-catalytic subunits (CK2beta). CK2alpha is a highly conserved member of the superfamily of eukaryotic protein kinases. The crystal structure of a C-terminal deletion mutant of human CK2alpha was solved and refined to 2.5A resolution. In the crystal the CK2alpha mutant exists as a monomer in agreement with the organization of the subunits in the CK2 holoenzyme. The refined structure shows the helix alphaC and the activation segment, two main regions of conformational plasticity and regulatory importance in eukaryotic protein kinases, in active conformations stabilized by extensive contacts to the N-terminal segment. This arrangement is in accordance with the constitutive activity of the enzyme. By structural superimposition of human CK2alpha in isolated form and embedded in the human CK2 holoenzyme the loop connecting the strands beta4 and beta5 and the ATP-binding loop were identified as elements of structural variability. This structural comparison suggests that the ATP-binding loop may be the key region by which the non-catalytic CK2beta dimer modulates the activity of CK2alpha. The beta4/beta5 loop was found in a closed conformation in contrast to the open conformation observed for the CK2alpha subunits of the CK2 holoenzyme. CK2alpha monomers with this closed beta4/beta5 loop conformation are unable to bind CK2beta dimers in the common way for sterical reasons, suggesting a mechanism to protect CK2alpha from integration into CK2 holoenzyme complexes. This observation is consistent with the growing evidence that CK2alpha monomers and CK2beta dimers can exist in vivo independently from the CK2 holoenzyme and may possess physiological roles of their own.     

Crystal structure of domain‐swapped STE20 OSR1 kinase domain

OSR1 (oxidative stress-responsive-1) and SPAK (Ste20/Sps1-related proline/alanine-rich kinase) belong to the GCK-VI subfamily of Ste20 group kinases. OSR1 and SPAK are key regulators of NKCCs (Na⁺/K⁺/2Cl⁻ cotransporters) and activated by WNK family members (with-no-lysine kinase), mutations of which are known to cause Gordon syndrome, an autosomal dominant form of inherited hypertension. The crystal structure of OSR1 kinase domain has been solved at 2.25 Å. OSR1 forms a domain-swapped dimer in an inactive conformation, in which P+1 loop and αEF helix are swapped between dimer-related monomers. Structural alignment with nonswapped Ste20 TAO2 kinase indicates that the integrity of chemical interactions in the kinase domain is well preserved in the domain-swapped interfaces. The OSR1 kinase domain has now been added to a growing list of domain-swapped protein kinases recently reported, suggesting that the domain-swapping event provides an additional layer of complexity in regulating protein kinase activity.     

Crystal structures of the Mnk2 kinase domain reveal an inhibitory conformation and a zinc binding site

Human mitogen-activated protein kinases (MAPK)-interacting kinases 1 and 2 (Mnk1 and Mnk2) target the translational machinery by phosphorylation of the eukaryotic initiation factor 4E (eIF4E). Here, we present the 2.1 A crystal structure of a nonphosphorylated Mnk2 fragment that encompasses the kinase domain. The results show Mnk-specific features such as a zinc binding motif and an atypical open conformation of the activation segment. In addition, the ATP binding pocket contains an Asp-Phe-Asp (DFD) in place of the canonical magnesium binding Asp-Phe-Gly (DFG) motif. The phenylalanine of this motif sticks into the ATP binding pocket and blocks ATP binding as observed with inhibitor bound and, thus, inactive p38 kinase. Replacement of the DFD by the canonical DFG motif affects the conformation of Mnk2, but not ATP binding and kinase activity. The results suggest that the ATP binding pocket and the activation segment of Mnk2 require conformational switches to provide kinase activity.