The subunit structure of chromatin from Physarum polycephalum.

Nucleosome DNA repeat lengths in Physarum chromatin, determined by nuclease digestion experiments, are shorter than those observed in most mammalian chromatin and longer than those reported for chromatin of certain other lower eukaryotes. After digestion with staphylococcal nuclease for short periods of time an average repeat length of 190 base pairs is measured. After more extensive digestion an average repeat length of 172 base pairs is measured. Upon prolonged digestion DNA is degraded to an average monomer subunit length of 160 base pairs, with only a small amount of DNA found in lengths of 130 base pairs or smaller. Mathematical analysis of the data suggests that the Physarum nucleosome DNA repeat comprises a protected DNA segment of about 159 base pairs with a nuclease-accessible interconnecting segment which ranges from 13 to 31 base pairs. The spacing data are compatible with measurements from electron micrographs of Physarum chromatin.     

Characterization of a telomere-binding protein from Physarum polycephalum.

We have partially purified a nuclear protein (PPT) from Physarum polycephalum that binds to the extrachromosomal ribosomal DNA telomeres of this acellular slime mold. Binding is specific for the (T2AG3)n telomere repeats, as evidenced by nitrocellulose filter binding assays, by gel mobility shift assays with both DNA fragments and double-stranded oligonucleotides, and by DNase I footprinting. PPT is remarkably heat stable, showing undiminished binding activity after incubation at 90 degrees C. It sediments at 1.2S, corresponding to a molecular weight of about 10,000 (for a globular protein), and its binding activity is undiminished by incubation with RNase, suggesting that it is not a ribonucleoprotein. We hypothesize that PPT plays a structural role in telomeres, perhaps preventing nucleolytic degradation or promoting telomere extension by a telomere-specific terminal transferase.     

Physarum amoebae express a distinct fragmin-like actin-binding protein that controls in vitro phosphorylation of actin by the actin-fragmin kinase.

Amoebae and plasmodia constitute the two vegetative growth phases of the Myxomycete Physarum. In vitro and in vivo phosphorylation of actin in plasmodia is tightly controlled by fragmin P, a plasmodium-specific actin-binding protein that enables actin phosphorylation by the actin-fragmin kinase. We investigated whether amoebal actin is phosphorylated by this kinase, in spite of the lack of fragmin P. Strong actin phosphorylation was detected only following addition of recombinant actin-fragmin kinase to cell-free extracts of amoebae, suggesting that amoebae contain a protein with properties similar to plasmodial fragmin. We purified the complex between actin and this protein to homogeneity. Using an antibody that specifically recognizes phosphorylated actin, we demonstrate that Thr203 in actin can be phosphorylated in this complex. A full-length amoebal fragmin cDNA was cloned and the deduced amino acid sequence shows 65% identity with plasmodial fragmin. However, the fragmins are encoded by different genes. Northern blots using RNA from a developing Physarum strain demonstrate that this fragmin isoform (fragmin A) is not expressed in plasmodia. In situ localization showed that fragmin A is present mainly underneath the plasma membrane. Our results indicate that Physarum amoebae express a fragmin P-like isoform which shares the property of binding actin and converting the latter into a substrate for the actin-fragmin kinase.     

Crystal structure of the atypical protein kinase domain of a TRP channel with phosphotransferase activity

Transient receptor potential (TRP) channels modulate calcium levels in eukaryotic cells in response to external signals. A novel transient receptor potential channel has the ability to phosphorylate itself and other proteins on serine and threonine residues. The catalytic domain of this channel kinase has no detectable sequence similarity to classical eukaryotic protein kinases and is essential for channel function. The structure of the kinase domain, reported here, reveals unexpected similarity to eukaryotic protein kinases in the catalytic core as well as to metabolic enzymes with ATP-grasp domains. The inclusion of the channel kinase catalytic domain within the eukaryotic protein kinase superfamily indicates a significantly wider distribution for this group of signaling proteins than suggested previously by sequence comparisons alone.     

