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We combine experiment and computer simulation to show how macromolecular crowding dramatically affects the structure, function, and folding landscape of phosphoglycerate kinase (PGK). Fluorescence labeling shows that compact states of yeast PGK are populated as the amount of crowding agents (Ficoll 70) increases. Coarse-grained molecular simulations reveal three compact ensembles: C (crystal structure), CC (collapsed crystal), and Sph (spherical compact). With an adjustment for viscosity, crowded wild-type PGK and fluorescent PGK are about 15 times or more active in 200 mg/ml Ficoll than in aqueous solution. Our results suggest a previously undescribed solution to the classic problem of how the ADP and diphosphoglycerate binding sites of PGK come together to make ATP: Rather than undergoing a hinge motion, the ADP and substrate sites are already located in proximity under crowded conditions that mimic the in vivo conditions under which the enzyme actually operates. We also examine T-jump unfolding of PGK as a function of crowding experimentally. We uncover a nonmonotonic folding relaxation time vs. Ficoll concentration. Theory and modeling explain why an optimum concentration exists for fastest folding. Below the optimum, folding slows down because the unfolded state is stabilized relative to the transition state. Above the optimum, folding slows down because of increased viscosity. enzymatic activity | FRET | folding kinetics | thermal denaturation | protein conformational changes doi/ 10.1073/pnas.1006760107

Compared to bacteria, archaea and eukaryotes employ an additional enzyme for the biosynthesis of selenocysteine (Sec), the 21st natural amino acid (aa). An essential RNA-dependent kinase, Ophosphoseryl-[tRNA.sup.Sec] kinase (PSTK), converts seryl-[tRNA.sup.Sec] to Ophosphoseryl-[tRNA.sup.Sec], the immediate precursor of selenocysteinyl-[tRNA.sup.Sec]. The sequence of Methanocaldococcusjannaschii PSTK (MjPSTK) suggests an N-terminal kinase domain (177 aa) followed by a presumed tRNA binding region (75 aa). The structures of MjPSTK complexed with ADP and AMPPNP revealed that this enzyme belongs to the P-loop kinase class, and that the kinase domain is closely related to gluconate kinase and adenylate kinase. ATP is bound by the P-loop domain (residues 11-18). Formed by antiparallel dimerization of two PSTK monomers, the enzyme structure shows a deep groove with positive electrostatic potential. Located in this groove is the enzyme's active site, which biochemical and genetic data suggest is composed of Asp-41, Arg-44, Glu-55, Tyr-82, Tyr-83, Met-86, and Met-132. Based on structural comparison with Escherichia coil adenylate kinase a docking model was generated that assigns these amino acids to the recognition of the terminal A76-Ser moieties of Ser-[tRNA.sup.Sec]. The geometry and electrostatic environment of the groove in MjPSTK are perfectly complementary to the unusually long acceptor helix of [tRNA.sup.Sec.]. amino acid conversion | aminoacyl-tRNA

Cyclin-dependent kinase 4 (CDK4)/cyclin D complexes are expressed early in the G1 phase of the cell cycle and stimulate the expression of genes required for G1 progression by phosphorylation of the product of the retinoblastoma gene, pRb. To elaborate the molecular pathway of CDK4 activation and substrate selection we have determined the structure of nonphosphorylated CDK4/ cyclin D3. This structure of an authentic CDK/cyclin complex shows that cyclin binding may not be sufficient to drive the CDK active site toward an active conformation. Phosphorylated CDK4/cyclin D3 is active as a pRb kinase and is susceptible to inhibition by [p27.sup.Kip1]. Unlike CDK2/cyclin A, CDK4/cyclin D3 can be inactivated by treatment with [lambda]-phosphatase, implying that phosphorylated T172 is accessible to a generic phosphatase while bound to a cyclin. Taken together, these results suggest that the structural mechanism of CDK4/cyclin D3 activation differs markedly from that of previously studied CDK/cyclin complexes. cell cycle | kinase | phosphorylation | X-ray crystallography | G1 phase

