The Mechanism of High MR Thioredoxin Reductase Investigated by Semisynthesis and Crystallography

The high Mr (~55 kDa) thioredoxin reductases (TR) characteristic of higher eukaryotes are members of the glutathione reductase (GR) family of pyridine nucleotide disulfide oxidoreductases. These homodimeric enzymes catalyze the reduction of a cognate disulfide substrate. During the enzymatic cycle, reducing equivalents pass from NADPH to the conserved active site disulfide via an enzyme-bound FAD and then to the cognate substrate. TRs are unique in the family as electrons are then transferred to the C-terminal active site of the adjacent molecule as part of a 16 amino acid extension (in place of the cognate GR substrate GSSG), prior to transfer to the substrate thioredoxin. Each electron transfer step occurs via thiol-disulfide exchange in a multi-step process mediated by a conserved catalytic acid/base. Mammalian TRs require selenocysteine (Sec) incorporated into the Gly-Cys-Sec-Gly-OH (GCUG) C-terminal tetrapeptide motif, while the TR from Drosophila melanogaster (DmTR) does not, and instead contains a Ser-Cys-Cys-Ser-OH (SCCS) tetrapeptide motif indicating that Sec is not universally necessary to catalyze the reduction of thioredoxin. This project has achieved three major objectives; 1) development of a semisynthetic method for production of mouse mitochondrial TR (mTR3) for structure-function studies, 2) establishment of a new method to study the mechanism of TR by using tetrapeptides in the oxidized form equivalent to the C-terminal active sites as substrates for the truncated forms of both enzymes, 3) determination of the crystal structure of DmTR. The results show that the structure of DmTR explains the biochemical data and has developed a new testable hypothesis in the field for the requirement of Sec in mammalian TR. We demonstrate that the tetrapeptides tested in Aim 2 were all better substrates for DmTR. The data also shows a far greater dependence on Sec for mTR3 than DmTR, which is in agreement with that observed for the collection full-length mutants produced for each enzyme in Aim 1. As this method of investigation is more analogous to the other enzymes of the GR family, the structures of the tetrapeptides determined by NMR spectroscopy were oriented in the active site of the both enzymes using the diglutathione bound in the structure of GR as template. DmTR appears to have a more open active site than observed in the known structure of mTR3. Residues from the helical face of the FAD-domain proximal to the FAD-associated active site are less bulky in DmTR to accommodate the hydroxyls of the serines. This is likely to make the enzyme more amenable for the conformational switching of the SCCS peptide necessary to protonate the leaving group cysteine by the proposed catalytic acid/base. In contrast, mTR3 shows a more restricted interface by incorporating bulkier residues at the interface in conjunction with the smaller Gly residues of the C-terminal sequence GCUG. The tetrapeptides display a conformational preference not suitable for protonation of the first leaving group in mTR3

Structural insights into recognition of acetylated histone ligands by the BRPF1 bromodomain

AbstractBromodomain-PHD finger protein 1 (BRPF1) is part of the MOZ HAT complex and contains a unique combination of domains typically found in chromatin-associated factors, which include plant homeodomain (PHD) fingers, a bromodomain and a proline-tryptophan-tryptophan-proline (PWWP) domain. Bromodomains are conserved structural motifs generally known to recognize acetylated histones, and the BRPF1 bromodomain preferentially selects for H2AK5ac, H4K12ac and H3K14ac. We solved the X-ray crystal structures of the BRPF1 bromodomain in complex with the H2AK5ac and H4K12ac histone peptides. Site-directed mutagenesis on residues in the BRPF1 bromodomain-binding pocket was carried out to investigate the contribution of specific amino acids on ligand binding. Our results provide critical insights into the molecular mechanism of ligand binding by the BRPF1 bromodomain, and reveal that ordered water molecules are an essential component driving ligand recognition

