Cytotoxic and antioxidant compounds from the stem bark of Goniothalamus tapisoides Mat Salleh.

Eleven compounds:goniomicin A (1), goniomicin B (2), goniomicin C (3), goniomicin D (4), tapisoidin (5), goniothalamin (6), 9-deoxygoniopypyrone (7), pterodondiol (8), liriodenine (9), benzamide (10) and cinnamic acid (11), were isolated from the stem bark of Goniothalamus tapisoides. All compounds were identified by spectroscopic analysis and, for known compounds, by comparison with published data. Goniothalamin (6) exhibited mild cytotoxic activity towards a colon cancer cell line (HT-29), with an IC(50)value of 64.17 ± 5.60 µM. Goniomicin B (2) give the highest antioxidant activity in the DPPH assay among all compounds tested, with an IC(50) of 0.207 µM.

Protein production and purification.

In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.

Capturing hammerhead ribozyme structures in action by modulating general base catalysis.

We have obtained precatalytic (enzyme-substrate complex) and postcatalytic (enzyme-product complex) crystal structures of an active full-length hammerhead RNA that cleaves in the crystal. Using the natural satellite tobacco ringspot virus hammerhead RNA sequence, the self-cleavage reaction was modulated by substituting the general base of the ribozyme, G12, with A12, a purine variant with a much lower pKa that does not significantly perturb the ribozyme's atomic structure. The active, but slowly cleaving, ribozyme thus permitted isolation of enzyme-substrate and enzyme-product complexes without modifying the nucleophile or leaving group of the cleavage reaction, nor any other aspect of the substrate. The predissociation enzyme-product complex structure reveals RNA and metal ion interactions potentially relevant to transition-state stabilization that are absent in precatalytic structures.

Structural and enzymatic characterization of DR1281: A calcineurin-like phosphoesterase from Deinococcus radiodurans.

We have determined the crystal structure of DR1281 from Deinococcus radiodurans. DR1281 is a protein of unknown function with over 170 homologs found in prokaryotes and eukaryotes. To elucidate the molecular function of DR1281, its crystal structure at 2.3 A resolution was determined and a series of biochemical screens for catalytic activity was performed. The crystal structure shows that DR1281 has two domains, a small alpha domain and a putative catalytic domain formed by a four-layered structure of two beta-sheets flanked by five alpha-helices on both sides. The small alpha domain interacts with other molecules in the asymmetric unit and contributes to the formation of oligomers. The structural comparison of the putative catalytic domain with known structures suggested its biochemical function to be a phosphatase, phosphodiesterase, nuclease, or nucleotidase. Structural analyses with its homologues also indicated that there is a dinuclear center at the interface of two domains formed by Asp8, Glu37, Asn38, Asn65, His148, His173, and His175. An absolute requirement of metal ions for activity has been proved by enzymatic assay with various divalent metal ions. A panel of general enzymatic assays of DR1281 revealed metal-dependent catalytic activity toward model substrates for phosphatases (p-nitrophenyl phosphate) and phosphodiesterases (bis-p-nitrophenyl phosphate). Subsequent secondary enzymatic screens with natural substrates demonstrated significant phosphatase activity toward phosphoenolpyruvate and phosphodiesterase activity toward 2',3'-cAMP. Thus, our structural and enzymatic studies have identified the biochemical function of DR1281 as a novel phosphatase/phosphodiesterase and disclosed key conserved residues involved in metal binding and catalytic activity.

Structural genomics of minimal organisms: pipeline and results.

The initial objective of the Berkeley Structural Genomics Center was to obtain a near complete three-dimensional (3D) structural information of all soluble proteins of two minimal organisms, closely related pathogens Mycoplasma genitalium and M. pneumoniae. The former has fewer than 500 genes and the latter has fewer than 700 genes. A semiautomated structural genomics pipeline was set up from target selection, cloning, expression, purification, and ultimately structural determination. At the time of this writing, structural information of more than 93% of all soluble proteins of M. genitalium is avail able. This chapter summarizes the approaches taken by the authors' center.

