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PubMed1999

Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus: the 1.85 A resolution crystal structure and its mechanistic implications.

Crystal structures of formaldehyde ferredoxin oxidoreductase (FOR), a tungstopterin-containing protein from the hyperthermophilic archaeon Pyrococcus furiosus, have been determined in the native state and as a complex with the inhibitor glutarate at 1.85 A and 2. 4 A resolution, respectively. The native structure was solved by molecular replacement using the structure of the homologous P. furiosus aldehyde ferredoxin oxidoreductase (AOR) as the initial model. Residues are identified in FOR that may be involved in either the catalytic mechanism or in determining substrate specificity. The binding site on FOR for the physiological electron acceptor, P. furiosus ferredoxin (Fd), has been established from an FOR-Fd cocrystal structure. Based on the arrangement of redox centers in this structure, an electron transfer pathway is proposed that begins at the tungsten center, leads to the (4Fe:4S) cluster of FOR via one of the two pterins that coordinate the tungsten, and ends at the (4Fe:4S) cluster of ferredoxin. This pathway includes two residues that coordinate the (4Fe:4S) clusters, Cys287 of FOR and Asp14 of ferredoxin. Similarities in the active site structures between FOR and the unrelated molybdoenzyme aldehyde oxidoreductase from Desulfovibrio gigas suggest that both enzymes utilize a common mechanism for aldehyde oxidation.

Aldehyde Oxidoreductases↗
PubMed1999

Crystal structures of thermostable xylose isomerases from Thermus caldophilus and Thermus thermophilus: possible structural determinants of thermostability.

The crystal structures of highly thermostable xylose isomerases from Thermus thermophilus (TthXI) and Thermus caldophilus (TcaXI), both with the optimum reaction temperature of 90 degrees C, have been determined by X-ray crystallography. The model of TcaXI has been refined to an R-factor of 17.8 % for data extending to 2.3 A and that of TthXI to 17.1 % for data extending to 2.2 A. The tetrameric arrangement of subunits characterized by the 222-symmetry and the tertiary fold of each subunit in both TcaXI and TthXI are basically the same as in other xylose isomerases. Each monomer is composed of two domains. Domain I (residues 1 to 321) folds into the (beta/alpha)8-barrel. Domain II (residues 322 to 387), lacking beta-strands, makes extensive contacts with domain I of an adjacent subunit. Each monomer of TcaXI contains ten beta-strands, 15 alpha-helices, and six 310-helices, while that of TthXI contains ten beta-strands, 16 alpha-helices, and five 310-helices. Although the electron density does not indicate the presence of bound metal ions in the present models of both TcaXI and TthXI, the active site residues show the conserved structural features. In order to understand the structural basis for thermostability of these enzymes, their structures have been compared with less thermostable XIs from Arthrobacter B3728 and Actinoplanes missouriensis (AXI and AmiXI), with the optimum reaction temperatures of 80 degrees C and 75 degrees C, respectively. Analyses of various factors that may affect protein thermostability indicate that the possible structural determinants of the enhanced thermostability of TcaXI/TthXI over AXI/AmiXI are (i) an increase in ion pairs and ion-pair networks, (ii) a decrease in the large inter-subunit cavities, (iii) a removal of potential deamidation/isoaspartate formation sites, and (iv) a shortened loop.

Aldose-Ketose Isomerases↗
PubMed1990

The hydrogen bonding in the crystal structure of raffinose pentahydrate.

The crystal structure of raffinose pentahydrate, O-alpha-D-galactopyranosyl-(1----6)-O-alpha-D-glucopyranosyl-(1----2)- beta-D- fructofuranose pentahydrate, C18H32O16.5H2O, has been redetermined using low-temperature, 119 K, CuK alpha X-ray data. All hydrogen atoms were unambiguously located on difference syntheses. The final R-factor is 0.036 for 2423 observed structure amplitudes. The hydrogen bonding is composed of infinite chains, which are linked through the water molecules to form a three-dimensional network containing a chain of five linked water molecules. Three of the infinite chains extend in the directions of the crystallographic axis of the space group P2(1)2(1)2(1). Four of the water molecules accept two hydrogen bonds and one accepts one. All the hydroxyls and the ring and glycosidic oxygen atoms are involved in the hydrogen bonding. With one exception, the ring and glycosidic oxygens are hydrogen-bonded by means of the minor components of unsymmetrical three-center bonds.

