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See detailStructure of PBP-A from Thermosynechococcus elongatus, a Penicillin-Binding Protein Closely Related to Class A β-Lactamases
Urbach, Carole; Evrard, Christine ULg; Pudzaitis, Vaidas et al

in Journal of Molecular Biology (2009), 386

Molecular evolution has always been a subject of discussions, and researchers are interested in understanding how proteins with similar scaffolds can catalyze different reactions. In the superfamily of ... [more ▼]

Molecular evolution has always been a subject of discussions, and researchers are interested in understanding how proteins with similar scaffolds can catalyze different reactions. In the superfamily of serine penicillin-recognizing enzymes, D-alanyl-D-alanine peptidases and β-lactamases are phylogenetically linked but feature large differences of reactivity towards their respective substrates. In particular, while β-lactamases hydrolyze penicillins very fast, leading to their inactivation, these molecules inhibit D-alanyl-D-alanine peptidases by forming stable covalent penicilloyl enzymes. In cyanobacteria, we have discovered a new family of penicillin-binding proteins (PBPs) presenting all the sequence features of class A β-lactamases but having a six-amino-acid deletion in the conserved Ω-loop and lacking the essential Glu166 known to be involved in the penicillin hydrolysis mechanism. With the aim of evolving a member of this family into a β-lactamase, PBP-A from Thermosynechococcus elongatus has been chosen because of its thermostability. Based on sequence alignments, introduction of a glutamate in position 158 of the shorter Ω-loop afforded an enzyme with a 50-fold increase in the rate of penicillin hydrolysis. The crystal structures of PBP-A in the free and penicilloylated forms at 1.9 Å resolution and of L158E mutant at 1.5 Å resolution were also solved, giving insights in the catalytic mechanism of the proteins. Since all the active-site elements of PBP-A-L158E, including an essential water molecule, are almost perfectly superimposed with those of a class A β-lactamase such as TEM-1, the question why our mutant is still 5 orders of magnitude less active as a penicillinase remains and our results emphasize how far we are from understanding the secrets of enzymes. Based on the few minor differences between the active sites of PBP-A and TEM-1,mutations were introduced in the L158E enzyme, but while activities on D-Ala-D-Ala mimicking substrates were severely impaired, further improvement in penicillinase activity was unsuccessful. [less ▲]

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See detailMolecular organization of selected prokaryotic S-Iayer proteins
Claus, Harald; Akça, Erol; Debaerdemaeker, Tony et al

in Canadian Journal of Microbiology (2005), 51

Regular crystalline surface layers (S-layers) are widespread among prokaryotes and probably represent the earliest cell wall structures. S-layer genes have been found in approximately 400 different ... [more ▼]

Regular crystalline surface layers (S-layers) are widespread among prokaryotes and probably represent the earliest cell wall structures. S-layer genes have been found in approximately 400 different species of the prokaryotic domains bacteria and archaea. S-layers usually consist of a single (glyco-rprotein species with molecular masses ranging from about 40 to 200 kDa that form lattices of oblique, tetragonal, or hexagonal architecture. The primary sequences of hyperthermophilic archaeal species exhibit some characteristic signatures, Further adaptations to their specific environments occur by various post-translational modifications, such as linkage of glycans, lipids, phosphate, and sulfate groups to the protein or by proteolytic processing. Specific domains direct the anchoring of the S-layer to the underlying cell wall components and transport across the cytoplasma memhrane. In addition to their presumptive original role as protective coats in archaea and bacteria, they have adapted new functions, e.g., as molecular sieves, attachment sites for extracellular enzymes, and virulence factors. [less ▲]

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See detailCrystal structures of oxidized and reduced forms of human mitochondrial thioredoxin 2
Smeets, Aude; Evrard, Christine ULg; Landtmeters, Marie et al

in Protein Science : A Publication of the Protein Society (2005), 14

Mammalian thioredoxin 2 is a mitochondrial isoform of highly evolutionary conserved thioredoxins. Thioredoxins are small ubiquitous protein–disulfide oxidoreductases implicated in a large variety of ... [more ▼]

