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P Brick

Publications and source records attributed to P Brick.

At least 19 recordsLinked to original sources
PubMed2001

Structure of an archaeal homolog of the eukaryotic RNA polymerase II RPB4/RPB7 complex.

The eukaryotic subunits RPB4 and RPB7 form a heterodimer that reversibly associates with the RNA polymerase II core and constitute the only two components of the enzyme for which no structural information is available. We have determined the crystal structure of the complex between the Methanococcus jannaschii subunits E and F, the archaeal homologs of RPB7 and RPB4. Subunit E has an elongated two-domain structure and contains two potential RNA binding motifs, while the smaller F subunit wraps around one side of subunit E, at the interface between the two domains. We propose a model for the interaction between RPB4/RPB7 and the core RNA polymerase in which the RNA binding face of RPB7 is positioned to interact with the nascent RNA transcript.

Amino Acid Motifs↗
PubMed2000

Quantum correlations in the nonperturbative regime of semiconductor microcavities.

The nonlinear optical response of semiconductor microcavities in the nonpertubative regime is studied in resonant single-beam-transmission and pump-probe experiments. In both cases a pronounced third transmission peak lying spectrally between the two normal modes is observed. A fully quantized theory is essential for the agreement with the experimental observations, demonstrating that quantum fluctuations leading to intraband polarizations are responsible for this effect.

Journal Article↗
PubMed2000

Structural studies of lysyl-tRNA synthetase: conformational changes induced by substrate binding.

Lysyl-tRNA synthetase is a member of the class II aminoacyl-tRNA synthetases and catalyses the specific aminoacylation of tRNA(Lys). The crystal structure of the constitutive lysyl-tRNA synthetase (LysS) from Escherichia coli has been determined to 2.7 A resolution in the unliganded form and in a complex with the lysine substrate. A comparison between the unliganded and lysine-bound structures reveals major conformational changes upon lysine binding. The lysine substrate is involved in a network of hydrogen bonds. Two of these interactions, one between the alpha-amino group and the carbonyl oxygen of Gly 216 and the other between the carboxylate group and the side chain of Arg 262, trigger a subtle and complicated reorganization of the active site, involving the ordering of two loops (residues 215-217 and 444-455), a change in conformation of residues 393-409, and a rotation of a 4-helix bundle domain (located between motif 2 and 3) by 10 degrees. The result of these changes is a closing up of the active site upon lysine binding.

Adenosine Triphosphate↗
PubMed2000

Active site of lysyl-tRNA synthetase: structural studies of the adenylation reaction.

Aminoacyl-tRNA synthetases play a key role in protein biosynthesis by catalyzing the specific aminoacylation of tRNA. The energy required for the formation of the ester bond between the amino acid carboxylate group and the tRNA acceptor stem is supplied by coupling the reaction to the hydrolysis of ATP. Lysyl-tRNA synthetase from Escherichia coli belongs to the family of class II synthetases and carries out a two-step reaction, in which lysine is activated by being attached to the alpha-phosphate of AMP before being transferred to the cognate tRNA. Crystals of the thermo-inducible E. coli lysyl-tRNA synthetase LysU which diffract to 2.1 A resolution have been used to determine crystal structures of the enzyme in the presence of lysine, the lysyl-adenylate intermediate, and the nonhydrolyzable ATP analogue AMP-PCP. Additional data have been obtained from crystals soaked in a solution containing ATP and Mn(2+). The refined crystal structures give "snapshots" of the active site corresponding to key steps in the aminoacylation reaction and provide the structural framework for understanding the mechanism of lysine activation. The active site of LysU is shaped to position the substrates for the nucleophilic attack of the lysine carboxylate on the ATP alpha-phosphate. No residues are directly involved in catalysis, but a number of highly conserved amino acids and three metal ions coordinate the substrates and stabilize the pentavalent transition state. A loop close to the catalytic pocket, disordered in the lysine-bound structure, becomes ordered upon adenine binding.