Structure of O67745_AQUAE, a hypothetical protein from Aquifex aeolicus.

Using single-wavelength anomalous dispersion data obtained from a gold-derivatized crystal, the X-ray crystal structure of the protein 067745_AQUAE from the prokaryotic organism Aquifex aeolicus has been determined to a resolution of 2.0 A. Amino-acid residues 1-371 of the 44 kDa protein were identified by Pfam as an HD domain and a member of the metal-dependent phosphohydrolase superfamily (accession No. PF01966). Although three families from this large and diverse group of enzymatic proteins are represented in the PDB, the structure of 067745_AQUAE reveals a unique fold that is unlike the others and that is likely to represent a new subfamily, further organizing the families and characterizing the proteins. Data are presented that provide the first insights into the structural organization of the proteins within this clan and a distal alternative GDP-binding domain outside the metal-binding active site is proposed.

Crystal structure of the DUF16 domain of MPN010 from Mycoplasma pneumoniae.

We have determined the crystal structure of the DUF16 domain of unknown function encoded by the gene MPN010 of Mycoplasma pneumoniae at 1.8 A resolution. The crystal structure revealed that this domain is composed of two separated homotrimeric coiled-coils. The shorter one consists of 11 highly conserved residues. The sequence comprises noncanonical heptad repeats that induce a right-handed coiled-coil structure. The longer one is composed of approximately nine heptad repeats. In this coiled-coil structure, there are three distinguishable regions that confer unique structural properties compared with other known homotrimeric coiled-coils. The first part, containing one stutter, is an unusual phenylalanine-rich region that is not found in any other coiled-coil structures. The second part is a highly conserved glutamine-rich region, frequently found in other trimeric coiled-coil structures. The last part is composed of prototype heptad repeats. The phylogenetic analysis of the DUF16 family together with a secondary structure prediction shows that the DUF16 family can be classified into five subclasses according to N-terminal sequences. Based on the structural comparison with other coiled-coil structures, a probable molecular function of the DUF16 family is discussed.

Crystal structure of ScpB from Chlorobium tepidum, a protein involved in chromosome partitioning.

Structural maintenance of chromosome (SMC) proteins are essential in chromosome condensation and interact with non-SMC proteins in eukaryotes and with segregation and condensation proteins (ScpA and ScpB) in prokaryotes. The highly conserved gene in Chlorobium tepidum gi 21646405 encodes ScpB (ScpB_ChTe). The high resolution crystal structure of ScpB_ChTe shows that the monomeric structure consists of two similarly shaped globular domains composed of three helices sided by beta-strands [a winged helix-turn-helix (HTH)], a motif observed in the C-terminal domain of Scc1, a functionally related eukaryotic ScpA homolog, as well as in many DNA binding proteins.

Crystal structures of an NAD kinase from Archaeoglobus fulgidus in complex with ATP, NAD, or NADP.

NAD kinase is a ubiquitous enzyme that catalyzes the phosphorylation of NAD to NADP using ATP or inorganic polyphosphate (poly(P)) as phosphate donor, and is regarded as the only enzyme responsible for the synthesis of NADP. We present here the crystal structures of an NAD kinase from the archaeal organism Archaeoglobus fulgidus in complex with its phosphate donor ATP at 1.7 A resolution, with its substrate NAD at 3.05 A resolution, and with the product NADP in two different crystal forms at 2.45 A and 2.0 A resolution, respectively. In the ATP bound structure, the AMP portion of the ATP molecule is found to use the same binding site as the nicotinamide ribose portion of NAD/NADP in the NAD/NADP bound structures. A magnesium ion is found to be coordinated to the phosphate tail of ATP as well as to a pyrophosphate group. The conserved GGDG loop forms hydrogen bonds with the pyrophosphate group in the ATP-bound structure and the 2' phosphate group of the NADP in the NADP-bound structures. A possible phosphate transfer mechanism is proposed on the basis of the structures presented.