Carbohydrate Conformation↗
PubMed1987

High-resolution crystal structure of cytochrome P450cam.

The crystal structure of Pseudomonas putida cytochrome P450cam with its substrate, camphor, bound has been refined to R = 0.19 at a normal resolution of 1.63 A. While the 1.63 A model confirms our initial analysis based on the 2.6 A model, the higher resolution structure has revealed important new details. These include a more precise assignment of sequence to secondary structure, the identification of three cis-proline residues, and a more detailed picture of substrate-protein interactions. In addition, 204 ordered solvent molecules have been found, one of which appears to be a cation. The cation stabilizes an unfavorable polypeptide conformation involved in forming part of the active site pocket, suggesting that the cation may be the metal ion binding site associated with the well-known ability of metal ions to enhance formation of the enzyme-substrate complex. Another unusual polypeptide conformation forms the proposed oxygen-binding pocket. A localized distortion and widening of the distal helix provides a pocket for molecular oxygen. An intricate system of side-chain to backbone hydrogen bonds aids in stabilizing the required local disruption in helical geometry. Sequence homologies strongly suggest a common oxygen-binding pocket in all P450 species. Further sequence comparisons between P450 species indicate common three-dimensional structures with changes focused in a region of the molecule postulated to be associated with the control of substrate specificity.

Amino Acid Sequence↗
PubMed1995

Acetylcholinesterase inhibition by fasciculin: crystal structure of the complex.

The crystal structure of the snake toxin fasciculin, bound to mouse acetylcholinesterase (mAChE), at 3.2 A resolution reveals a synergistic three-point anchorage consistent with the picomolar dissociation constant of the complex. Loop II of fasciculin contains a cluster of hydrophobic residues that interact with the peripheral anionic site of the enzyme and sterically occlude substrate access to the catalytic site. Loop I fits in a crevice near the lip of the gorge to maximize the surface area of contact of loop II at the gorge entry. The fasciculin core surrounds a protruding loop on the enzyme surface and stabilizes the whole assembly. Upon binding of fasciculin, subtle structural rearrangements of AChE occur that could explain the observed residual catalytic activity of the fasciculin-enzyme complex.

Animals↗
PubMed1984

Hydroperoxides as inactivators of aromatase: 10 beta-hydroperoxy-4-estrene-3,17-dione, crystal structure and inactivation characteristics.

The crystal structure of 10 beta-hydroperoxy-4-estrene-3,17-dione (10 beta-OOH) was determined, and its inhibition of human placental aromatase was investigated. In the absence of added NADPH, 10 beta-OOH caused a time-dependent loss of aromatase activity (e.g., 50% loss after 90 s with 2.16 microM 10 beta-OOH). Protection against this loss of activity was provided when a substrate, androstenedione, was included in the incubation. Centrifugation and resuspension of the 10 beta-OOH-treated microsomes in fresh buffer failed to restore the activity, but partial recovery could be effected by dithiothreitol. Experiments to detect destruction of aromatase protoheme were done but were inconclusive. In the presence of NADPH, 10 beta-OOH did not cause a time-dependent loss of activity but was instead a competitive inhibitor (Ki = 330 nM) of androstenedione (Km = 21 nM) aromatization. The added NADPH was not utilized for the aromatization of 10 beta-OOH to estrogens, and enhanced reduction of 10 beta-OOH to 10 beta-hydroxy-4-estrene-3,17-dione could not be detected. In addition, microsomes alone were incapable of using 10 beta-OOH to support the aromatization of androstenedione. Cumene hydroperoxide and H2O2 were also investigated as inactivators of aromatase. Losses of activity comparable to those found for 10 beta-OOH could only be observed at 500-1000-fold higher concentrations of these agents, and no protection was provided by either androstenedione or NADPH. Extensive destruction of microsomal protoheme was found with these nonsteroidal agents.