Adenosine Triphosphate↗
PubMed2000

Crystal structure of RPB5, a universal eukaryotic RNA polymerase subunit and transcription factor interaction target.

Eukaryotic nuclei contain three different types of RNA polymerases (RNAPs), each consisting of 12-18 different subunits. The evolutionarily highly conserved RNAP subunit RPB5 is shared by all three enzymes and therefore represents a key structural/functional component of all eukaryotic RNAPs. Here we present the crystal structure of the RPB5 subunit from Saccharomyces cerevisiae. The bipartite structure includes a eukaryote-specific N-terminal domain and a C-terminal domain resembling the archaeal RNAP subunit H. RPB5 has been implicated in direct protein-protein contacts with transcription factor IIB, one of the components of the RNAP(II) basal transcriptional machinery, and gene-specific activator proteins, such as the hepatitis B virus transactivator protein X. The experimentally mapped regions of RPB5 involved in these interactions correspond to distinct and surface-exposed alpha-helical structures.

Amino Acid Sequence↗
PubMed1999

Fatty acid binding to human serum albumin: new insights from crystallographic studies.

Human serum albumin possesses multiple fatty acid binding sites of varying affinities, but the precise locations of these sites have remained elusive. The determination of the crystal structure of human serum albumin complexed with myristic acid recently revealed the positions and architecture of six binding sites on the protein. While the structure of the complex is consistent with a great deal of the biochemical and biophysical data on fatty acid binding, it is not yet possible to provide a completely rigorous correlation between the structural and binding data. The challenge now is to use the new structural information to design experiments that will identify the physiologically important binding sites on HSA and provide a much richer description of fatty acid interactions with the protein.

Binding Sites↗
PubMed1999

Identification and characterization of a DNA primase from the hyperthermophilic archaeon Methanococcus jannaschii.

We report the identification and characterisation of a DNA primase from the thermophilic methanogenic archaeon Methanococcus jannaschii (Mjpri). The analysis of the complete genome sequence of this organism has identified an open reading frame coding for a protein with sequence similarity to the small subunit of the eukaryotic DNA primase (the p50 subunit of the polymerase alpha-primase complex). This protein has been overexpressed in Escherichia coli and purified to near homogeneity. Recombinant Mjpri is able to synthesise oligoribonucleotides on various pyrimidine single-stranded DNA templates [poly(dT) and poly(dC)]. This activity requires divalent cations such Mg(2+), Mn(2+)or Zn(2+), and is additionally stimulated by the monovalent cation K(+). A multiple sequence alignment has revealed that most of the regions that are conserved in eukaryotic p50 subunits are also present in the archaeal primases, including the conserved negatively charged residues, which have been shown to be essential for catalysis in the mouse primase. Of the four cysteine residues that have been postulated to make up a putative Zn-binding motif, two are not present in the archaeal homologue. This is the first report on the biochemical characterisation of an archaeal DNA primase.

Amino Acid Sequence↗
PubMed1998

Structure of the Kunitz-type soybean trypsin inhibitor (STI): implication for the interactions between members of the STI family and tissue-plasminogen activator.

The Kunitz-type soybean trypsin inhibitor (STI) has played a key role in the early study of proteinases, having been used as the main substrate in the biochemical and kinetic work that led to the definition of the standard mechanism of action of proteinase inhibitors. A partial structure of STI complexed with porcine trypsin has previously been reported, in which the first 93 residues of the inhibitor, including the region of contact with trypsin, were relatively well defined, whereas for the remaining part of the peptide chain only some Calpha atoms were located. The structure of the inhibitor in its free form has now been determined by molecular replacement to 2.5 A, using the coordinates of the homologous Erythrina trypsin inhibitor as a search model. When the refined atomic coordinates of STI are compared with the partial model previously available, the conformation of the reactive-site loop and its position with respect to the main body of the molecule does not change when the inhibitor interacts with trypsin. There are instead, despite the high similarity in the overall tertiary structure, significant differences between STI and Erythrina trypsin inhibitor (ETI) in the region which is in contact with the enzyme in the STI:trypsin crystal structure. Some of these differences can explain the unique specificity of ETI and its ability to inhibit the fibrinolytic enzyme tissue-type plasminogen activator.

