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Janet L Smith

Publications and source records attributed to Janet L Smith.

34 records · Page 2Linked to original sources
PubMed2004

Design, expression, and purification of a Flaviviridae polymerase using a high-throughput approach to facilitate crystal structure determination.

Bovine viral diarrhea virus (BVDV) nonstructural protein 5B is an RNA-dependent RNA polymerase, essential for viral replication. Initial attempts to crystallize a soluble form of the 695-residue BVDV polymerase did not produce any crystals. Limited proteolysis, homology modeling, and mutagenesis data were used to aid the design of polymerase constructs that might crystallize more readily. Limited proteolysis of the polymerase with trypsin identified a domain boundary within the protein. Homology modeling of the polymerase, based on the structure of hepatitis C virus polymerase, indicated that the two polymerases share a 23% identical "core," although overall sequence identity is low. Eighty-four expression clones of the BVDV polymerase were designed by fine-sampling of chain termini at the boundaries of domain and of active truncated forms of the polymerase. The resulting constructs were expressed in Escherichia coli and purified using high-throughput methods. Soluble truncated proteins were subjected to crystallization trials in a 96-well format, and two of these proteins were successfully crystallized.

Amino Acid Sequence↗
PubMed2003

Regulatory mechanism of Dictyostelium myosin light chain kinase A.

In this study, we examined the activation mechanism of Dictyostelium myosin light chain kinase A (MLCK-A) using constitutively active Ca2+/calmodulin-dependent protein kinase kinase as a surrogate MLCK-A kinase. MLCK-A was phosphorylated at Thr166 by constitutively active Ca2+/calmodulin-dependent protein kinase kinase, resulting in an approximately 140-fold increase in catalytic activity, using intact Dictyostelium myosin II. Recombinant Dictyostelium myosin II regulatory light chain and Kemptamide were also readily phosphorylated by activated MLCK-A. Mass spectrometry analysis revealed that MLCK-A expressed by Escherichia coli was autophosphorylated at Thr289 and that, subsequent to Thr166 phosphorylation, MLCK-A also underwent a slow rate of autophosphorylation at multiple Ser residues. Using site-directed mutagenesis, we show that autophosphorylation at Thr289 is required for efficient phosphorylation and activation by an upstream kinase. By performing enzyme kinetics analysis on a series of MLCK-A truncation mutants, we found that residues 283-288 function as an autoinhibitory domain and that autoinhibition is fully relieved by Thr166 phosphorylation. Simple removal of this region resulted in a significant increase in the kcat of MLCK-A; however, it did not generate maximum enzymatic activity. Together with the results of our kinetic analysis of the enzymes, these findings demonstrate that Thr166 phosphorylation of MLCK-A by an upstream kinase subsequent to autophosphorylation at Thr289 results in generation of maximum MLCK-A activity through both release of an autoinhibitory domain from its catalytic core and a further increase (15-19-fold) in the kcat of the enzyme.

Amino Acid Sequence↗
PubMed2003

Structure of the cytochrome b6f complex of oxygenic photosynthesis: tuning the cavity.

The cytochrome b6f complex provides the electronic connection between the photosystem I and photosystem II reaction centers of oxygenic photosynthesis and generates a transmembrane electrochemical proton gradient for adenosine triphosphate synthesis. A 3.0 angstrom crystal structure of the dimeric b6f complex from the thermophilic cyanobacterium Mastigocladus laminosus reveals a large quinone exchange cavity, stabilized by lipid, in which plastoquinone, a quinone-analog inhibitor, and a novel heme are bound. The core of the b6f complex is similar to the analogous respiratory cytochrome bc1 complex, but the domain arrangement outside the core and the complement of prosthetic groups are strikingly different. The motion of the Rieske iron-sulfur protein extrinsic domain, essential for electron transfer, must also be different in the b6f complex.

Cell Membrane↗
PubMed2003

Substrate-induced changes in the ammonia channel for imidazole glycerol phosphate synthase.

IGP synthase is a glutamine amidotransferase that incorporates ammonia derived from glutamine into the unusual nucleotide, N(1)-[(5'-phosphoribulosyl)-formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR) to form 5'-(5-aminoimidazole-4-carboxamide) ribonucleotide (AICAR) and imidazole glycerol phosphate (IGP). A common feature of all glutamine amidotransferases is the upregulation of glutamine hydrolysis in the presence of an acceptor substrate. A refined assay system was developed to establish that Saccharomyces cerevisae IGP synthase shows a 4900-fold stimulation of glutaminase in the presence of the substrate acceptor PRFAR. The structure and function of IGP synthase acceptor substrate binding site were probed with competitive inhibitors that are nucleotide substrate and product analogues. In addition, these analogues were also used to establish that the normal steady-state turnover cycle involves a random sequential mechanism. Upregulation of the glutaminase active site occurs when these competitive inhibitors bind in the nucleotide site over 30 A away. One of the key structural features of IGP synthase is that the transfer of ammonia from the glutaminase site occurs through the (beta/alpha)(8) core of the protein. Upon the basis of the recent substrate-occupied structure for yeast IGP synthase (1), kinetic investigations of site-directed mutants revealed that a conserved K258 residue is key to productive binding and the overall stoichiometry of the reaction. The binding of the ribulosyl phosphate portion of the substrate PRFAR appears to be transduced through reorientation of K258 resulting in a conformational switch at the base of the (beta/alpha)(8) core that enables the passage of ammonia through the core of the protein. The overall analysis also leads to further discussion of how the residues that cover the opening of the (beta/alpha)(8) in the closed state may assist the channeling of ammonia at the interface of the two functional domains in the open state.