The cyclin D1-cyclin-dependent kinase 4 (CDK4) complex is a key regulator of the transition through the [G.sub.1] phase of the cell cycle. Among the cyclin/CDKs, CDK4 and cyciin D1 are the most frequently activated by somatic genetic alterations in multiple tumor types. Thus, aberrant regulation of the CDK4/cyclin D1 pathway plays an essential role in oncogenesis; hence, CDK4 is a genetically validated therapeutic target. Although X-ray crystallographic structures have been determined for various CDK/cyclin complexes, CDK4/cyclin D1 has remained highly refractory to structure determination. Here, we report the crystal structure of CDK4 in complex with cyclin D1 at a resolution of 2.3 [Angstrom]. Although CDK4 is bound to cyclin D1 and has a phosphorylated T-loop, CDK4 is in an inactive conformation and the conformation of the heterodimer diverges from the previously known CDK/cyclin binary complexes, which suggests a unique mechanism for the process of CDK4 regulation and activation. cell cycle | kinase | X-ray | CDK4 | cyclinD

The phosphoinositide phosphatidylinositol 4-phosphate (Ptdlns4P) is an essential signaling lipid that regulates secretion and polarization of the actin cytoskeleton. In Saccharomyces cerevisiae, the Ptdlns 4-kinase Stt4 catalyzes the synthesis of Ptdlns4P at the plasma membrane (PM). In this paper, we identify and characterize two novel regulatory components of the Stt4 kinase complex, Ypp1 and Efr3. The essential gene YPP1 encodes a conserved protein that colocalizes with Stt4 at cortical punctate structures and regulates the stability of this lipid kinase. Accordingly, Ypp1 interacts with distinct regions on Stt4 that are necessary for the assembly and recruitment of multiple copies of the kinase into phosphoinositide kinase (PIK) patches. We identify the membrane protein Efr3 as an additional component of Stt4 PIK patches. Efr3 is essential for assembly of both Yppl and Stt4 at PIK patches. We conclude that Yppl and Efr3 are required for the formation and architecture of Stt4 PIK patches and ultimately PM-based Ptdlns4P signaling.

Structure-based design methods are used for designing and synthesizing a new series of 3-benzofuranyl-4-indolylmaleimides as potent and relatively selective glycogen synthase kinase-3[beta] (GSK-3[beta]) inhibitors. The analysis has demonstrated the efficacy of a GSK-3[beta] inhibitor in a novel mouse model of mania.

Enzymatic analysis of recombinant proteins was carried out and Saccharomyces cervisiae was subjected to feedback inhibition by proline. It was seen that yeast cell expressing mutant [gamma]-glutamyl kinase were more tolerant to freezing stress than cells expressing the Asp154Asn mutant.

The study describes the design, synthesis and phosphorylation pattern of the new bicyclo[3.1.0]hexane template that exhibits highly strong and increased chemical and enzymatic stability due to the absence of a true glycosyl bond. The template is found to be extremely rigid that makes it useful for sculpting other carbocyclic nucleosides, which also makes it easier to interact with the herpes simplex thymidine kinase.

The remote SH2 domain in each of the substrates is required to achieve efficient tyrosine phosphorylation by Itk and extend this observation to two other Tec family kinases, Btk and Tec. At the same time a stable interaction between the substrate SH2 domains and the kinase domain of Itk is detected and found that addition of specific, exogenous SH2 domains to the in vitro kinase assay competes directly with substrate phosphorylation.

Aminoglycoside 2'-Ib phosphotransferase [APH(2')-Ib] produces resistance to several clinically important aminoglycosides in Gram-positive and Gram-negative bacteria. Nuclear magnetic resonance analysis of the product of kanamycin A phosphorylation revealed that modification occurs at the 2'-hydroxyl of the aminoglycoside.