Mammalian thioredoxin 2 is a mitochondrial isoform of highly evolutionary conserved thioredoxins. Thioredoxins are small ubiquitous protein–disulfide oxidoreductases implicated in a large variety of biological functions. In mammals, thioredoxin 2 is encoded by a nuclear gene and is targeted to mitochondria by a N-terminal mitochondrial presequence. Recently, mitochondrial thioredoxin 2 was shown to interact with components of the mitochondrial respiratory chain and to play a role in the control of mitochondrial membrane potential, regulating mitochondrial apoptosis signaling pathway. Here we report the first crystal structures of a mammalian mitochondrial thioredoxin 2. Crystal forms of reduced and oxidized human thioredoxin 2 are described at 2.0 and 1.8A ˚ resolution. Though the folding is rather similar to that of human cytosolic/nuclear thioredoxin 1, important differences are observed during the transition between the oxidized and the reduced states of human thioredoxin 2, compared with human thioredoxin 1. In spite of the absence of the Cys residue implicated in dimer formation in human thioredoxin 1, dimerization still occurs in the crystal structure of human thioredoxin 2, mainly mediated by hydrophobic contacts, and the dimers are associated to form two-dimensional polymers. Interestingly, the structure of human thioredoxin 2 reveals possible interaction domains with human peroxiredoxin 5, a substrate protein of human thioredoxin 2 in mitochondria. [less ▲]

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See detailCrystal structure of a dimeric oxidized form of human peroxiredoxin 5
Evrard, Christine ULg; Capron, Arnaud; Marchand, Cécile et al

in Journal of Molecular Biology (2004), 337

Peroxiredoxin 5 is the last discovered mammalian member of an ubiquitous family of peroxidases widely distributed among prokaryotes and eukaryotes. Mammalian peroxiredoxin 5 has been recently classified ... [more ▼]

Peroxiredoxin 5 is the last discovered mammalian member of an ubiquitous family of peroxidases widely distributed among prokaryotes and eukaryotes. Mammalian peroxiredoxin 5 has been recently classified as an atypical 2-Cys peroxiredoxin due to the presence of a conserved peroxidatic N-terminal cysteine (Cys47) and an unconserved resolving C-terminal cysteine residue (Cys151) forming an intramolecular disulfide intermediate in the oxidized enzyme. We have recently reported the crystal structure of human peroxiredoxin 5 in its reduced form. Here, a new crystal form of human peroxiredoxin 5 is described at 2.0 Ǻ resolution. The asymmetric unit contains three polypeptide chains. Surprisingly, beside two reduced chains, the third one is oxidized although the enzyme was crystallized under initial reducing conditions in presence of 1 mM 1,4-dithio-DL-threitol. The oxidized polypeptide chain forms an homodimer with a symmetry related one through intermolecular disulfide bonds between Cys47 and Cys151. The formation of these disulfide bonds is accompanied by the partial unwinding of the N-terminal parts of the a2 helix, which in the reduced form, contains the peroxidatic Cys47 and the α6 helix, which is sequentially close to the resolving residue Cys151. In each monomer of the oxidized chain, the C-terminal part including the α6 helix is completely reorganized and is isolated from the rest of the protein on an extended arm. In the oxidized dimer, the arm belonging to the first monomer now appears at the surface of the second subunit and vice versa. [less ▲]

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See detailCrystal structure of the C47S mutant of human peroxiredoxin 5
Evrard, Christine ULg; Smeets, Aude; Knoops, Bernard et al

in Journal of Chemical Crystallography (2004), 34

In the crystal structure of the reduced form of the wild-type human peroxiredoxin 5, the presence of a benzoate ion in direct interaction with the peroxidatic cysteine (Cys 47) appeared as a rather ... [more ▼]

In the crystal structure of the reduced form of the wild-type human peroxiredoxin 5, the presence of a benzoate ion in direct interaction with the peroxidatic cysteine (Cys 47) appeared as a rather intriguing feature since it is known that the benzoate ion can play the role of a specific hydroxyl radical scavenger. The crystal structure of the C47S mutant of the same enzyme has been crystallized in the tetragonal system, space group P41212, with a = 65.65 Å, c = 122.04 Å. It confirms the presence of this benzoate ion in spite of the mutation into a serine of the Cys 47 residue to which the benzoate ion was directly linked in the wild-type structure. The benzoate ion seems to be stabilized by hydrophobic contacts on both sides of the aromatic ring. In this matter, the α5 helix, which is specific to peroxiredoxin 5 among mammalian peroxiredoxins, plays an important role. These hydrophobic contacts also allow to suggest why the benzoate ion disappears when the molecule is oxidized. [less ▲]

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See detailPrimary Structure of Selected Archaeal Mesophilic and Extremely Thermophilic Outer Surface Layer Proteins
Claus, Harald; Akça, Erol; Debaerdemaeker, Tony et al

in Systematic & Applied Microbiology (2002), 25

The archaea are recognized as a separate third domain of life together with the bacteria and eucarya. The archaea include the methanogens, extreme halophiles, thermoplasmas, sulfate reducers and sulfur ... [more ▼]