Amino Acid Sequence↗
PubMed1998

X-ray and spectrophotometric studies of the binding of proflavin to the S1 specificity pocket of human alpha-thrombin.

Proflavin can be used to study the interactions of inhibitors and substrates with thrombin by monitoring the changes in the visible absorption spectrum that occur on dye displacement. We have used microspectrophotometric methods to investigate the binding of proflavin to crystals of an alpha-thrombin-hirugen complex and have determined the structure by X-ray crystallography. The proflavin molecule binds in the S1 pocket of the enzyme with one of the amino groups hydrogen bonded to the carboxylate of Asp-189 while the protonated ring nitrogen is hydrogen bonded to the carbonyl of Gly-219. This result indicates that the proflavin displacement assay can be used to specifically monitor the binding of inhibitors to the S1 pocket.

Absorption↗
PubMed1998

Structural basis for the inhibition of firefly luciferase by a general anesthetic.

The firefly luciferase enzyme from Photinus pyralis is probably the best-characterized model system for studying anesthetic-protein interactions. It binds a diverse range of general anesthetics over a large potency range, displays a sensitivity to anesthetics that is very similar to that found in animals, and has an anesthetic sensitivity that can be modulated by one of its substrates (ATP). In this paper we describe the properties of bromoform acting as a general anesthetic (in Rana temporaria tadpoles) and as an inhibitor of the firefly luciferase enzyme at high and low ATP concentrations. In addition, we describe the crystal structure of the low-ATP form of the luciferase enzyme in the presence of bromoform at 2.2-A resolution. These results provide a structural basis for understanding the anesthetic inhibition of the enzyme, as well as an explanation for the ATP modulation of its anesthetic sensitivity.

Adenosine Triphosphate↗
PubMed1998

Crystal structure of human serum albumin complexed with fatty acid reveals an asymmetric distribution of binding sites.

Human serum albumin (HSA) is the most abundant protein in the circulatory system. Its principal function is to transport fatty acids, but it is also capable of binding a great variety of metabolites and drugs. Despite intensive efforts, the detailed structural basis of fatty acid binding to HSA has remained elusive. We have now determined the crystal structure of HSA complexed with five molecules of myristate at 2.5 A resolution. The fatty acid molecules bind in long, hydrophobic pockets capped by polar side chains, many of which are basic. These pockets are distributed asymmetrically throughout the HSA molecule, despite its symmetrical repeating domain structure.

Amino Acid Sequence↗
PubMed1997

Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S.

The non-ribosomal synthesis of the cyclic peptide antibiotic gramicidin S is accomplished by two large multifunctional enzymes, the peptide synthetases 1 and 2. The enzyme complex contains five conserved subunits of approximately 60 kDa which carry out ATP-dependent activation of specific amino acids and share extensive regions of sequence similarity with adenylating enzymes such as firefly luciferases and acyl-CoA ligases. We have determined the crystal structure of the N-terminal adenylation subunit in a complex with AMP and L-phenylalanine to 1.9 A resolution. The 556 amino acid residue fragment is folded into two domains with the active site situated at their interface. Each domain of the enzyme has a similar topology to the corresponding domain of unliganded firefly luciferase, but a remarkable relative domain rotation of 94 degrees occurs. This conformation places the absolutely conserved Lys517 in a position to form electrostatic interactions with both ligands. The AMP is bound with the phosphate moiety interacting with Lys517 and the hydroxyl groups of the ribose forming hydrogen bonds with Asp413. The phenylalanine substrate binds in a hydrophobic pocket with the carboxylate group interacting with Lys517 and the alpha-amino group with Asp235. The structure reveals the role of the invariant residues within the superfamily of adenylate-forming enzymes and indicates a conserved mechanism of nucleotide binding and substrate activation.