Interleukin-2 tyrosine kinase (Itk) is used as a model system to gain insight into the regulatory apparatus of the Tec kinases. Use of a quantitative in vitro kinase assay uncovered an essential role for the short linker region flanked by the SH2 and kinase domains of Itk in positively regulating Itk catalytic activity.

Polo-like kinases (Plks) play crucial roles in mitosis and cell division. Whereas lower eukaryotes typically contain a single PIk, mammalian cells express several closely related but functionally distinct Plks. We describe here a chemical genetic system in which a single PIk family member, Plkl, can be inactivated with high selectivity and temporal resolution by using an allele-specific, small-molecule inhibitor, as well as the application of this system to dissect Plk1's role in cytokinesis. To do this, we disrupted both copies of the PLK1 locus in human ceils through homologous recombination and then reconstituted Plk1 activity by using either the wild-type kinase ([Plk1.sup.wt]) or a mutant version whose catalytic pocket has been enlarged to accommodate bulky purine analogs ([Plk1.sup.as]). When cultured in the presence of these analogs, [Plk1.sup.as] cells accumulate in prometaphase with defects that parallel those found in [Plk1.sup.[DELTA]/[DELTA]] cells. In addition, acute treatment of [Plk1.sup.as] cells during anaphase prevents recruitment of both Plk1 itself and the Rho guanine nucleotide exchange factor (RhoGEF) Ect2 to the central spindle, abolishes RhoA GTPase localization to the equatorial cortex, and suppresses cleavage furrow formation and cell division. Our studies define and illuminate a late mitotic function of Plk1 that, although difficult or impossible to detect in Plk1-depleted cells, is readily revealed with chemical genetics. cell division | Ect2 | knockout | mitosis

Mice lacking p63, a single gene that encodes a group of transcription factors that either contain (TA) or lack (AN) a transactivation domain, fail to develop stratified epithelia as well as epithelial appendages and limbs. [DELTA]Np63 isoforms are predominantly expressed during late embryonic and postnatal epidermal development, however, the function of these proteins remains elusive. Using an epidermal-specific inducible knockdown mouse model, we demonstrate that [DELTA]Np63 proteins are essential for maintaining basement membrane integrity and terminal differentiation of keratinocytes. Furthermore, we have identified two [DELTA]Np63[alpha] target genes that mediate these processes. We propose that [DELTA]Np63[alpha] initially induces expression of the extracellular matrix component Fras1, which is required for maintaining the integrity of the epidermal-dermal interface at the basement membrane. Subsequently, induction of I[kappa]B kinase-[alpha] by [DELTA]Np63[alpha] initiates epidermal terminal differentiation resulting in the formation of the spinous layer. Our data provide insights into the role of [DELTA]Np63[alpha] in epidermal morphogenesis and homeostasis, and may contribute to our understanding of the pathogenic mechanisms underlying disorders caused by p63 mutations. I[kappa]B kinase-[alpha]

The Escherichia coli mannitol transporter ([II.sup.Mtl]) comprises three domains connected by flexible linkers: a transmembrane domain (C) and two cytoplasmic domains (A and B). [II.sup.Mtl] catalyzes three successive phosphoryl-transfer reactions: one intermolecular (from histidine phosphocarrier protein to the A domain) and two intramolecular (from the A to the B domain and from the B domain to the incoming sugar bound to the C domain). A key functional requirement of [II.sup.Mtl] is that the A and B cytoplasmic domains be able to rapidly associate and dissociate while maintaining reasonably high occupancy of an active stereospecific AB complex to ensure effective phosphoryl transfer along the pathway. We have investigated the rate of intramolecular domain-domain association and dissociation in [IIBA.sup.Mtl] by using [sup.1]H relaxation dispersion spectroscopy in the rotating frame. The open, dissociated state (comprising an ensemble of states) and the closed, associated state (comprising the stereospecific complex) are approximately equally populated. The first-order rate constants for intramolecular association and dissociation are 1,7 ([+ or -]03) x [10.sup.4] and 1.8 ([+ or -] 0.4) x [10.sup.4] [s.sup.-1], respectively. These values compare to rate constants of [approximately equal to] 500 [s.sup.-1] for A [right arrow] B and B [right arrow] A phosphoryl transfer, derived from qualitative line-shape analysis of [sup.1]H-[sup.15]N correlation spectra taken during the course of active catalysis. Thus, on average, [approximately equal to] 80 association/ dissociation events are required to effect a single phosphoryl-transfer reaction. We conclude that intramolecular phosphoryl transfer between the A and B domains of [II.sup.Mtl] is rate-limited by chemistry and not by the rate of formation or dissociation of a stereospecific complex in which the active sites are optimally apposed. domain motion | NMR | protein dynamics | relaxation dispersion | phosphotransferase system