Characterization of the thymidine kinase of Physarum polycephalum

Thymidine kinase [ATP: thymidine 5'-phosphotransferase, EC 2.7.1.21] has been purified more than 3,500 fold from microplasmodia of Physarum polycephalum. Properties of the enzyme were determined on preparations purified 1,400 fold. Thymidine was transformed to dTMP while a stoichiometric quantity of ATP was transformed to ADP. 5-Iododeoxyuridine, 5-bromodeoxyuridine, and 5-fluorodeoxyuridine acted as competitive inhibitors for the thymidine substrate while 5-bromodeoxyuridine could be used as a substrate. In contrast uridine did not inhibit the enzymatic activity while deoxyuridine was a very poor competitive inhibitor in agreement with the observation that deoxyuridine could not be used as a substrate. Two apparent Michaelis constants were found for thymidine. Only the highest Michaelis constant could be decreased in the presence of increasing concentrations of ATP. Among the various nucleoside mono, di, or triphosphates studied only ATP and to a less extent dATP could be used as phosphate donors. A non competitive inhibition for thymidine was observed with dTTP. dTMP, dTDP, and dTTP acted as competitive inhibitors for ATP. None of the nucleoside mono, di, or triphosphates studied showed an activatory effect at low concentrations of ATP, even in the presence of dTTP. However, dUTP and dGDP acted as competitive inhibitors for ATP.     

A novel Physarum polycephalum SR protein kinase specifically phosphorylates the RS domain of the human SR protein,ASF/SF2

A 1591-bp cDNA of a serine-rich protein kinase （SRPK）-Iike protein has been identified in Physarum polycephalum （GenBank accession No. DQ140379）. The cDNA contains two repeat sequences at bp 1-153 and bp 395-547. The encoding sequence is 56% homologous to human SRPK1 and is named Physarum SRPK （PSRPK）. Consistent with other SRPKs, the consensus motifs of PSRPK are within the two conserved domains （CDs）. However, divergent motifs between the N-terminal and CDs are much shorter than the corresponding sequences of other SRPKs. To study the structure and function of this protein, we performed co-expression experiment in Escherichia coli and in vitro phosphorylation assay to investigate the phosphorylation effect of recombinant PSRPK on the human SR protein, ASF/SF2. Western blot analysis showed that PSRPK could phosphorylate ASF/SF2 in E. coil cells. Autoradiographic examination showed that both recombinant PSRPK and a truncated form of PSRPK with a 28-aa deletion at the N-terminus could phosphorylate ASF/SF2 and a truncated form of ASF/SF2 that contains the RS domain. However, these two forms of PSRPK could not phosphorylate a truncated form ASF/SF2 that lacks the RS domain. A truncated form of PSRPK that lacks either of CDs does not have any phosphorylation activity. These results indicated that, like other SRPKs, the phosphorylation site in PSRPK is located within the RS domain of the SR protein and that its phosphorylation activity is closely associated with the two CDs. This study on the structure and function of PSRPK demonstrates that it is a new member of the SRPK family.     

The crystal structure of the substrate-binding protein OpuBC from Bacillus subtilis in complex with choline

Bacillus subtilis can synthesize the compatible solute glycine betaine as an osmoprotectant from an exogenous supply of the precursor choline. Import of choline is mediated by two osmotically inducible ABC transport systems: OpuB and OpuC. OpuC catalyzes the import of various osmoprotectants, whereas OpuB is highly specific for choline. OpuBC is the substrate-binding protein of the OpuB transporter, and we have analyzed the affinity of the OpuBC/choline complex by intrinsic tryptophan fluorescence and determined a K_d value of about 30 μM. The X-ray crystal structure of the OpuBC/choline complex was solved at a resolution of 1.6 Å and revealed a fold typical of class II substrate-binding proteins. The positively charged trimethylammonium head group of choline is wedged into an aromatic cage formed by four tyrosine residues and is bound via cation-pi interactions. The hydroxyl group of choline protrudes out of this aromatic cage and makes a single interaction with residue Gln19. The substitution of this residue by Ala decreases choline binding affinity by approximately 15-fold. A water network stabilizes choline within its substrate-binding site and promotes indirect interactions between the two lobes of the OpuBC protein. Disruption of this intricate water network by site-directed mutagenesis of selected residues in OpuBC either strongly reduces choline binding affinity (between 18-fold and 25-fold) or abrogates ligand binding. The crystal structure of the OpuBC/choline complex provides a rational for the observed choline specificity of the OpuB ABC importer in vivo and explains its inability to catalyze the import of glycine betaine into osmotically stressed B. subtilis cells.     

Ribosomes of Physarum polycephalum. Subunits and protein composition.