Structural and biochemical insights into NEIL2’s preference for abasic sites

Cellular DNA is subject to damage from a multitude of sources and repair or bypass of sites of damage utilize an array of context or cell cycle dependent systems. The recognition and removal of oxidatively damaged bases is the task of DNA glycosylases from the base excision repair pathway utilizing two structural families that excise base lesions in a wide range of DNA contexts including duplex, single-stranded and bubble structures arising during transcription. The mammalian NEIL2 glycosylase of the Fpg/Nei family excises lesions from each of these DNA contexts favoring the latter two with a preference for oxidized cytosine products and abasic sites. We have determined the first liganded crystal structure of mammalian NEIL2 in complex with an abasic site analog containing DNA duplex at 2.08 Å resolution. Comparison to the unliganded structure revealed a large interdomain conformational shift upon binding the DNA substrate accompanied by local conformational changes in the C-terminal domain zinc finger and N-terminal domain void-filling loop necessary to position the enzyme on the DNA. The detailed biochemical analysis of NEIL2 with an array of oxidized base lesions indicates a significant preference for its lyase activity likely to be paramount when interpreting the biological consequences of variants

Structural and Functional Analysis of the CspB Protease Required for <em>Clostridium</em> Spore Germination

<div><p>Spores are the major transmissive form of the nosocomial pathogen <em>Clostridium difficile</em>, a leading cause of healthcare-associated diarrhea worldwide. Successful transmission of <em>C. difficile</em> requires that its hardy, resistant spores germinate into vegetative cells in the gastrointestinal tract. A critical step during this process is the degradation of the spore cortex, a thick layer of peptidoglycan surrounding the spore core. In <em>Clostridium</em> sp., cortex degradation depends on the proteolytic activation of the cortex hydrolase, SleC. Previous studies have implicated Csps as being necessary for SleC cleavage during germination; however, their mechanism of action has remained poorly characterized. In this study, we demonstrate that CspB is a subtilisin-like serine protease whose activity is essential for efficient SleC cleavage and <em>C. difficile</em> spore germination. By solving the first crystal structure of a Csp family member, CspB, to 1.6 Å, we identify key structural domains within CspB. In contrast with all previously solved structures of prokaryotic subtilases, the CspB prodomain remains tightly bound to the wildtype subtilase domain and sterically occludes a catalytically competent active site. The structure, combined with biochemical and genetic analyses, reveals that Csp proteases contain a unique jellyroll domain insertion critical for stabilizing the protease <em>in vitro</em> and in <em>C. difficile</em>. Collectively, our study provides the first molecular insight into CspB activity and function. These studies may inform the development of inhibitors that can prevent clostridial spore germination and thus disease transmission.</p> </div

Structural Studies of a Bacterial tRNA^HIS Guanylyltransferase (Thg1)-Like Protein, with Nucleotide in the Activation and Nucleotidyl Transfer Sites

<div><p>All nucleotide polymerases and transferases catalyze nucleotide addition in a 5′ to 3′ direction. In contrast, tRNA^His guanylyltransferase (Thg1) enzymes catalyze the unusual reverse addition (3′ to 5′) of nucleotides to polynucleotide substrates. In eukaryotes, Thg1 enzymes use the 3′–5′ addition activity to add G₋₁ to the 5′-end of tRNA^His, a modification required for efficient aminoacylation of the tRNA by the histidyl-tRNA synthetase. Thg1-like proteins (TLPs) are found in Archaea, Bacteria, and mitochondria and are biochemically distinct from their eukaryotic Thg1 counterparts TLPs catalyze 5′-end repair of truncated tRNAs and act on a broad range of tRNA substrates instead of exhibiting strict specificity for tRNA^His. Taken together, these data suggest that TLPs function in distinct biological pathways from the tRNA^His maturation pathway, perhaps in tRNA quality control. Here we present the first crystal structure of a TLP, from the gram-positive soil bacterium <i>Bacillus thuringiensis</i> (BtTLP). The enzyme is a tetramer like human THG1, with which it shares substantial structural similarity. Catalysis of the 3′–5′ reaction with 5′-monophosphorylated tRNA necessitates first an activation step, generating a 5′-adenylylated intermediate prior to a second nucleotidyl transfer step, in which a nucleotide is transferred to the tRNA 5′-end. Consistent with earlier characterization of human THG1, we observed distinct binding sites for the nucleotides involved in these two steps of activation and nucleotidyl transfer. A BtTLP complex with GTP reveals new interactions with the GTP nucleotide in the activation site that were not evident from the previously solved structure. Moreover, the BtTLP-ATP structure allows direct observation of ATP in the activation site for the first time. The BtTLP structural data, combined with kinetic analysis of selected variants, provide new insight into the role of key residues in the activation step.</p></div