The archaea are recognized as a separate third domain of life together with the bacteria and eucarya. The archaea include the methanogens, extreme halophiles, thermoplasmas, sulfate reducers and sulfur metabolizing thermophiles, which thrive in different habitats such as anaerobic niches, salt lakes, and marine hydrothermals systems and continental solfataras. Many of these habitats represent extreme environments in respect to temperature, osmotic pressure and pH-values and remind on the conditions of the early earth. The cell envelope structures were one of the first biochemical characteristics of archaea studied in detail. The most common archaeal cell envelope is composed of a single crystalline protein or glycoprotein surface layer (S-layer), which is associated with the outside of the cytoplasmic membrane. The S-layers are directly exposed to the extreme environment and can not be stabilized by cellular components. Therefore, from comparative studies of mesophilic and extremely thermophilic S-layer proteins hints can be obtained about the molecular mechanisms of protein stabilization at high temperatures. First crystallization experiments of surface layer proteins under microgravity conditions were successful. Here, we report on the biochemical features of selected mesophilic and extremely archaeal S-layer (glyco-) proteins. [less ▲]

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See detailCrystal Structure of Human Peroxiredoxin 5, a Novel Type of Mammalian Peroxiredoxin at 1.5 Ǻ Resolution
Declercq, Jean-Paul; Evrard, Christine ULg; Clippe, André et al

in Journal of Molecular Biology (2001), 311

The peroxiredoxins define an emerging family of peroxidases able to reduce hydrogen peroxide and alkyl hydroperoxides with the use of reducing equivalents derived from thiol-containing donor molecules ... [more ▼]

The peroxiredoxins define an emerging family of peroxidases able to reduce hydrogen peroxide and alkyl hydroperoxides with the use of reducing equivalents derived from thiol-containing donor molecules such as thioredoxin, glutathione, trypanothione and AhpF. Peroxiredoxins have been identified in prokaryotes as well as in eukaryotes. Peroxiredoxin 5 (PRDX5) is a novel type of mammalian thioredoxin peroxidase widely expressed in tissues and located cellularly to mitochondria, peroxisomes and cytosol. Functionally, PRDX5 has been implicated in antioxidant protective mechanisms as well as in signal transduction in cells. We report here the 1.5 Ǻ resolution crystal structure of human PRDX5 in its reduced form. The crystal structure reveals that PRDX5 presents a thioredoxin-like domain. Interestingly, the crystal structure shows also that PRDX5 does not form a dimer like other mammalian members of the peroxiredoxin family. In the reduced form of PRDX5, Cys47 and Cys151 are distant of 13.8 Ǻ although these two cysteine residues are thought to be involved in peroxide reductase activity by forming an intramolecular disul®de intermediate in the oxidized enzyme. These data suggest that the enzyme would necessitate a conformational change to form a disulfide bond between catalytic Cys47 and Cys151 upon oxidation according to proposed peroxide reduction mechanisms. Moreover, the presence of a benzoate ion, a hydroxyl radical scavenger, was noted close to the active-site pocket. The possible role of benzoate in the antioxidant activity of PRDX5 is discussed. [less ▲]

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See detailA twinned monoclinic crystal form of human peroxiredoxin 5 with eight molecules in the asymmetric unit
Declercq, Jean-Paul; Evrard, Christine ULg

in Acta Crystallographica Section D-Biological Crystallography (2001), D57

The monoclinic crystal form of human peroxiredoxin 5 with eight molecules in the asymmetric unit was obtained under exactly the same conditions as the tetragonal form with one molecule in the asymmetric ... [more ▼]

The monoclinic crystal form of human peroxiredoxin 5 with eight molecules in the asymmetric unit was obtained under exactly the same conditions as the tetragonal form with one molecule in the asymmetric unit, except that the latter was briefly cryosoaked with halide for derivatization. A merohedral twinning was observed, which is rather unusual in the monoclinic system and only possible with particular unit-cell dimensions. After detwinning the native and a mercury derivative, the structure was solved by the SIR method with the help of the non-crystallographic symmetry. The packing of the monoclinic and tetragonal forms are compared, with special attention to the role of bromide ions in the change of space group after crystallization. The availability of nine (eight monoclinic plus one tetragonal) independent molecules allows an analysis of the mobility. The two Cys residues implicated in the peroxide-reduction mechanism are located in rigid regions but are covered by mobile loops. [less ▲]