Amino Acid Substitution↗
PubMed2003

Toward understanding the mechanism of the complex cyclization reaction catalyzed by imidazole glycerolphosphate synthase: crystal structures of a ternary complex and the free enzyme.

Imidazole glycerol phosphate synthase catalyzes formation of the imidazole ring in histidine biosynthesis. The enzyme is also a glutamine amidotransferase, which produces ammonia in a glutaminase active site and channels it through a 30-A internal tunnel to a cyclase active site. Glutaminase activity is impaired in the resting enzyme, and stimulated by substrate binding in the cyclase active site. The signaling mechanism was investigated in the crystal structure of a ternary complex in which the glutaminase active site was inactivated by a glutamine analogue and the unstable cyclase substrate was cryo-trapped in the active site. The orientation of N(1)-(5'-phosphoribulosyl)-formimino-5-aminoimidazole-4-carboxamide ribonucleotide in the cyclase active site implicates one side of the cyclase domain in signaling to the glutaminase domain. This side of the cyclase domain contains the interdomain hinge. Two interdomain hydrogen bonds, which do not exist in more open forms of the enzyme, are proposed as molecular signals. One hydrogen bond connects the cyclase domain to the substrate analogue in the glutaminase active site. The second hydrogen bond connects to a peptide that forms an oxyanion hole for stabilization of transient negative charge during glutamine hydrolysis. Peptide rearrangement induced by a fully closed domain interface is proposed to activate the glutaminase by unblocking the oxyanion hole. This interpretation is consistent with biochemical results [Myers, R. S., et al., (2003) Biochemistry 42, 7013-7022, the accompanying paper in this issue] and with structures of the free enzyme and a binary complex with a second glutamine analogue.

Aminohydrolases↗
PubMed2003

Differential functional properties of calmodulin-dependent protein kinase IIgamma variants isolated from smooth muscle.

Six variants of calmodulin-dependent protein kinase IIgamma were isolated from a ferret-aorta smooth-muscle cDNA library. Variant G-2 is generated by a novel alternative polyadenylation, utilizing a site contained in an intron. The last 77 residues of the association domain are replaced with 99 residues of a unique sequence containing Src homology 3-domain-binding motifs, which alter catalytic activity. Variant C-2 has an eight-residue deletion in an ATP-binding motif and does not autophosphorylate Thr(286), but does phosphorylate exogenous substrate. Two variants, B and J, autodephosphorylate. Four variants differing only in the variable domain have differing catalytic activities, despite identical sequences in the catalytic domains. Thus structural features determined by variable and association domains are important for the catalytic activity of calmodulin-dependent protein kinase II.

Alternative Splicing↗
PubMed2003

A defined protein-detergent-lipid complex for crystallization of integral membrane proteins: The cytochrome b6f complex of oxygenic photosynthesis.

The paucity of integral membrane protein structures creates a major bioinformatics gap, whose origin is the difficulty of crystallizing these detergent-solubilized proteins. The problem is particularly formidable for hetero-oligomeric integral membrane proteins, where crystallization is impeded by the heterogeneity and instability of the protein subunits and the small lateral pressure imposed by the detergent micelle envelope that surrounds the hydrophobic domain. In studies of the hetero (eight subunit)-dimeric 220,000 molecular weight cytochrome b(6)f complex, derived from the thermophilic cyanobacterium, Mastigocladus laminosus, crystals of the complex in an intact state could not be obtained from highly purified delipidated complex despite exhaustive screening. Crystals of proteolyzed complex could be obtained that grew very slowly and diffracted poorly. Addition to the purified lipid-depleted complex of a small amount of synthetic nonnative lipid, dioleolyl-phosphatidylcholine, resulted in a dramatic improvement in crystallization efficiency. Large crystals of the intact complex grew overnight, whose diffraction parameters are as follows: 94% complete at 3.40 A spacing; R(merge) = 8.8% (38.5%), space group, P6(1)22; and unit cell parameters, a = b = 156.3 A, c = 364.0 A, alpha = beta = 90 degrees, gamma = 120 degrees. It is proposed that the methodology of augmentation of a well-defined lipid-depleted integral membrane protein complex with synthetic nonnative lipid, which can provide conformational stability to the protein complex, may be of general use in the crystallization of integral membrane proteins.