CspB undergoes autoprocessing in a position-dependent manner.

<p>(<b>a</b>) Coomassie staining of recombinant <i>C. perfringens</i> and <i>C. difficile</i> CspB variants. 7.5 µg of each purified CspB variant was resolved by SDS-PAGE on a 4–12% Bis-Tris gel and visualized by Coomassie staining. The P3-P1 residues of the prodomain were mutated to Ala for the YTS/AAA and QTQ/AAA mutants, while the P3-P1 residues were deleted from CspB <i>perfringens</i> in the ΔYTS mutant. The products resulting from autoprocessing are indicated. (<b>b</b>) Sequence alignment of Csp prodomain cleavage sites mapped by Edman sequencing; the Csp <i>perfringens</i> cleavage sites were mapped in a previous study <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003165#ppat.1003165-Shimamoto1" target="_blank">[25]</a>. Completely conserved identical residues are blocked in black with white text, conserved identical residues in grey with white text, and conserved similar residues in light grey.</p

The jellyroll domain and catalytic serine of CspBA are required for efficient germination.

<p>(<b>a</b>) Schematic of CspBA variants produced by <i>cspBAC</i> complementation constructs. “Pro” denotes the prodomain; black rectangle demarcates the jellyroll domain; a thin white rectangle represents the jellyroll deletion; and white star indicates S461A mutation. (<b>b</b>) Western blot analyses of sporulating cells expressing <i>cspBAC</i> complementation constructs and (<b>c</b>) germinating spores expressing <i>cspBAC</i> complementation constructs. Purified spores of the indicated strain were either untreated (−) or exposed to 0.2% w/v sodium taurocholate (+, germinant) for 15 min at 37°C and analyzed by Western blotting with the indicted antibodies. Germination efficiency was determined via colony forming unit (cfu) determination. Representative clones of each construct are shown, but more than two clones of each complementation construct were tested. m-CspBA reflects the mature form of CspBA following autoprocessing, and m-CspB reflects the mature form of CspB following autoprocessing. The different mutant CspB variants are indicated.</p

CspBA activity downstream of autoprocessing is required for efficient SleC cleavage.

<p>(<b>a</b>) Schematic of CspBA variants produced by <i>cspBAC</i> transcomplementation constructs. “Pro” denotes the prodomain; black rectangle demarcates the jellyroll domain; a thin white rectangle represents the jellyroll deletion; and white star indicates S461A mutation. (<b>b</b>) Western blot analyses of sporulating cells expressing <i>cspBAC</i> transcomplementation constructs and (<b>c</b>) germinating spores expressing transcomplementation constructs. Purified spores of the indicated strain were either untreated (−) or exposed to 0.2% w/v sodium taurocholate (+, germinant) for 15 min at 37°C and analyzed by Western blotting with the indicated antibody. Germination efficiency was determined via colony forming unit (cfu) determination. Representative clones of each construct are shown, but more than two clones of each complementation construct were tested. m-CspBA reflects the mature form of CspBA following autoprocessing, and m-CspB reflects the mature form of CspB following autoprocessing.</p