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See detailThe first successful crystallization of a prokaryotic extremely thermophilic outer surface layer glycoprotein
Evrard, Christine ULg; Declercq, Jean-Paul; Debaerdemaeker, Tony et al

in Zeitschrift für Kristallographie (1999), 214

Methanothermus fervidus belongs to the group of hyperthermophilic Archaea. The Archaea comprise organisms that live under environmental extremes. like high temperature, low pH value or high salt ... [more ▼]

Methanothermus fervidus belongs to the group of hyperthermophilic Archaea. The Archaea comprise organisms that live under environmental extremes. like high temperature, low pH value or high salt concentration. The outer surface of the pseudomurein sacculi of the cells of Methanothermus fervidus is covered by glycoprotein subunits (S-Iayer) directly exposed to the extreme environment. The elucidation of the crystal structure of this surface glycoprotein may provide important information on the survival strategies of these unusual micro-organisms. Before our investigations neither three-dimensional crystals have been obtained nor X-ray analyses were performed. Only electron microscopic and computer image enhancement techniques have been applied to obtain structural information from crystalline two-dimensional S-layer sheets. We describe here the successful crystallization of this surface glycoprotein under microgravity conditions. [less ▲]

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See detailA crystal of a typical EF-hand protein grown under microgravity diffracts X-rays beyond 0.9 Å resolution
Declercq, Jean-Paul; Evrard, Christine ULg; Carter, Daniel et al

in Journal of Crystal Growth (1999), 196

We report on our recent observation that crystals of a typical EF-hand protein (parvalbumin or Pa; Ca-loaded component from pike muscle with isoelectric point 4.10) grown under microgravity conditions ... [more ▼]

We report on our recent observation that crystals of a typical EF-hand protein (parvalbumin or Pa; Ca-loaded component from pike muscle with isoelectric point 4.10) grown under microgravity conditions diffract X-rays to a resolution better than 0.9 Å. The crystals were grown in the US space shuttle and characterized at 100 K, using an X-ray synchrotron beam. An effective atomic resolution has been achieved and substates in the conformation of the protein are observed. Large crystals up to 3 mm were also obtained. [less ▲]

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See detailThe incorporation of a non-natural amino acid (aza-tryptophan) may help to crystallize a protein and to solve its crystal structure. Application to bacteriophage lambda lysozyme.
Evrard, Christine ULg; Fastrez, Jacques; Declercq, Jean-Paul

in Acta Crystallographica Section D-Biological Crystallography (1999), D55

Until now, wild-type bacteriophage lambda lysozyme had been impossible to crystallize. This difficulty could be overcome by the replacement of the four tryptophan residues by azatryptophans. Analysis of ... [more ▼]

Until now, wild-type bacteriophage lambda lysozyme had been impossible to crystallize. This difficulty could be overcome by the replacement of the four tryptophan residues by azatryptophans. Analysis of the intermolecular and intramolecular contacts in this modification allows understanding of the differences in behaviour between the native and modified molecules. Furthermore, this mutation was very useful for the creation of new heavy-atom binding sites and for the solution of the non-crystallographic symmetry, which is extremely important for phase improvement. This procedure seems to be generally applicable, at least in the search for new possibilities for heavy-atom binding sites. [less ▲]

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See detailCrystal structure of the EF-hand parvalbumin at atomic resolution (0.91 Å) and at low temperature (100 K). Evidence for conformational multistates within the hydrophobic core.
Declercq, Jean-Paul; Evrard, Christine ULg; Lamzin, Victor et al

in Protein Science : A Publication of the Protein Society (1999), 8

Several crystal structures of parvalbumin (Parv), a typical EF-hand protein, have been reported so far for different species with the best resolution achieving 1.5 Å. Using a crystal grown under ... [more ▼]