Polo-like kinase (PIk1) is crucial for cell cycle progression through mitosis. Here we present the molecular and structural mechanisms that regulate the substrate recognition of PIk1 and influence its centrosomal localization and activity. Our work shows that PIk1 localization is controlled not only by the polo box domain (PBD); remarkably, the kinase domain is also involved in PIk1 targeting mechanism to the centrosome. The crystal structures of the PBD in complex with Cdc25C and Cdc25C-P target peptides reveal that Trp-414 is fundamental in their recognition regardless of its phosphorylation status. Binding measurements demonstrate that W414F mutation abolishes molecular recognition and diminishes centrosomal localization. Therefore, PIk1 centrosomal localization is not controlled by His-538 and Lys-540, the residues involved in phosphorylated target binding. The different conformations of the loop, which connects the polo boxes in the apo and the PBD-Cdc25C and PBD-Cdc25C-P complex structures, together with changes in the proline adjacent to the phosphothreonine in the target peptide, suggest a regulatory mechanism to detect binding of unphosphorylated or phosphorylated target substrates. Altogether, these data propose a model for the interaction between PIk1 and Cdc25C. cell cycle | enzymes | kinase | protein structure | polo box domain

A novel mode of interaction between ubiquitin and the cyclin-dependent kinase subunit of Saccharomyces cerevisae, Cks1p, an adaptor protein involved in transcriptional regulation through recruitment of proteasomal subunits to gene promoters is reported. Cks1p interacts specifically with monoubiquitin and tetraubiquitin with an affinity several orders of magnitude greater than that of other ubiquitin-binding domains.

In silico analysis of the Bifidobacterium breve UCC2003 genome has helped in identifying the four genetic loci, each of which has specified a putative enzyme II (EII) protein of a phosphoenolpyruvate:sugar phosphotransferase system. Biochemical studies have shown that FruA has phosphorylated fructose at the C-6 position.

The origins of a bacterial phosphotriesterase (PTE), an enzyme thought to have evolved for the purpose of degradation of a synthetic insecticide introduced in the 20th century is traced to establish a role for promiscuity in the divergence of natural enzymes. Three genes are identified that define a new group of microbial lactonases dubbed PTE-like lactonases.

The human homologue of rat eukaryotic initiation factor 2-associated glycoprotein, p67/MetAP2 was ectopically expressed in EGF-treated mouse embryonic NIH3T3 fibroblasts and C2C12 myoblasts and NIH3T3 cell lines expressing the constitutively active form of MAP kinase (MEK) to inhibit the activation and activity of extracellular signal-regulated kinases (ERK1/2) MAP kinases. The ability of ectopic expression of rat p67/MetAP2 in transformed cells to inhibit the tumorigenic phenotype by inhibiting the activation and activity of ERK1/2 MAP kinases confirmed the tumor suppression activity of p67/MetAP2

The significance of zinc in the assembly of the sterile alpha motif (SAM) domain of diacylglycerol kinase [delta] is analyzed. Zinc is found to be extremely significant for the assembly, as well as the physiology of the compound.

The article discusses the various roles and applications of serum amyloid A (SAA), an acute phase protein. SAA is shown to be extremely beneficial for activating the peroxisome proliferator-activated receptor [gamma] through extracellularly regulated kinase 1/2 and COX-2 expression in hepatocytes.

