Aude Smeets

The crystal structures of oxidized forms of human peroxiredoxin 5 with an intramolecular disulfide bond confirm the proposed enzymatic mechanism for atypical 2-Cys peroxiredoxins.

Peroxiredoxin 5 (PRDX5) belongs to the PRDX superfamily of thiol-dependent peroxidases able to reduce hydrogen peroxide, alkyl hydroperoxides and peroxynitrite. PRDX5 is classified in the atypical 2-Cys subfamily of PRDXs. In this subfamily, the oxidized form of the enzyme is characterized by the presence of an intramolecular disulfide bridge between the peroxidatic and the resolving cysteine residues. We report here three crystal forms in which this intramolecular disulfide bond is indeed observed. The structures are characterized by the expected local unfolding of the peroxidatic loop, but also by the unfolding of the resolving loop. A new type of interface between PRDX molecules is described. The three crystal forms were not oxidized in the same way and the influence of the oxidizing conditions is discussed.

Crystal Structures of Oxidized and Reduced Forms of Human Mitochondrial Thioredoxin 2.

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 1.8 Å 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.

The Crystal Structure of the C45S Mutant of Annelid Arenicola Marina Peroxiredoxin 6 Supports its Assignment to the Mechanistically Typical 2- Cys Subfamily without Any Formation of Toroid- Shaped Decamers.

The peroxiredoxins (PRDXs) define a superfamily of thiol‐dependent peroxidases able to reduce hydrogen peroxide, alkyl hydroperoxides, and peroxynitrite. Besides their cytoprotective antioxidant function, PRDXs have been implicated in redox signaling and chaperone activity, the latter depending on the formation of decameric high‐molecular‐weight structures. PRDXs have been mechanistically divided into three major subfamilies, namely typical 2‐Cys, atypical 2‐Cys, and 1‐Cys PRDXs, based on the number and position of cysteines involved in the catalysis. We report the structure of the C45S mutant of annelid worm Arenicola marina PRDX6 in three different crystal forms determined at 1.6, 2.0, and 2.4 Å resolution. Although A. marina PRDX6 was cloned during the search of annelid homologs of mammalian 1‐Cys PRDX6s, the crystal structures support its assignment to the mechanistically typical 2‐Cys PRDX subfamily. The protein is composed of two distinct domains: a C‐terminal domain and an N‐terminal domain exhibiting a thioredoxin fold. The subunits are associated in dimers compatible with the formation of intersubunit disulfide bonds between the peroxidatic and the resolving cysteine residues in the wild‐type enzyme. The packing of two crystal forms is very similar, with pairs of dimers associated as tetramers. The toroid‐shaped decamers formed by dimer association and observed in most typical 2‐Cys PRDXs is not present. Thus, A. marina PRDX6 presents structural features of typical 2‐Cys PRDXs without any formation of toroid‐shaped decamers, suggesting that it should function more like a cytoprotective antioxidant enzyme or a modulator of peroxide‐dependent cell signaling rather than a molecular chaperone.

Possible interactions between human mitochondrial thioredoxin 2 and human peroxiredoxin 5.

Intermediate filaments (IFs) are principal components of the cytoskeleton in higher eukaryotic cells. The way the elementary IF dimers consisting of a head, coiled-coiled rod and tail domains assemble into a filament is currently poorly understood. However, mutations in these proteins are known to be responsible for a number of currently uncurable human diseases such as myopathies, cardiopathies, neuronal and skin diseases. We are currently working on the 3D structure of several different types of IFs, as well as on the mechanism of the disease-related point mutations that distort the IF structure. With desmin present in muscle cells, we are focussing on mutations in the 2B coil of its rod domain that are linked to myopathies. Most of these are mutations to proline expected to distort the α-helical structure. Multiple 2B coil fragments of about 60 amino acids were purified and screened towards obtaining crystals suitable for X-ray analysis. We are also trying to obtain the crystal structure of the desmin fragment carrying the E401S mutation which disrupts a predicted interhelical salt bridge. The assembly of lamins representing the nuclear IF proteins starts with the longitudinal association of dimers with a short N-C overlap of their rod domains (rather than with a lateral association typical for cytoplasmic IFs). To investigate the assembly of lamins A, B1 and B2 at the molecular level, we have prepared 13 recombinant Nand C-terminal fragments of these proteins. These fragments assumed to represent the minimal regions of the longitudinal association were analysed using analytical ultracentrifugation and circular dichroism. We show that the most of the studied N-terminal fragments from lamins A and B1 form dimers. Furthermore, we demonstrate that the Nand C-terminal fragments mixed at 1:1 ratio are able to form heterotetramers, mimicking the N-C overlap. Remarkably, N-terminal constructs from lamin A may associate with C-terminal constructs from lamin B1 and vice versa. This is of considerable importance for the understanding of lamin assembly within the nuclear lamina. Moreover, the N-terminal fragments lacking the short head domain can still form stable tetramers with the C-terminal ones, suggesting that the head is not involved in the complex formation. Crystallisation trials with lamin fragments and their heterotetrameric complexes are in progress. m06.p11