Adenosine Monophosphate↗
PubMed1997

Crystal structure of carboxypeptidase G2, a bacterial enzyme with applications in cancer therapy.

BACKGROUND: Carboxypeptidase G enzymes hydrolyze the C-terminal glutamate moiety from folic acid and its analogues, such as methotrexate. The enzyme studied here, carboxypeptidase G2 (CPG2), is a dimeric zinc-dependent exopeptidase produced by Pseudomonas sp. strain RS-16. CPG2 has applications in cancer therapy: following its administration as an immunoconjugate, in which CPG2 is linked to an antibody to a tumour-specific antigen, it can enzymatically convert subsequently administered inactive prodrugs to cytotoxic drugs selectively at the tumour site. CPG2 has no significant amino acid sequence homology with proteins of known structure. Hence, structure determination of CPG2 was undertaken to identify active-site residues, which may in turn provide ideas for protein and/or substrate modification with a view to improving its therapeutic usefulness. RESULTS: We have determined the crystal structure of CPG2 at 2.5 A resolution using multiple isomorphous replacement methods and non-crystallographic symmetry averaging. Each subunit of the molecular dimer consists of a larger catalytic domain containing two zinc ions at the active site, and a separate smaller domain that forms the dimer interface. The two active sites in the dimer are more than 60 A apart and are presumed to be independent; each contains a symmetric distribution of carboxylate and histidine ligands around two zinc ions which are 3.3 A apart. This distance is bridged by two shared zinc ligands, an aspartic acid residue and a hydroxyl ion. CONCLUSIONS: We find that the CPG2 catalytic domain has structural homology with other zinc-dependent exopeptidases, both those with a single zinc ion and those with a pair of zinc ions in the active site. The closest structural homology is with the aminopeptidase from Aeromonas proteolytica, where the similarity includes superposable zinc ligands but does not extend to the rest of the active-site residues, consistent with the different substrate specificities. The mechanism of peptide cleavage is likely to be very similar in these two enzymes and may involve the bridging hydroxyl ion ligand acting as a primary nucleophile.

Aminopeptidases↗
PubMed1996

Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes.

BACKGROUND: Firefly luciferase is a 62 kDa protein that catalyzes the production of light. In the presence of MgATP and molecular oxygen, the enzyme oxidizes its substrate, firefly luciferin, emitting yellow-green light. The reaction proceeds through activation of the substrate to form an adenylate intermediate. Firefly luciferase shows extensive sequence homology with a number of enzymes that utilize ATP in adenylation reactions. RESULTS: We have determined the crystal structure of firefly luciferase at 2.0 A resolution. The protein is folded into two compact domains. The large N-terminal domain consists of a beta-barrel and two beta-sheets. The sheets are flanked by alpha-helices to form an alphabetaalphabetaalpha five-layered structure. The C-terminal portion of the molecule forms a distinct domain, which is separated from the N-terminal domain by a wide cleft. CONCLUSIONS: Firefly luciferase is the first member of a superfamily of homologous enzymes, which includes acyl-coenzyme A ligases and peptide synthetases, to have its structure characterized. The residues conserved within the superfamily are located on the surfaces of the two domains on either side of the cleft, but are too far apart to interact simultaneously with the substrates. This suggests that the two domains will close in the course of the reaction. Firefly luciferase has a novel structural framework for catalyzing adenylate-forming reactions.

Adenosine↗
PubMed1996

Collagenase: a key enzyme in collagen turnover.

The primary agents responsible for cartilage and bone destruction in joint diseases are active proteinases that degrade collagen and proteoglycan. All four main classes of proteolytic enzymes are involved in either the normal turnover of connective tissue or its pathological destruction. These proteinases are made by different cells found within the joints. Both extracellular and intracellular pathways exist and individual enzymes can be inhibited by specific proteinaceous inhibitors that block their activity. Recent research has implicated the matrix metalloproteinases (MMPs) in many of the processes involved in joint diseases. The metalloproteinases are capable of degrading all components of the extracellular matrix. This family of proteinases contains a group of at least three collagenases that are capable of degrading native fibrillar collagen. Collagen degradation within joint disease is recognized as the irreversible step in the destruction of cartilage that leads to a failure in joint function. The collagenases are the enzymes necessary to initiate collagen turnover in normal connective tissue turnover and in disease.