Several studies are conducted to explain the structure and the properties of the Mistic-fused histidine kinase receptor, EnvZ, an Escherichia coli two-component system. The study has been shown to be extremely beneficial for analyzing the properties of the different integral membrane proteins.

FLAG-M2 affinity chromatography is used for expressed and purified human mammalian target of rapamycin (mTOR1) and mTORC2 from HEK-293 cells. All inhibitors have displayed similar inhibitory potencies between mTORC1 and truncated mTOR containing the kinase domain and they have provided the kinetic studies of a recombinant mTOR complex.

Residual dipolar couplings and small- and wide-angle X-ray scattering are used to solve the solution structures of free 128 kDa Enzyme I (EI), the first enzyme in the bacterial phosphotransferase system and its 146 kDa complex with HPr. The large scale interdomain motins observed for the free EI and the EI-HPr complex when compared to that of the crystal structure of a trapped phosphorylated EI intermediate offer insight into the structural transitions that accompany the catalytic cycle of EI.

Molecular dynamics simulations and high-resolution X-ray structure studies are performed to experimentally identify the correlations between structural changes in the active site and determine kinetic parameters for stereoselective substrate hydrolysis by phosphotriesterase (PTE). The differences in the computed angle of attack for the nucleophilic hydroxide relative to the phenolic leaving group of the substrate are found to correlate well with the experimental stereoselectivity.

Deletional and site-directed mutagenesis approaches were used to systematically study the regions of TRUSS that interact with TNF receptor-1 (TNF-R1) and TRAF2 and the ability of TRUSS to self-associate to form higher-order complexes. Data suggested that the interaction between TNF-R1 and TRAF2 requires sequences located in the entire N-terminal half (residues 1-440) of TRUSS and the binding interface for TNF-R1 is closely linked with the TRAF2 binding interface.

The article helps in the identification of various different small-molecules that leads to the selection of DNA-templated small-molecule library. The application of these molecules for modulating the activity of different macrocyclic kinase inhibitors is also analyzed.

Steady-state kinetic analysis of focal adhesion kinase-1 (FAK1) is performed by using radiometric measurement of phosphorylation of a synthetic peptide substrate that has corresponded to the sequence of an autophosphorylation site in FAK1. The positional isotope exchange, viscosity and structural data have shown that enzyme turnover is rate-limited by both reversible phosphoryl group transfer and a slow step that is the opening of the activation loop after phosphoryl group transfer but preceding product release.

Near-optimal catalytic properties are seen for the integral membrane enzyme diacylglycerol kinase (DAGK) in micelles composed of lysomyristoylphosphatidylcholine (LMPC), along with important activity observed in micelles composed of lysomyristoylphosphatidylglycerol and tetradecylphosphocholine. The results have shown that some integral membrane proteins can maintain native-like properties in lipid-free detergent micelles and [C.sub.14]-based detergents are useful in studies of membrane proteins.

The article discusses the development of new C-1 alkyl analogs of AI-2 antagonists, which can be used for the quenching of the quorum sensing (QS) response. New approaches for the tailoring the communication network among bacterial species are also presented.

The crystal structures of the unphosphorylated human anaplastic lymphoma kinase (ALK) domain in complex with the ATP competitive ligands PHA-E429 and NVP-TAE684 are described. The mapping of the activating mutations associated with neuroblastoma onto these structures has explained the roles of the residues in the activation process.

The subunits of homologous [alpha] and [beta] phosphorylase kinase (PhK) are shown to be multidomain structures and the predictions based on their sequences have shown the presence of functional glucoamylase (GH15)-like domains near their amino termini. The experimental evidence of the domain in PhK is presented by showing that the glucoamylase inhibitor acarbose binds PhK, perturbs its structure and stimulates its kinase activity.

