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 disulfide 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.

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features of lysozymes from different classes.The crystal structure of the enzyme in which all tryptophan residues have been replaced by aza-tryptophan has been solved by X-ray crystallography at 2.3 Å using a combination of multiple isomorphous replacement, noncrystallographic symmetry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 C α atoms with a rms distance close to 2 Å. The best alignments concern the helix preceding the catalytic residue, some parts of the β 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.

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 the presence of 1 mM 1,4-dithio-DL-threitol. The oxidized polypeptide chain forms an homo-dimer 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 α2 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.Keywords: antioxidant enzyme; peroxiredoxin; thioredoxin fold; thioredoxin peroxidase; crystal structure Abbreviations used: PRDX, human peroxiredoxin; Prdx, mouse and rat peroxiredoxin; 1hd2, RCSB Protein Data Bank code of the tetragonal form of human PRDX5; ROS, reactive oxygen species; RNS, reactive nitrogen species; MALDI, matrix-assisted laser desorption/ ionization.

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-)protein 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 membrane. 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.

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 penicillinrecognizing enzymes, D-alanyl-D-alanine peptidases and β-lacta-mases 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 penicillinbinding 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.

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 Ca 2ϩ -loaded form of pike~component pI 4.10! Parv.Ca 2 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 Å 3 . 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.

Atopic dermatitis (AD) is a complex inflammatory skin condition that is not fully understood. Epidermal barrier defects and Th2 immune response dysregulations are thought to play crucial roles in the pathogenesis of the disease. A vicious circle takes place between these alterations, and it can further be complicated by additional genetic and environmental factors. Studies investigating in more depth the etiology of the disease are thus needed in order to develop functional treatments. In recent years, there have been significant advances regarding in vitro models reproducing important features of AD. However, since a lot of models have been developed, finding the appropriate experimental setting can be difficult. Therefore, herein, we review the different types of in vitro models mimicking features of AD. The simplest models are two-dimensional culture systems composed of immune cells or keratinocytes, whereas three-dimensional skin or epidermal equivalents reconstitute more complex stratified tissues exhibiting barrier properties. In those models, hallmarks of AD are obtained, either by challenging tissues with interleukin cocktails overexpressed in AD epidermis or by silencing expression of pivotal genes encoding epidermal barrier proteins. Tissue equivalents cocultured with lymphocytes or containing AD patient cells are also described. Furthermore, each model is placed in its study context with a brief summary of the main results obtained. In conclusion, the described in vitro models are useful tools to better understand AD pathogenesis, but also to screen new compounds in the field of AD, which probably will open the way to new preventive or therapeutic strategies.

A quantitative study using laser confocal microscopy combined with differential interference microscopy on the kinetics and thermodynamics of the crystallization of glucose isomerase is presented. Fundamental crystallization parameters are determined from the kinetics of step advancement and rates of two-dimensional (2D) nucleation. The ruling mass transfer pathway and accompanying activation barriers are discussed. In brief, the solubility exhibits normal temperature dependence and the crystallization enthalpy is the thermodynamic driving force. The diminishing entropic cost for higher PEG concentrations is attributed to water structuring and a decrease in water activity. The prominent step generation mechanism is homogeneous 2D nucleation for high supersaturations. At low driving forces 2D nucleation occurs on anomalously hyperactive sites and the step edge free energies for homogeneous and heterogeneous nucleation are determined. The number of nucleation centers for both mechanisms are estimated and from the density of nucleation centers we obtain for the activation barrier of adsorption ∼3.8 kJ mol−1. No step-step interaction is observed for interstep distances >70 nm. Theoretical fits of step velocity data suggest surface diffusion makes a non-negligible contribution to surface kinetics. From the temperature dependence of the step kinetic coefficient the activation barrier for crystallization was determined to be <22.4 kJ mol−1.