Androstenedione↗
PubMed1992

The crystal structure of diphtheria toxin.

The crystal structure of the diphtheria toxin dimer at 2.5 A resolution reveals a Y-shaped molecule of three domains. The catalytic domain, called fragment A, is of the alpha + beta type. Fragment B actually consists of two domains. The transmembrane domain consists of nine alpha-helices, two pairs of which are unusually apolar and may participate in pH-triggered membrane insertion and translocation. The receptor-binding domain is a flattened beta-barrel with a jelly-roll-like topology. Three distinct functions of the toxin, each carried out by a separate structural domain, can be useful in designing chimaeric proteins, such as immunotoxins, in which the receptor-binding domain is substituted with antibodies to target other cell types.

Bacterial Toxins↗
PubMed1995

Extension of the four-stranded intercalated cytosine motif by adenine.adenine base pairing in the crystal structure of d(CCCAAT).

The crystal structure of d(CCCAAT), refined at 2.0 A resolution, shows a four stranded molecule in which two parallel duplexes intercalate with opposite polarity, using cytosine.protonated cytosine base pairs. The intercalation motif in this structure is extended by adenine.adenine base pairs. Two topologically distinct broad grooves are found in the lath-like central part of the molecule with the phosphate groups on one side bent over towards each other, stabilized by bridging water molecules. At the 3' ends, two arrangements of intermolecular A.A.T base triplets are found, involving both asymmetric and symmetric A.A base pairs joined to thymine residues by Watson-Crick and reverse Hoogsteen base pairing, respectively.

Adenine↗
PubMed1994

Crystal structure of PvuII endonuclease reveals extensive structural homologies to EcoRV.

The crystal structure of the dimeric PvuII restriction endonuclease (R.PvuII) has been determined at a resolution of 2.4A. The protein has a mixed alpha/beta architecture and consists of two subdomains. Despite a lack of sequence homology, extensive structural similarities exist between one R.PvuII subdomain and the DNA-binding subdomain of EcoRV endonuclease (R.EcoRV); the dimerization subdomains are unrelated. Within the similar domains, flexible segments of R.PvuII are topologically equivalent to the DNA-binding turns of R.EcoRV; potential catalytic residues can be deduced from the structural similarities to R.EcoRV. Conformational flexibility is important for the interaction with DNA. A possible classification of endonuclease structures on the basis of the positions of the scissile phosphates is discussed.

Bacterial Proteins↗
PubMed1994

Crystal structure of peanut lectin, a protein with an unusual quaternary structure.

The x-ray crystal structure of the tetrameric T-antigen-binding lectin from peanut, M(r) 110,000, has been determined by using the multiple isomorphous replacement method and refined to an R value of 0.218 for 22,155 reflections within the 10- to 2.95-A resolution range. Each subunit has essentially the same characteristic tertiary fold that is found in other legume lectins. The structure, however, exhibits an unusual quaternary arrangement of subunits. Unlike other well-characterized tetrameric proteins with identical subunits, peanut lectin has neither 222 (D2) nor fourfold (C4) symmetry. A noncrystallographic twofold axis relates two halves of the molecule. The two monomers in each half are related by a local twofold axis. The mutual disposition of the axes is such that they do not lead to a closed point group. Furthermore, the structure of peanut lectin demonstrates that differences in subunit arrangement in legume lectins could be due to factors intrinsic to the protein molecule and, contrary to earlier suggestions, are not necessarily caused by interactions involving covalently linked sugar. The structure provides a useful framework for exploring the structural basis and the functional implications of the variability in the subunit arrangement in legume lectins despite all of them having nearly the same subunit structure, and also for investigating the general problem of "open" quaternary assembly in oligomeric proteins.