Amino Acid Sequence↗
PubMed1995

Structure of full-length porcine synovial collagenase reveals a C-terminal domain containing a calcium-linked, four-bladed beta-propeller.

BACKGROUND: The collagenases are members of the family of zinc-dependent enzymes known as the matrix metalloproteinases (MMPs). They are the only proteinases that specifically cleave the collagen triple helix, and are important in a large number of physiological and pathological processes. Structures are known for the N-terminal catalytic' domain of collagenases MMP-1 and MMP-8 and of stromelysin (MMP-3). This catalytic domain alone, which comprises about 150 amino acids, has no activity against collagen. A second domain, of 200 amino acids, is homologous to haemopexin, a haem-binding glycoprotein. RESULTS: The crystal structure of full-length MMP-1 at 2.5 A resolution gives an R-factor of 21.7%. Two domains are connected by an exposed proline-rich linker of 17 amino acids, which is probably flexible and has no secondary structure. The catalytic domain resembles those previously observed, and contains three calcium-binding sites. The haemopexin-like domain contains four units of four-stranded antiparallel beta sheet stabilized on its fourfold axis by a cation, which is probably calcium. The domain constitutes a four-bladed beta-propeller structure in which the blades are scarcely twisted. CONCLUSIONS: The exposed linker accounts for the difficulty in purifying full-length collagenase. The C-terminal domain provides a structural model for haemopexin and its homologues. It controls the specificity of MMPs, affecting both substrate and inhibitor binding, although its role remains obscure. These structural results should aid the design of site-specific mutants which will reveal further details of the specificity mechanism.

Amino Acid Sequence↗
PubMed1995

The crystal structure of the lysyl-tRNA synthetase (LysU) from Escherichia coli.

BACKGROUND: Lysyl-tRNA synthetase catalyzes the attachment of the amino acid lysine to the cognate tRNA. The enzyme is a member of the class II amino-acyl-tRNA synthetases; the crystal structures of the seryl- and aspartyl-tRNA synthetases from this class are already known. Lysyl-tRNA synthetase shows extensive sequence homology with aspartyl-tRNA synthetase. In Escherichia coli there are two isoforms of the enzyme, LysS and LysU. Unlike LysS, which is synthesized under normal growth conditions, LysU is the product of a normally silent gene which is overexpressed under extreme physiological conditions (such as heat-shock), and can synthesize a number of adenyl dinucleotides (in particular AppppA). These dinucleotides have been proposed to act as modulators of the heat-shock response and stress response. RESULTS: The crystal structure of E. coli LysU has been determined to 2.8 A resolution, with lysine bound to the active site. The protein is a homodimer, with a rather extended dimer interface spanning the entire length of the molecule. Each monomer consists of two domains: a smaller N-terminal domain which binds the tRNA anticodon, and a larger C-terminal domain with the topology characteristic of the catalytic domain found in class II synthetases. CONCLUSIONS: A comparison of the LysU crystal structure with the structures of seryl- and aspartyl-tRNA synthetases enables a conserved core to be identified. The structural homology with the aspartyl-tRNA synthetase extends to include the anticodon-binding domain. When the active sites of lysyl-, aspartyl- and seryl-tRNA synthetases are compared, a number of catalytically important residues are conserved and a similar extended network of hydrogen bonds can be observed in the amino acid binding pocket in all three structures, although the details may differ. The lysine substrate is involved in an extended network of hydrogen bonds and polar interactions, with the side chain amino group forming a salt bridge with Glu428. The binding of ATP to LysU can be modelled on the basis of the aspartyl-tRNA synthetase-ATP complex, but the tRNA acceptor stem interaction for LysU cannot be easily modelled by similar extrapolation.

Amino Acid Sequence↗