Stopped-flow fluorescence experiments that monitored binding of ATP and TNP-ATP to the catalytic domain (P4) of CheA was used to study the mechanism of nucleotide binding to the active site of Thermotoga maritima CheA. The results provide insight into the remarkable effect of the TNP moieties on CheA-nucleotide interactions and the kinetic mechanism underlying interaction of a protein histidine kinase TNP-ATP tight-binding inhibitor.

A more comprehensive, transparent, and easy-to-use approach that facilitates a detailed and accurate interpretation of variety of function-related conformational changes in proteins is presented. Almost all monomeric apo chains, containing stable fragments and/or domains which indicated instability of the entire protein molecule emphasized the importance of fragments and domains motions prior to stabilization by substrate binding or crystallization.

The signal transduction pathway is identified through which protein kinase C[epsilon] (PKC[epsilon]) has regulated translocator protein TSPO gene expression. The results have shown that PKC[epsilon] has regulated Tspo gene expression through a MAPK signal transduction pathway, which has acted in part through c-Jun and STAT3 transcription factors.

The structural underpinnings of LOV-mediated signal transduction in the context of a full-length histidine kinase is described. The studies have provided evidence for a model in which small changes in the tertiary/quaternary structure of a two-component signaling protein LovK that is triggered by photon detection in the N-terminal LOV sensory domain is enough for regulating histidine kinase activity.

A high-throughput yeast-based assay was developed to screen for chemical inhibitors of [Ca.sup.2+]/calmodulin-dependent kinase pathways and identify the novel antagonist 125-C9, a substituted ethyleneamine. The results not only defined a novel [Ca.sup.2+]/CaM inhibitor but also revealed a chemically unique CaM antagonists that could bind CaM by distinct mechanisms but similarly inhibit cellular actions of CaM.

Ammonium sulfate fractionation and divalent cation precipitation were applied to purify major contractile proteins from relaxed actomyosin extracted from molluscan catch muscle myofibrils, as a supramolecular complex composed of twitchin (TW), myosin (MY), and myorod (MR). The phosphorylation of twitchin by PKA-like kinase and the phosphorylation of myosin and myorod by myosin-associated kinase were found to play crucial role in the development of the catch state.

A study was conducted to gain insight into the oxidative stress-induced regulation of phosphorylating MKK6 in presence of apoptosis signal-regulating kinase 1 (ASK1) which is a serine/threonine kinase that responds to stress-inducing signals. The result data suggested that oxidative stress rapidly increased ASK1 catalytic efficiency for MKK6 phosphorylation by increasing MKK6 binding affinity within the ASK1 signalosome prior to induction of inactivation and degradation of the complex.

A protein tyrosine kinase-inducible domain is designed, which is a small, genetically encodable protein motif whose structure is dependent on its tyrosine phosphorylation state. The results have shown that tyrosine kinase-inducible domains are used as sensors of tyrosine kinase and tyrosine phosphatase activity and in the detection of tyrosine kinase inhibitors.

The leucine-rich repeat kinase 2 (LRRK2) G20 19S mutant are activated by [Mn.sup.2+] in vitro to demonstrate the phosphorylation of MBP and LRRKtide. The apparent [K.sub.i] values of a small set of LRRK2 ATP-competitive inhibitors are found to be within 5-fold between [Mg.sup.2+]- and [Mn.sup.2+]-mediated reactions except AMP-CPP, an ATP analogue.

The structural homology and molecular dynamics methods are used to construct various all-atom representation models of smooth muscle myosin light chain kinase (smMLCK) and its two fragments which are a calcium-calmodulin complex-dependent enzyme that activates contraction of smooth muscle. The predicted and observed sedimentation coefficients for models were in agreement with each other for the independently folded catalytic domain, Fn3, and Ig domains which are aligned consecutively on the long axis of the molecule.

The application of the ERK/MAPK effector p90 ribosomal S6 kinase 2 (RSK2) in reducing the signaling of the G protein-coupled receptor superfamily (GPCR) kinase is discussed. The mechanisms involved in the activation of multiple endogeneous receptor tyrosine kinases (RTKs) in different types of cells are also explained.