Several crystal structures of parvalbumin (Parv), a typical EF-hand protein, have been reported so far for different species with the best resolution achieving 1.5 Å. Using a crystal grown under microgravity conditions, cryotechniques (100 K), and synchrotron radiation, it has now been possible to determine the crystal structure of the fully Ca2+ loaded form of pike (component pI 4.10) Parv.Ca2 at atomic resolution (0.91 Å). The availability of such a high quality structure offers the opportunity to contribute to the definition of the validation tools useful for the refinement of protein crystal structures determined to lower resolution. Besides a better definition of most of the elements in the protein threedimensional structure than in previous studies, the high accuracy thus achieved allows the detection of well-defined alternate conformations, which are observed for 16 residues out of 107 in total. Among them, six occupy an internal position within the hydrophobic core and converge toward two small buried cavities with a total volume of about 60 Å3. There is no indication of any water molecule present in these cavities. It is probable that at temperatures of physiological conditions there is a dynamic interconversion between these alternate conformations in an energy-barrier dependent manner. Such motions for which the amplitudes are provided by the present study will be associated with a timedependent remodeling of the void internal space as part of a slow dynamics regime (millisecond timescales) of the parvalbumin molecule. The relevance of such internal dynamics to function is discussed. [less ▲]

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See detailPCAM: a multi-user facility-based protein crystallization apparatus for microgravity
Carter, Daniel; Wright, Brenda; Miller, Teresa et al

in Journal of Crystal Growth (1999), 196

A facility-based protein crystallization apparatus for microgravity (PCAM) has been constructed and flown on a series of Space Shuttle Missions. The hardware development was undertaken largely because of ... [more ▼]

A facility-based protein crystallization apparatus for microgravity (PCAM) has been constructed and flown on a series of Space Shuttle Missions. The hardware development was undertaken largely because of the many important examples of quality improvements gained from crystal growth in the diffusion-limited environment in space. The concept was based on the adaptation for microgravity of a commonly available crystallization tray to increase sample density, to facilitate co-investigator participation and to improve flight logistics and handling. A co-investigator group representing scientists from industry, academia, and government laboratories has been established. Microgravity applications of the hardware have produced improvements in a number of structure-based crystallographic studies and include examples of enabling research. Additionally, the facility has been used to support fundamental research in protein crystal growth which has delineated factors contributing to the effect of microgravity on the growth and quality of protein crystals. [less ▲]

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See detailCrystal Structure of the Lysozyme from Bacteriophage Lambda and its Relationship with V and C-type Lysozymes
Evrard, Christine ULg; Fastrez, Jacques; Declercq, Jean-Paul

in Journal of Molecular Biology (1998), 276

Like other lysozymes, the bacteriophage lambda lysozyme is involved in the digestion of bacterial walls. This enzyme is remarkable in that its mechanism of action is different from the classical lysozyme ... [more ▼]

Like other lysozymes, the bacteriophage lambda lysozyme is involved in the digestion of bacterial walls. This enzyme is remarkable in that its mechanism of action is different from the classical lysozyme's mechanism. From the point of view of protein evolution, it shows features of lysozymes from different classes. The crystal structure of the enzyme in which all tryptophan residues have been replaced by aza-tryptophan has been solved by X-ray crystallography at 2.3 Å using a combination of multiple isomorphous replacement, non-crystallographic symmetry averaging and density modification techniques. There are three molecules in the asymmetric unit. The characteristic structural elements of lysozymes are conserved: each molecule is organized in two domains connected by a helix and the essential catalytic residue (Glu19) is located in the depth of a cleft between the two domains. This cleft shows an open conformation in two of the independent molecules, while access to the cavity is much more restricted in the last one. A structural alignment with T4 lysozyme and hen egg white lysozyme allows us to superpose about 60 Cα atoms with a rms distance close to 2 Å. The best alignments concern the helix preceding the catalytic residue, some parts of the beta sheets and the helix joining the two domains. The results of sequence alignments with the V and C lysozymes, in which weak local similarities had been detected, are compared with the structural results. [less ▲]

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See detailCrystallization and preliminary X-ray analysis of bacteriophage lambda lysozyme in which all tryptophans have been replaced by aza-tryptophans
Evrard, Christine ULg; Declercq, Jean-Paul; Fastrez, Jacques

in Acta Crystallographica Section D-Biological Crystallography (1997), D53

After many unsuccessful attempts to crystallize the bacteriophage lambda lysozyme, a mutant where all the tryptophan residues have been replaced by aza-tryptophans has been crystallized by the vapor ... [more ▼]

After many unsuccessful attempts to crystallize the bacteriophage lambda lysozyme, a mutant where all the tryptophan residues have been replaced by aza-tryptophans has been crystallized by the vapor-diffusion method. The crystals are orthorhombic and belong to space group P212121 with cell dimensions a = 73.01, b = 78.80, c = 82.31 Å. Diffraction data were collected using synchrotron radiation sources. Crystals diffract to a resolution of 2.3 Å. Data from two different platinum derivatives were also recorded to 2.8 and 2.5 Å, respectively. [less ▲]

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