Thioredoxin reductases (TrxRs) from mammalian cells contain an essential selenocysteine residue in the conserved C-terminal sequence Gly-Cys-SeCys-Gly forming a selenenylsulfide in the oxidized enzyme. Reduction by NADPH generates a selenolthiol, which is the active site in reduction of Trx. The three-dimensional structure of the SeCys498Cys mutant of rat TrxR in complex with NADP ؉ has been determined to 3.0-Å resolution by x-ray crystallography. The overall structure is similar to that of glutathione reductase (GR), including conserved amino acid residues binding the cofactors FAD and NADPH. Surprisingly, all residues directly interacting with the substrate glutathione disulfide in GR are conserved despite the failure of glutathione disulfide to act as a substrate for TrxR. The 16-residue C-terminal tail, which is unique to mammalian TrxR, folds in such a way that it can approach the active site disulfide of the other subunit in the dimer. A model of the complex of TrxR with Trx suggests that electron transfer from NADPH to the disulfide of the substrate is possible without large conformational changes. The C-terminal extension typical of mammalian TrxRs has two functions: (i) it extends the electron transport chain from the catalytic disulfide to the enzyme surface, where it can react with Trx, and (ii) it prevents the enzyme from acting as a GR by blocking the redox-active disulfide. Our results suggest that mammalian TrxR evolved from the GR scaffold rather than from its prokaryotic counterpart. This evolutionary switch renders cell growth dependent on selenium.

The Ras-GAP SH3 domain–binding proteins (G3BP) are essential regulators of the formation of stress granules (SG), cytosolic aggregates of proteins and RNA that are induced upon cellular stress, such as virus infection. Many viruses, including Semliki Forest virus (SFV), block SG induction by targeting G3BP. In this work, we demonstrate that the G3BP-binding motif of SFV nsP3 consists of two FGDF motifs, in which both phenylalanine and the glycine residue are essential for binding. In addition, we show that binding of the cellular G3BP-binding partner USP10 is also mediated by an FGDF motif. Overexpression of wt USP10, but not a mutant lacking the FGDF-motif, blocks SG assembly. Further, we identified FGDF-mediated G3BP binding site in herpes simplex virus (HSV) protein ICP8, and show that ICP8 binding to G3BP also inhibits SG formation, which is a novel function of HSV ICP8. We present a model of the three-dimensional structure of G3BP bound to an FGDF-containing peptide, likely representing a binding mode shared by many proteins to target G3BP.

Selenoproteins contain a highly reactive 21st amino acid selenocysteine (Sec) encoded by recoding of a predefined UGA codon. Because of a lack of selenoprotein supply, high chemical reactivity of Sec, and intricate translation machineries, selenoprotein crystal structures are difficult to obtain. Structural prerequisites for Sec involvement in enzyme catalysis are therefore sparsely known. Here we present the crystal structure of catalytically active rat thioredoxin reductase 1 (TrxR1), revealing surprises at the C-terminal Sec-containing active site in view of previous literature. The oxidized enzyme presents a selenenylsulfide motif in trans-configuration, with the selenium atom of Sec-498 positioned beneath the side chain of Tyr-116, thereby located far from the redox active moieties proposed to be involved in electron transport to the Sec-containing active site. Upon reduction to a selenolthiol motif, the Sec residue moved toward solvent exposure, consistent with its presumed role in reduction of TrxR1 substrates or as target of electrophilic agents inhibiting the enzyme. A Y116I mutation lowered catalytic efficiency in reduction of thioredoxin, but surprisingly increased turnover using 5-hydroxy-1,4-naphthoquinone (juglone) as substrate. The same mutation also decreased sensitivity to inhibition by cisplatin. The results suggest that Tyr-116 plays an important role for catalysis of TrxR1 by interacting with the selenenylsulfide of oxidized TrxR1, thereby facilitating its reduction in the reductive half-reaction of the enzyme. The interaction of a selenenylsulfide with the phenyl ring of a tyrosine, affecting turnover, switch of substrate specificity, and modulation of sensitivity to electrophilic agents, gives important clues into the mechanism of TrxR1, which is a selenoprotein that plays a major role for mammalian cell fate and function. The results also demonstrate that a recombinant selenoprotein TrxR can be produced in high amount and sufficient purity to enable crystal structure determination, which suggests that additional structural studies of these types of proteins are feasible.

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The nirA gene of Mycobacterium tuberculosis is upregulated in the persistent state of the bacteria, suggesting that it is a potential target for the development of antituberculosis agents particularly active against the pathogen in its dormant phase. This gene encodes a ferredoxin-dependent sulfite reductase, and the structure of the enzyme has been determined using