Crystal structure of a heat-inducible transcriptional repressor HrcA from Thermotoga maritima: structural insight into DNA binding and dimerization.

All cells have a defense mechanism against a sudden heat-shock stress. Commonly, they express a set of proteins that protect cellular proteins from being denatured by heat. Among them, GroE and DnaK chaperones are representative defending systems, and their transcription is regulated by a heat-shock repressor protein HrcA. HrcA repressor controls the transcription of groE and dnaK operons by binding the palindromic CIRCE element, presumably as a dimer, and the activity of HrcA repressor is modulated by GroE chaperones. Here, we report the first crystal structure of a heat-inducible transcriptional repressor, HrcA, from Thermotoga maritima at 2.2A resolution. The Tm_HrcA protein crystallizes as a dimer. The monomer is composed of three domains: an N-terminal winged helix-turn-helix domain (WH), a GAF-like domain, and an inserted dimerizing domain (IDD). The IDD shows a unique structural fold with an anti-parallel beta-sheet composed of three beta-strands sided by four alpha-helices. The Tm_HrcA dimer structure is formed through hydrophobic contact between the IDDs and a limited contact that involves conserved residues between the GAF-like domains. In the overall dimer structure, the two WH domains are exposed, but the conformation of these two domains seems to be incompatible with DNA binding. We suggest that our structure may represent an inactive form of the HrcA repressor. Structural implication on how the inactive form of HrcA may be converted to the active form by GroEL binding to a conserved C-terminal sequence region of HrcA is discussed.

Crystal structure of MJ1247 protein from M. jannaschii at 2.0 A resolution infers a molecular function of 3-hexulose-6-phosphate isomerase.

The crystal structure of the hypothetical protein MJ1247 from Methanococccus jannaschii at 2 A resolution, a detailed sequence analysis, and biochemical assays infer its molecular function to be 3-hexulose-6-phosphate isomerase (PHI). In the dissimilatory ribulose monophosphate (RuMP) cycle, ribulose-5-phosphate is coupled to formaldehyde by the 3-hexulose-6-phosphate synthase (HPS), yielding hexulose-6-phosphate, which is then isomerized to fructose-6-phosphate by the enzyme 3-hexulose-6-phosphate isomerase. MJ1247 is an alpha/beta structure consisting of a five-stranded parallel beta sheet flanked on both sides by alpha helices, forming a three-layered alpha-beta-alpha sandwich. The fold represents the nucleotide binding motif of a flavodoxin type. MJ1247 is a tetramer in the crystal and in solution and each monomer has a folding similar to the isomerase domain of glucosamine-6-phosphate synthase (GlmS).

Structural and functional characterization of a 5,10-methenyltetrahydrofolate synthetase from Mycoplasma pneumoniae (GI: 13508087).

Mycoplasma pneumoniae 5,10-methenyltetrahydrofolate synthetase [MTHFS; also known as 5-formyltetrahydrofolate cycloligase; Enzyme Commission (EC) 6.3.3.2] belongs to a large cycloligase protein family with 97 sequence homologues from bacteria to human. To help define the molecular (biochemical and biophysical) function of the M. pneumoniae MTHFS, we have previously determined its crystal structure at 2.2 A resolution (Chen et al., Proteins 2004;56:839-843). In this current study, activity assays confirmed the functionality of the recombinant protein, with K(m) = 165 microM for 5-formyltetrahydrofolate (5-FTHF) and K(m) = 166 microM for MgATP. The methenyltetrahydrofolate activity of M. pneumoniae MTHFS has a requirement for divalent metal ions with Mg2+ being most effective, and an absolute requirement for nucleoside 5'-triphosphates with adenosine triphosphate (ATP) being most effective. Crystallization in the presence of substrates (MgATP, with or without 5-FTHF) produced the complex structures of the protein with adenosine diphosphate (ADP) and phosphate at 2.2 A resolution; with ADP, phosphate, and 5-FTHF at 2.5 A resolution. These structures directly demonstrated that the role of Mg2+ in the reaction is to form the ATP--Mg2+-enzyme complex.