Arachis↗
PubMed1995

Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5-A resolution: structural basis for thermostability.

The crystal structure of the large fragment of the Thermus aquaticus DNA polymerase (Klentaq1), determined at 2.5-A resolution, demonstrates a compact two-domain architecture. The C-terminal domain is identical in fold to the equivalent region of the Klenow fragment of Escherichia coli DNA polymerase I (Klenow pol I). Although the N-terminal domain of Klentaq1 differs greatly in sequence from its counterpart in Klenow pol I, it has clearly evolved from a common ancestor. The structure of Klentaq1 reveals the strategy utilized by this protein to maintain activity at high temperatures and provides the structural basis for future improvements of the enzyme.

Amino Acid Sequence↗
PubMed2006

DNA octaplex formation with an I-motif of water-mediated A-quartets: reinterpretation of the crystal structure of d(GCGAAAGC).

The crystal structure of the tetragonal form of d(gcGAAAgc) has been revised and reasonably refined including the disordered residues. The two DNA strands form a base-intercalated duplex, and the four duplexes are assembled according to the crystallographic 222 symmetry to form an octaplex. In the central region, the eight strands are associated by I-motif of double A-quartets. Furthermore, eight hydrated-magnesium cations link the four duplexes to support the octaplex formation. Based on these structural features, a proposal that folding of d(GAAA)n, found in the non-coding region of genomes, into an octaplex can induce slippage during replication to facilitate length polymorphism is presented.

Base Pairing↗
PubMed2006

Multiplex formation of repetitive sequences d([G]nA)m through non-WC pairings: crystal structure of d(gcGAGGGAgc).

The crystal structure of a DNA fragment with the sequence d(gcGA[G](3)Agc), a shortened and mutated derivative of the VNTR sequence d(ccGA[G](4)Agg), has been determined by X-ray analyses at 1.8 A resolution to investigate structures and mechanisms of a non-coding DNA. This sequence forms an antiparallel duplex with two WC pairings at the both ends and six consecutive non-WC pairings in the major part. Based on this structure, d(GA[G](n)A) (n=3-6) and d([G](n)A)(m) (n>1 and m>1) were designed. It is expected that all bases form non-WC pairs when the DNA fragments adapt antiparallel duplex. Electrophoresis experiments showed that these DNA fragments form not only duplexes, but also multiplexes such as quadruplexes, octaplexes, and so on.

Base Pairing↗
PubMed2000

Bonding effects and the crystal structures of (NH4)2

The crystal structures of the Tutton salts (NH4)2[Cu(H2O)6](SO4)2, diammonium hexaaquacopper disulfate, formed with normal water and isotopically substituted H2(18)O, have been determined by X-ray diffraction at 9.5 K and are very similar, with Cu-O(7) the longest of the Cu-O bonds of the Jahn-Teller distorted octahedral [Cu(H2O)6]2+ complex. It is known that structural differences accompany deuteration of (NH4)2[Cu(H2O)6](SO4)2, the most dramatic of which is a switch to Cu-O(8) as the longest such bond. The present result suggests that the structural differences are associated with hydrogen-bonding effects rather than with increased mass of the water ligands affecting the Jahn-Teller coupling. The Jahn-Teller distortions and hydrogen-bonding contacts in the compounds are compared with those reported for other Tutton salts at ambient and high pressure.

Journal Article↗
PubMed2000

Architecture of crystal structures from square planes.

The crystal structures of ordered b.c.c. (body-centered cubic), f.c. c. (face-centered cubic) or primitive cubic alloys AxBy and related NaCl, ZnS or CaF(2) derivative structures are characterized by the self-coordination numbers T(1), T(2) of the A atoms with A atoms. Structures with identical T(1) and T(2) values for all A atoms are at the corners of T(1) and T(2) structure maps, and can be analyzed for attractive or repulsive interactions of A atoms. Most observed structures are at the borders of the structure map and can be obtained by approximately 10 different combinations of structural units. The different combination mechanisms explain e.g. the shear structures of CuAu II or Nb(2)O(5) and the occurrence of vacancies in NaCl-related structures like NbO.