MARCO is a trimeric class A scavenger receptor of macrophages and dendritic cells that recognizes polyanionic particles and pathogens. The distal, scavenger receptor cysteine-rich (SRCR) domain of the extracellular part of this receptor has been implicated in ligand binding. To provide a structural basis for understanding the ligand-binding mechanisms of MARCO, we have determined the crystal structure of the mouse MARCO SRCR domain. The recombinant SRCR domain purified as monomeric and dimeric forms, and their structures were determined at 1.78 and 1.77 Å resolution, respectively. The monomer has a compact globular fold with a twisted five-stranded antiparallel ␤-sheet and a long loop covering a single ␣-helix, whereas the dimer is formed via ␤-strand swapping of two monomers, thus containing a large eight-stranded ␤-sheet. Calculation of the surface electrostatic potential revealed that the ␤-sheet region with several arginines forms a basic cluster. Unexpectedly, an acidic cluster was found in the long loop region. In the monomer, the acidic cluster is involved in metal ion binding. Studies with cells expressing various SRCR domain mutants showed that all of the arginines of the basic cluster are involved in ligand binding, suggesting a cooperative binding mechanism. Ligand binding is also dependent on the acidic cluster and Ca 2؉ ions whose depletion appears to affect ligand binding at least by modulating the electrostatic potential or relative domain orientation. We propose that the SRCR domain dimerization can contribute to the recognition of large ligands by providing a means for the MARCO receptor oligomerization.MARCO belongs to a diverse group of scavenger receptors (SRs) 3 expressed by macrophages, dendritic cells, and certain endothelial cells (1). These germ line-encoded receptors, also known as pattern recognition receptors due to their ability to recognize conserved pathogen-associated molecular patterns, are considered as an important part of innate immunity, the evolutionarily conserved, first line host defense mechanism. In addition to pathogen-associated molecular patterns, a long list of SR ligands, often polyanionic in nature, includes pollution particles, polyribonucleotides, bacterial lipopolysaccharides, modified host molecules such as oxidized low density lipoprotein (LDL), and unmodified endogenous proteins (1, 2). The SRs are classified into several subgroups, of which class A SRs have primarily been associated with innate immunity. This class consists of five members: SR-A (SR-AI, -II, and -III/ SCARA1) (3, 4), MARCO (macrophage receptor with collagenous domain)/SCARA2 (5), CSR1 (cellular stress response 1) and CSR2/SCARA3 (6), SRCL (scavenger receptor with C-type lectin) I and II/SCARA4 (7, 8), and Tesr (testis-expressed scavenger receptor)/SCARA5 (class A scavenger receptor 5) (9, 10). All of these are trimeric type II membrane proteins with a similar predicted tertiary structure consisting of a short intracellular domain, a transmembrane domain, and a large extracellular domain with...

Based on a structure-assisted sequence alignment we designed 11 focused libraries at residues in the active site of transaldolase B from Escherichia coli and screened them for their ability to synthesize fructose 6-phosphate from dihydroxyacetone and glyceraldehyde 3-phosphate using a newly developed color assay. We found one positive variant exhibiting a replacement of Phe 178 to Tyr. This mutant variant is able not only to transfer a dihydroxyacetone moiety from a ketose donor, fructose 6-phosphate, onto an aldehyde acceptor, erythrose 4-phosphate (14 units/mg), but to use it as a substrate directly in an aldolase reaction (7 units/mg). With a single amino acid replacement the fructose-6-phosphate aldolase activity was increased considerably (>70-fold compared with wild-type). Structural studies of the wild-type and mutant protein suggest that this is due to a different H-bond pattern in the active site leading to a destabilization of the Schiff base intermediate. Furthermore, we show that a homologous replacement has a similar effect in the human transaldolase Taldo1 (aldolase activity, 14 units/mg). We also demonstrate that both enzymes TalB and Taldo1 are recognized by the same polyclonal antibody. Transaldolase (Tal)2 is a ubiquitous enzyme that is present in all domains of life. It is part of the non-oxidative path of the pentose phosphate pathway. Here, it catalyzes the reversible transfer of a dihydroxyacetone (DHA) moiety from a ketose donor, e.g. fructose 6-phosphate (Fru-6-P), onto an aldehyde acceptor, e.g. erythrose 4-phosphate (Ery-4-P). The best studied example is transaldolase B (TalB) from Escherichia coli (1). A number of structural and mechanistic studies have been published elucidating its reaction mechanism (1-6). Similar to class I aldolases the reaction proceeds via a Schiff base intermediate. TalB is a homo-dimer and the monomer exhibits a (␤/␣) 8 -barrel fold where the C-terminal helix lies across the barrel opening at one site (5). A similar structure had been determined for the human transaldolase (7).Recently, a fructose-6-phosphate aldolase (FSA) of E. coli has been discovered by our group (8) that uses DHA as donor substrate and catalyzes the reversible formation of Fru-6-P from DHA and GAP (Fig. 1). Multiple sequence alignments of different Tal sequences demonstrate that FSA resides within the family of transaldolases (8, 9) and does show little similarity to DHAP-dependent aldolases, such as class I fructose-1,6-bisphosphate aldolase. Also the FSA monomer is highly similar to TalB exhibiting a similar (␤/␣) 8 -barrel fold (9). But in contrast to TalB, FSA forms a homo-decamer, which is arranged by two doughnut-shaped pentameric rings where the C-terminal helix of one subunit interacts with the adjacent subunit (9). With 220 amino acids compared with 317 amino acids FSA is smaller than TalB and shows about 20% sequence identity to TalB. Mechanistically, it is a class I aldolase, i.e. the reaction proceeds via a Schiff base intermediate.In this study, we addressed the differences b...