Design of a data model for developing laboratory information management and analysis systems for protein production.

Data management has emerged as one of the central issues in the high-throughput processes of taking a protein target sequence through to a protein sample. To simplify this task, and following extensive consultation with the international structural genomics community, we describe here a model of the data related to protein production. The model is suitable for both large and small facilities for use in tracking samples, experiments, and results through the many procedures involved. The model is described in Unified Modeling Language (UML). In addition, we present relational database schemas derived from the UML. These relational schemas are already in use in a number of data management projects.

Structural analyses of peptide release factor 1 from Thermotoga maritima reveal domain flexibility required for its interaction with the ribosome.

We have determined the crystal structure of peptide chain release factor 1 (RF1) from Thermotoga maritima (gi 4981173) at 2.65 Angstrom resolution by selenomethionine single-wavelength anomalous dispersion (SAD) techniques. RF1 is a protein that recognizes stop codons and promotes the release of a nascent polypeptide from tRNA on the ribosome. Selenomethionine-labeled RF1 crystallized in space group P2(1) with three monomers per asymmetric unit. It has approximate dimensions of 75 Angstrom x 70 Angstrom x 45 Angstrom and is composed of four domains. The overall fold of each RF1 domain shows almost the same topology with Escherichia coli RF2, except that the RF1 N-terminal domain is shorter and the C-terminal domain is longer than that of RF2. The N-terminal domain of RF1 indicates a rigid-body movement relative to that of RF2 with an angle of approximately 90 degrees. Including these features, RF1 has a tripeptide anticodon PVT motif instead of the SPF motif of RF2, which confers the specificity towards the stop codons. The analyses of three molecules in the asymmetric unit and comparison with RF2 revealed the presence of dynamic movement of domains I and III, which are anchored to the central domain by hinge loops. The crystal structure of RF1 elucidates the intrinsic property of this family of having large domain movements for proper function with the ribosome.

Structural characterization of the reaction pathway in phosphoserine phosphatase: crystallographic "snapshots" of intermediate states.

Phosphoserine phosphatase (PSP) is a member of a large class of enzymes that catalyze phosphoester hydrolysis using a phosphoaspartate-enzyme intermediate. PSP is a likely regulator of the steady-state d-serine level in the brain, which is a critical co-agonist of the N-methyl-d-aspartate type of glutamate receptors. Here, we present high-resolution (1.5-1.9 A) structures of PSP from Methanococcus jannaschii, which define the open state prior to substrate binding, the complex with phosphoserine substrate bound (with a D to N mutation in the active site), and the complex with AlF3, a transition-state analog for the phospho-transfer steps in the reaction. These structures, together with those described for the BeF3- complex (mimicking the phospho-enzyme) and the enzyme with phosphate product in the active site, provide a detailed structural picture of the full reaction cycle. The structure of the apo state indicates partial unfolding of the enzyme to allow substrate binding, with refolding in the presence of substrate to provide specificity. Interdomain and active-site conformational changes are identified. The structure with the transition state analog bound indicates a "tight" intermediate. A striking structure homology, with significant sequence conservation, among PSP, P-type ATPases and response regulators suggests that the knowledge of the PSP reaction mechanism from the structures determined will provide insights into the reaction mechanisms of the other enzymes in this family.

Structure of a NAD kinase from Thermotoga maritima at 2.3 A resolution.