Ribosomes from Physarum polycephalum were purified. Optimal conditions for preparation and stability of subunits were determined. KCl concentration above 200 mM induced protein dissociation from the subunits. It was observed that dissociated ribosomes were more stable in a low ionic strength buffer than in 200 mM KCl, where the 40 S was preferentially degraded by ribonucleases. Ribosomal proteins were analyzed by two-dimensional gel electrophoresis. The first dimension was carried out at pH 8.6 while the second was run at pH 4.6. The monosome contained sixty seven proteins, of which six were acidic. Two proteins were lost after subunit dissociation. Twenty six basic and two acidic proteins were observed in the 40 S subunit while the largest subunit gave thirty nine spots on the basic part of the gel and three additional spots on the acidic side. Five proteins were shared by 40 S and 60 S.     

Catalytic domain crystal structure of protein kinase C-theta (PKCtheta)

A member of the novel protein kinase C (PKC) subfamily, PKC, is an essential component of the T cell synapse and is required for optimal T cell activation and interleukin-2 production. Selective involvement of PKC in TCR signaling makes this enzyme an attractive therapeutic target in T cell-mediated disease processes. In this report we describe the crystal structure of the catalytic domain of PKC at 2.0-A resolution. Human recombinant PKC kinase domain was expressed in bacteria as catalytically active phosphorylated enzyme and co-crystallized with its subnanomolar, ATP site inhibitor staurosporine. The structure follows the classic bilobal kinase fold and shows the enzyme in its active conformation and phosphorylated state. Inhibitory interactions between conserved features of staurosporine and the ATP-binding cleft are accompanied by closing of the glycine-rich loop, which also maintains an inhibitory arrangement by blocking the phosphate recognition subsite. The two major phosphorylation sites, Thr-538 in the activation loop and Ser-695 in the hydrophobic motif, are both occupied in the structure, playing key roles in stabilizing active conformation of the enzyme and indicative of PKC autocatalytic phosphorylation and activation during bacterial expression. The PKC-staurosporine complex represents the first kinase domain crystal structure of any PKC isotypes to be determined and as such should provide valuable insight into PKC specificity and into rational drug design strategies for PKC selective leads.     

Purification of an alkaline nuclease from Physarum polycephalum.

An alkaline nuclease was purified from microplasmodia of Physarum polycephalum. The nuclease, active on denatured DNA and RNA and free of contamination by other nucleolytic activities, appeared to be a zinc-metallo protein. The enzyme was only active under conditions, where Zn2+ were retained in the enzyme. Loss of zinc occurred by the chelating action of EDTA, EGTA or ampholines, by acid of highly alkaline pH conditions or by high ionic strength. The addition of ZnCl2 to compensate losses, restored all activity, while all other divalent cations caused inhibition. The nuclease, with a molecular weight of 32 000, was stable at neutral pH at high temperatures with a half-life of 20 min at 80 degrees C. It was inhibited by any salt of buffer concentration above the level of zero ionic strength and showed a special sensitivity towards phosphate ions. The possible similarity of this enzyme to nuclease S1 from Aspergillus oryzae is pointed out.     

X-ray structure of HPr kinase: a bacterial protein kinase with a P-loop nucleotide-binding domain

HPr kinase/phosphatase (HprK/P) is a key regulatory enzyme controlling carbon metabolism in Gram- positive bacteria. It catalyses the ATP-dependent phosphorylation of Ser46 in HPr, a protein of the phosphotransferase system, and also its dephosphorylation. HprK/P is unrelated to eukaryotic protein kinases, but contains the Walker motif A characteristic of nucleotide-binding proteins. We report here the X-ray structure of an active fragment of Lactobacillus casei HprK/P at 2.8 A resolution, solved by the multiwavelength anomalous dispersion method on a seleniated protein (PDB code 1jb1). The protein is a hexamer, with each subunit containing an ATP-binding domain similar to nucleoside/nucleotide kinases, and a putative HPr-binding domain unrelated to the substrate-binding domains of other kinases. The Walker motif A forms a typical P-loop which binds inorganic phosphate in the crystal. We modelled ATP binding by comparison with adenylate kinase, and designed a tentative model of the complex with HPr based on a docking simulation. The results confirm that HprK/P represents a new family of protein kinases, first identified in bacteria, but which may also have members in eukaryotes.     