Crystallization↗
PubMed2003

The purine repressor of Bacillus subtilis: a novel combination of domains adapted for transcription regulation.

The purine repressor from Bacillus subtilis, PurR, represses transcription from a number of genes with functions in the synthesis, transport, and metabolism of purines. The 2.2-A crystal structure of PurR reveals a two-domain protein organized as a dimer. The larger C-terminal domain belongs to the PRT structural family, in accord with a sequence motif for binding the inducer phosphoribosylpyrophosphate (PRPP). The PRT domain is fused to a smaller N-terminal domain that belongs to the winged-helix family of DNA binding proteins. A positively charged surface on the winged-helix domain likely binds specific DNA sequences in the recognition site. A second positively charged surface surrounds the PRPP site at the opposite end of the PurR dimer. Conserved amino acids in the sequences of PurR homologs in 21 gram-positive bacteria cluster on the proposed recognition surface of the winged-helix domain and around the PRPP binding site at the opposite end of the molecule, supporting a common function of DNA and PRPP binding for all of the proteins. The structure supports a binding mechanism in which extended regions of DNA interact with extensive protein surface. Unlike most PRT proteins, which are phosphoribosyltransferases (PRTases), PurR lacks catalytic activity. This is explained by a tyrosine side chain that blocks the site for a nucleophile cosubstrate in PRTases. Thus, B. subtilis has adapted an enzyme fold to serve as an effector-binding domain and has used it in a novel combination with the DNA-binding winged-helix domain as a repressor of purine genes.

Adaptation, Physiological↗
PubMed2003

Functional dissection of the Bacillus subtilis pur operator site.

Bacillus subtilis PurR represses transcription of several genes involved in purine synthesis, metabolism, and transport and cofactor synthesis. PurR binds specifically to DNAs containing an inverted repeat of a 14-nucleotide "PurBox" located in the upstream control regions of genes in the PurR regulon. Further biochemical investigation of the interaction of PurR with a series of shortened upstream DNA fragments of the pur operon determined the minimum length and specificity elements of the operator. The relative affinities of the two PurBoxes differ significantly, such that upstream PurBox1 (-81 to -68 relative to the transcription start site) is designated "strong" and downstream PurBox2 (-49 to -36) is designated "weak." Two PurBoxes are required for high-affinity PurR binding, and one of these must be strong. The shortest DNA construct with high affinity for PurR is a 74-bp perfect palindrome in which weak PurBox2 and its flanking sequences are replaced by strong PurBox1 and flanking sequences. Two PurR dimers bind to this symmetric construct. Phosphoribosylpyrophosphate (PRPP), the effector molecule that reduces affinity of PurR for DNA, requires one weak PurBox in the DNA construct to inhibit PurR binding. PRPP binds, as expected, to a PRPP-motif in PurR. A tracks outside the central conserved CGAA sequence of the PurBox may facilitate DNA bending, leading to a proposal for strong and weak designations of PurBoxes in the control regions of other genes regulated by PurR.

Bacillus subtilis↗
PubMed2002

Dictyostelium discoideum has a single diacylglycerol kinase gene with similarity to mammalian theta isoforms.

Diacylglycerol kinases (DGKs) phosphorylate the neutral lipid diacylglycerol (DG) to produce phosphatidic acid (PA). In mammalian systems DGKs are a complex family of at least nine isoforms that are thought to participate in down-regulation of DG-based signalling pathways and perhaps activation of PA-stimulated signalling events. We report here that the simple protozoan amoeba Dictyostelium discoideum appears to contain a single gene encoding a DGK enzyme. This gene, dgkA, encodes a deduced protein that contains three C1-type cysteine-rich repeats, a DGK catalytic domain most closely related to the theta subtype of mammalian DGKs and a C-terminal segment containing a proline/glutamine-rich region and a large aspargine-repeat region. This gene corresponds to a previously reported myosin II heavy chain kinase designated myosin heavy chain-protein kinase C (MHC-PKC), but our analysis clearly demonstrates that this protein does not, as suggested by earlier data, contain a protein kinase catalytic domain. A FLAG-tagged version of DgkA expressed in Dictyostelium displayed robust DGK activity. Earlier studies indicating that disruption of this locus alters myosin II assembly levels in Dictyostelium raise the intriguing possibility that DG and/or PA metabolism may play a role in controlling myosin II assembly in this system.

Algorithms↗
PubMed2002

A novel cGMP signalling pathway mediating myosin phosphorylation and chemotaxis in Dictyostelium.