Journal Article↗
PubMed1996

Mutant Met121Ala of Pseudomonas aeruginosa azurin and its azide derivative: crystal structures and spectral properties.

The crystal structures of the azurin mutant Met121Ala and its azide derivative Met121Ala-azide from Pseudomonas aeruginosa have been determined. The final crystallographic R values are 21.3 and 19.4% for the two structures, respectively. In the Met121Ala mutant, the distance between the copper ion and His117 increases by 0.34 A compared with the wild-type structure. The removal of the methionine in the apical position induces a shortening of the distance from the copper ion to the carbonyl O atom of Gly45 from 2.97 to 2.74 A. In the Met121Ala-azide structure, the azide anion occupies the cavity created by replacing the Met121 side chain with the smaller methyl group of Ala. The azide anion binds with a terminal N atom to the copper ion at a distance of about 2.04 A. In addition, the copper ion has moved out of the trigonal plane by about 0.26 A towards the azide anion. Thus, the copper site in this structure has a distorted tetrahedral arrangement. The spectroscopic characteristics show, in addition, that the copper sites in the two structures are distinctively different. The Met121Ala mutant still maintains the properties of an ordinary type 1 copper site while the Met121Ala-azide derivative has an absorption maximum at about 409 nm and the copper hyperfine coupling has increased to a value intermediate between those of type 2 copper and the wild-type azurin.

Journal Article↗
PubMed2001

Detailed analysis of the atrial natriuretic factor receptor hormone-binding domain crystal structure.

The X-ray crystal structure of the dimerized atrial natriuretic factor (ANF) receptor hormone-binding domain has provided a first structural view of this anti-hypertensive receptor. The structure reveals a surprising evolutionary link to the periplasmic-binding protein fold family. Furthermore, the presence of a chloride ion in the membrane distal domain and the presence of a second putative effector pocket suggests that the extracellular domain of this receptor is allosterically regulated. The scope of this article is to extensively review the data published on this receptor and to correlate it with the hormone-binding domain structure. In addition, a more detailed description is provided of the important features of this structure including the different binding sites for the ANF hormone, chloride ion, putative effector pocket, glycosylation sites, and dimer interface.

Animals↗
PubMed1999

Structural basis for cold adaptation. Sequence, biochemical properties, and crystal structure of malate dehydrogenase from a psychrophile Aquaspirillium arcticum.

Aquaspillium arcticum is a psychrophilic bacterium that was isolated from arctic sediment and grows optimally at 4 degrees C. We have cloned, purified, and characterized malate dehydrogenase from A. arcticum (Aa MDH). We also have determined the crystal structures of apo-Aa MDH, Aa MDH.NADH binary complex, and Aa MDH.NAD.oxaloacetate ternary complex at 1.9-, 2.1-, and 2.5-A resolutions, respectively. The Aa MDH sequence is most closely related to the sequence of a thermophilic MDH from Thermus flavus (Tf MDH), showing 61% sequence identity and over 90% sequence similarity. Stability studies show that Aa MDH has a half-life of 10 min at 55 degrees C, whereas Tf MDH is fully active at 90 degrees C for 1 h. Aa MDH shows 2-3-fold higher catalytic efficiency compared with a mesophilic or a thermophilic MDH at the temperature range 4-10 degrees C. Structural comparison of Aa MDH and Tf MDH suggests that the increased relative flexibility of active site residues, favorable surface charge distribution for substrate and cofactor, and the reduced intersubunit ion pair interactions may be the major factors for the efficient catalytic activity of Aa MDH at low temperatures.

Adaptation, Physiological↗