The crystal structure of choline kinase reveals a eukaryotic protein kinase fold

Choline kinase catalyzes the ATP-dependent phosphorylation of choline, the first committed step in the CDP-choline pathway for the biosynthesis of phosphatidylcholine. The 2.0 A crystal structure of a choline kinase from C. elegans (CKA-2) reveals that the enzyme is a homodimeric protein with each monomer organized into a two-domain fold. The structure is remarkably similar to those of protein kinases and aminoglycoside phosphotransferases, despite no significant similarity in amino acid sequence. Comparisons to the structures of other kinases suggest that ATP binds to CKA-2 in a pocket formed by highly conserved and catalytically important residues. In addition, a choline binding site is proposed to be near the ATP binding pocket and formed by several structurally flexible loops.     

A novel type of protein kinase phosphorylates actin in the actin-fragmin complex.

Actin-fragmin kinase (AFK) from Physarum polycephalum specifically phosphorylates actin in the EGTA-resistant 1:1 actin-fragmin complex. The cDNA deduced amino acid sequence reveals two major domains of approximately 35 kDa each that are separated by a hinge-like proline/serine-rich segment of 50 residues. Whereas the N-terminal domain does not show any significant similarity to protein sequences from databases, there are six complete kelch repeats in the protein that comprise almost the entire C-terminal half of the molecule. To prove the intrinsic phosphorylation activity of AFK, full-length or partial cDNA fragments were expressed both in a reticulocyte lysate and in Escherichia coli. In both expression systems, we obtained specific actin phosphorylation and located the catalytic domain in the N-terminal half. Interestingly, this region did not contain any of the known protein kinase consensus sequences. The only known sequence motif present that could have been involved in nucleotide binding was a nearly perfect phosphate binding loop (P-loop). However, introduction of two different point mutations into this putative P-loop sequence did not alter the catalytic activity of the kinase, which indicates an as yet unknown mechanism for phosphate transfer. Our data suggest that AFK belongs to a new class of protein kinases and that this actin phosphorylation might be the first example of a widely distributed novel type of regulation of the actin cytoskeleton in non-muscle cells.     

The 2.7 A crystal structure of the autoinhibited human c-Fms kinase domain

c-Fms, a member of the Platelet-derived Growth Factor (PDGF) receptor family of receptor tyrosine kinases (RTKs), is the receptor for macrophage colony stimulating factor (CSF-1) that regulates proliferation, differentiation and survival of cells of the mononuclear phagocyte lineage. Abnormal expression of c-fms proto-oncogene is associated with a significant number of human pathologies, including a variety of cancers and rheumatoid arthritis. Accordingly, c-Fms represents an attractive therapeutic target. To further understand the regulation of c-Fms, we determined the 2.7 A resolution crystal structure of the cytosolic domain of c-Fms that comprised the kinase domain and the juxtamembrane domain. The structure reveals the crucial inhibitory role of the juxtamembrane domain (JM) that binds to a hydrophobic site immediately adjacent to the ATP binding pocket. This interaction prevents the activation loop from adopting an active conformation thereby locking the c-Fms kinase into an autoinhibited state. As observed for other members of the PDGF receptor family, namely c-Kit and Flt3, three JM-derived tyrosine residues primarily drive the mechanism for autoinhibition in c-Fms, therefore defining a common autoinhibitory mechanism within this family. Moreover the structure provides an understanding of c-Fms inhibition by Gleevec as well as providing a platform for the development of more selective inhibitors that target the inactive conformation of c-Fms kinase.     

Purification of a novel Ca-binding protein that inhibits myosin light chain kinase activity in lower eukaryote Physarum polycephalum

Myosin light chain kinase (MLCK) was partially purified from the lower eukaryote Physarum polycephalum. The activity to phosphorylate Physarum myosin was maximal in the absence of Ca2+ and decreased with an increase in Ca2+ concentration with a microM-level Kd. The Ca-binding protein contained in the MLCK preparation was purified to homogeneity. The native protein had a molecular mass of 75 kDa, while under denaturing conditions, it was 38 kDa. Ca-dependent changes in the intensities of intrinsic fluorescence showed that the Kd of the protein for Ca2+ was also in the microM-range. Our results suggest that the Ca-binding protein would play a key role in the effects of Ca2+ in the MLCK preparation.