NAD kinase is the only known enzyme that catalyzes the formation of NADP, a coenzyme involved in most anabolic reactions and in the antioxidant defense system. Despite its importance, very little is known regarding the mechanism of catalysis and only recently have several NAD kinase structures been deposited in the PDB. Here, an independent investigation of the crystal structure of inorganic polyphosphate/ATP-NAD kinase, PPNK_THEMA, a protein from Thermotoga maritima, is reported at a resolution of 2.3 A. The crystal structure was solved using single-wavelength anomalous diffraction (SAD) data collected at the Se absorption-peak wavelength in a state in which no cofactors or substrates were bound. It revealed that the 258-amino-acid protein is folded into two distinct domains, similar to recently reported NAD kinases. The N-terminal alpha/beta-domain spans the first 100 amino acids and the last 30 amino acids of the polypeptide and has several topological matches in the PDB, whereas the other domain, which spans the middle 130 residues, adopts a unique beta-sandwich architecture and only appreciably matches the recently deposited PDB structures of NAD kinases.

Crystal structure of a phosphatase with a unique substrate binding domain from Thermotoga maritima.

We have determined the crystal structure of a phosphatase with a unique substrate binding domain from Thermotoga maritima, TM0651 (gi 4981173), at 2.2 A resolution by selenomethionine single-wavelength anomalous diffraction (SAD) techniques. TM0651 is a member of the haloacid dehalogenase (HAD) superfamily, with sequence homology to trehalose-6-phosphate phosphatase and sucrose-6(F)-phosphate phosphohydrolase. Selenomethionine labeled TM0651 crystallized in space group C2 with three monomers per asymmetric unit. Each monomer has approximate dimensions of 65 x 40 x 35 A(3), and contains two domains: a domain of known hydrolase fold characteristic of the HAD family, and a domain with a new tertiary fold consisting of a six-stranded beta-sheet surrounded by four alpha-helices. There is one disulfide bond between residues Cys35 and Cys265 in each monomer. One magnesium ion and one sulfate ion are bound in the active site. The superposition of active site residues with other HAD family members indicates that TM0651 is very likely a phosphatase that acts through the formation of a phosphoaspartate intermediate, which is supported by both NMR titration data and a biochemical assay. Structural and functional database searches and the presence of many aromatic residues in the interface of the two domains suggest the substrate of TM0651 is a carbohydrate molecule. From the crystal structure and NMR data, the protein likely undergoes a conformational change upon substrate binding.

Crystal structure of a protein associated with cell division from Mycoplasma pneumoniae (GI: 13508053): a novel fold with a conserved sequence motif.

UPF0040 is a family of proteins implicated in a cellular function of bacteria cell division. There is no structure information available on protein of this family. We have determined the crystal structure of a protein from Mycoplasma pneumoniae that belongs to this family using X-ray crystallography. Structural homology search reveals that this protein has a novel fold with no significant similarity to any proteins of known three-dimensional structure. The crystal structures of the protein in three different crystal forms reveal that the protein exists as a ring of octamer. The conserved protein residues, including a highly conserved DXXXR motif, are examined on the basis of crystal structure.

Crystal structure of a hypothetical protein, TM841 of Thermotoga maritima, reveals its function as a fatty acid-binding protein.

We determined the three-dimensional (3D) crystal structure of protein TM841, a protein product from a hypothetical open-reading frame in the genome of the hyperthermophile bacterium Thermotoga maritima, to 2.0 A resolution. The protein belongs to a large protein family, DegV or COG1307 of unknown function. The 35 kDa protein consists of two separate domains, with low-level structural resemblance to domains from other proteins with known 3D structures. These structural homologies, however, provided no clues for the function of TM841. But the electron density maps revealed clear density for a bound fatty-acid molecule in a pocket between the two protein domains. The structure indicates that TM841 has the molecular function of fatty-acid binding and may play a role in the cellular functions of fatty acid transport or metabolism.

Expression of soluble recombinant proteins in a cell-free system using a 96-well format.

For structural and functional genomics programs, new high-throughput methods to obtain well-expressing and highly soluble proteins are essential. Here, we describe a rapid procedure to express recombinant proteins in an Escherichia coli cell-free system using a 96-well format. The identification of soluble proteins is performed by the Dot Blot procedure using an anti-His tag antibody. The applications and the automation of this method are described.