Chemotactic stimulation of Dictyostelium cells results in a transient increase in cGMP levels, and transient phosphorylation of myosin II heavy and regulatory light chains. In Dictyostelium, two guanylyl cyclases and four candidate cGMP-binding proteins (GbpA- GbpD) are implicated in cGMP signalling. GbpA and GbpB are homologous proteins with a Zn2+-hydrolase domain. A double gbpA/gbpB gene disruption leads to a reduction of cGMP-phosphodiesterase activity and a 10-fold increase of basal and stimulated cGMP levels. Chemotaxis in gbpA(-)B(-) cells is associated with increased myosin II phosphorylation compared with wild-type cells; formation of lateral pseudopodia is suppressed resulting in enhanced chemotaxis. GbpC is homologous to GbpD, and contains Ras, MAPKKK and Ras-GEF domains. Inactivation of the gbp genes indicates that only GbpC harbours high affinity cGMP-binding activity. Myosin phosphorylation, assembly of myosin in the cytoskeleton as well as chemotaxis are severely impaired in mutants lacking GbpC and GbpD, or mutants lacking both guanylyl cyclases. Thus, a novel cGMP signalling cascade is critical for chemotaxis in Dictyostelium, and plays a major role in myosin II regulation during this process.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2002

Identification of four candidate cGMP targets in Dictyostelium.

In Dictyostelium, a transient increase in intracellular cGMP is important for cytoskeletal rearrangements during chemotaxis. There must be cGMP-binding proteins in Dictyostelium that regulate key cytoskeletal components after treatment with chemoattractants, but to date, no such proteins have been identified. Using a bioinformatics approach, we have found four candidate cGMP-binding proteins (GbpA-D). GbpA and -B have two tandem cGMP-binding sites downstream of a metallo beta-lactamase domain, a superfamily that includes cAMP phosphodiesterases. GbpC contains the following nine domains (in order): leucine-rich repeats, Ras, MEK kinase, Ras guanine nucleotide exchange factor N-terminal (RasGEF-N), DEP, RasGEF, cGMP-binding, GRAM, and a second cGMP-binding domain. GbpD is related to GbpC, but is much shorter; it begins with the RasGEF-N domain, and lacks the DEP domain. Disruption of the gbpC gene results in loss of all high-affinity cGMP-binding activity present in the soluble cellular fraction. GbpC mRNA levels increase dramatically 8 h after starvation is initiated. GbpA, -B, and -D mRNA levels show less dramatic changes, with gbpA mRNA levels highest 4 h into starvation, gbpB mRNA levels highest in vegetative cells, and gbpD levels highest at 8 h. The identification of these genes is the first step in a molecular approach to studying downstream effects of cGMP signaling in Dictyostelium.

Amino Acid Sequence↗
PubMed2002

A biologist's guide to synchrotron facilities: the BioSync web resource.

Research at synchrotron radiation facilities, once the domain of high energy physics, now has a major impact on fields as diverse as immunology, neurobiology, physiology, molecular biology, medicine and biotechnology. This article describes the development of a comprehensive synchrotron portal and informational website (http://www.biosync.sdsc.edu) for biologists engaged in research at synchrotrons. The site automatically provides timely and accurate information in a unified format by gathering technical descriptions of synchrotron beamlines using modern information management practices.

Internet↗
PubMed2002

Signaling pathways regulating Dictyostelium myosin II.

Dictyostelium myosin II is a conventional, two-headed myosin that consists of two copies each of a myosin heavy chain (MHC), an essential light chain (ELC) and a regulatory light chain (RLC). The MHC is comprised of an amino-terminal motor domain, a neck region that binds the RLC and ELC and a carboxyl-terminal alpha-helical coiled-coil tail. Electrostatic interactions between the tail domains mediate the self-assembly of myosin II into bipolar filaments that are capable of interacting with actin filaments to generate a contractile force. In this review we discuss the regulation of Dictyostelium myosin II by a myosin light chain kinase (MLCK-A) that phosphorylates the RLC and increases motor activity and by MHC kinases (MHCKs) that phosphorylate the tail and prevent filament assembly. Dictyostelium may express as many as four MHCKs (MHCK A-D) consisting of an atypical alpha-kinase catalytic domain and a carboxyl-terminal WD repeat domain that targets myosin II filaments. A previously reported MHCK, termed MHC-PKC, now seems more likely to be a diacylglycerol kinase (DgkA). The relationship of the MHCKs to the larger family of alpha-kinases is discussed and key features of the structure of the alpha-kinase catalytic domain are reviewed. Potential upstream regulators of myosin II are described, including DgkA, cGMP, cAMP and PAKa, a target for Rac GTPases. Recent results point to a complex network of signaling pathways responsible for controling the activity and localization of myosin II in the cell.

Amino Acid Sequence↗