Crystal structure of the yeast cytoplasmic thioredoxin Trx2.
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Amino Acid Sequence↗
Biomedical subjects
Cong-Zhao Zhou
Publications and source records attributed to Cong-Zhao Zhou.
Expression, purification, crystallization and preliminary X-ray diffraction analysis of mitochondrial thioredoxin Trx3 from Saccharomyces cerevisiae.
There are three thioredoxin isoforms in the yeast Saccharomyces cerevisiae: two cytosolic/nuclear thioredoxins, Trx1 and Trx2, and one mitochondrial thioredoxin, Trx3. In the present work, S. cerevisiae Trx3 overexpressed in Escherichia coli was purified and crystallized. The Trx3 crystals were obtained by the hanging-drop vapour-diffusion method. A data set diffracting to 2.0 A resolution was collected from a single crystal. The crystal belongs to space group P3(1), with unit-cell parameters a = b = 49.57, c = 94.55 A, alpha = beta = 90, gamma = 120 degrees. The asymmetric unit is assumed to contain two subunits of Trx3, with a V(M) value of 2.62 A(3) Da(-1) and a solvent content of 53%.
Cloning, Molecular↗
Purification, crystallization and preliminary X-ray analysis of glutathione peroxidase Gpx3 from Saccharomyces cerevisiae.
The glutathione peroxidase Gpx3 from the yeast Saccharomyces cerevisiae has been overexpressed, purified and crystallized. Both gel-filtration and dynamic light-scattering (DLS) results indicate that Gpx3 is a monomer in solution at a concentration of about 2 mg ml(-1), whereas glutathione peroxidases are normally tetrameric or dimeric. X-ray diffraction data from a single crystal of Gpx3 have been collected to 2.6 A resolution. The crystals are triclinic and belong to space group P1, with unit-cell parameters a = 38.187, b = 43.372, c = 56.870 A, alpha = 71.405, beta = 73.376, gamma = 89.633 degrees. There are two Gpx3 monomers in a crystallographic asymmetric unit. Preliminary analyses show that the yeast Gpx3 is quite different from those of mammals.
Crystallization↗
Catalytic mechanism and structure of viral flavin-dependent thymidylate synthase ThyX.
By using biochemical and structural analyses, we have investigated the catalytic mechanism of the recently discovered flavin-dependent thymidylate synthase ThyX from Paramecium bursaria chlorella virus-1 (PBCV-1). Site-directed mutagenesis experiments have identified several residues implicated in either NADPH oxidation or deprotonation activity of PBCV-1 ThyX. Chemical modification by diethyl pyrocarbonate and mass spectroscopic analyses identified a histidine residue (His53) crucial for NADPH oxidation and located in the vicinity of the redox active N-5 atom of the FAD ring system. Moreover, we observed that the conformation of active site key residues of PBCV-1 ThyX differs from earlier reported ThyX structures, suggesting structural changes during catalysis. Steady-state kinetic analyses support a reaction mechanism where ThyX catalysis proceeds via formation of distinct ternary complexes without formation of a methyl enzyme intermediate.
Amino Acid Sequence↗
Crystal structure and confirmation of the alanine:glyoxylate aminotransferase activity of the YFL030w yeast protein.
We have determined the three-dimensional crystal structure of the protein encoded by the open reading frame YFL030w from Saccharomyces cerevisiae to a resolution of 2.6 A using single wavelength anomalous diffraction. YFL030w is a 385 amino-acid protein with sequence similarity to the aminotransferase family. The structure of the protein reveals a homodimer adopting the fold-type I of pyridoxal 5'-phosphate (PLP)-dependent aminotransferases. The PLP co-factor is covalently bound to the active site in the crystal structure. The protein shows close structural resemblance with the human alanine:glyoxylate aminotransferase (EC 2.6.1.44), an enzyme involved in the hereditary kidney stone disease primary hyperoxaluria type 1. In this paper we show that YFL030w codes for an alanine:glyoxylate aminotransferase, highly specific for its amino donor and acceptor substrates.
Carbohydrate Conformation↗
Solution structure of Kti11p from Saccharomyces cerevisiae reveals a novel zinc-binding module.
Kti11p is a small, highly conserved CSL zinc finger-containing protein found in many eukaryotes. It was first identified as one of the factors required for maintaining the sensitivity of Saccharomyces cerevisiae to Kluyveromyces lactis zymocin. Then, it was found to be identical to Dph3, a protein required for diphthamide biosynthesis on eEF-2, the target of diphtheria toxin and Pseudomonas exotoxin A, in both yeast and higher eukaryotes. Furthermore, Kti11p/Dph3 was found to physically interact with core-Elongator, ribosomal proteins, eEF-2, two other proteins required for diphthamide modification on eEF-2, and DelGEF. Here, we determined the solution structure of Kti11p using NMR, providing the first structure of the CSL-class zinc-binding protein family. We present the first experimental evidence that Kti11p can bind a single Zn(2+) ion by its four conserved cysteine residues. The major structure of Kti11p comprises a beta sandwich as well as an alpha helix. Moreover, a structure-based similarity search suggests that it represents a novel structure and may define a new family of the zinc ribbon fold group. Therefore, our work provides a molecular basis for further understanding the multiple functions of Kti11p/Dph3 in different biological processes.
Amino Acid Sequence↗
Activation of the LicT transcriptional antiterminator involves a domain swing/lock mechanism provoking massive structural changes.
The transcriptional antiterminator protein LicT regulates the expression of Bacillus subtilis operons involved in beta-glucoside metabolism. It consists of an N-terminal RNA-binding domain (co-antiterminator (CAT)) and two phosphorylatable phosphotransferase system regulation domains (PRD1 and PRD2). In the activated state, each PRD forms a dimeric unit with the phosphorylation sites totally buried at the dimer interface. Here we present the 1.95 A resolution structure of the inactive LicT PRDs as well as the molecular solution structure of the full-length protein deduced from small angle x-ray scattering. Comparison of native (inactive) and mutant (constitutively active) PRD crystal structures shows massive tertiary and quaternary rearrangements of the entire regulatory domain. In the inactive state, a wide swing movement of PRD2 results in dimer opening and brings the phosphorylation sites to the protein surface. This movement is accompanied by additional structural rearrangements of both the PRD1-PRD1 ' interface and the CAT-PRD1 linker. Small angle x-ray scattering experiments indicate that the amplitude of the PRD2 swing might even be wider in solution than in the crystals. Our results suggest that PRD2 is highly mobile in the native protein, whereas it is locked upon activation by phosphorylation.
Bacillus subtilis↗
Crystal structure of the YML079w protein from Saccharomyces cerevisiae reveals a new sequence family of the jelly-roll fold.
We determined the three-dimensional crystal structure of the protein YML079wp, encoded by a hypothetical open reading frame from Saccharomyces cerevisiae to a resolution of 1.75 A. The protein has no close homologs and its molecular and cellular functions are unknown. The structure of the protein is a jelly-roll fold consisting of ten beta-strands organized in two parallel packed beta-sheets. The protein has strong structural resemblance to the plant storage and ligand binding proteins (canavalin, glycinin, auxin binding protein) but also to some plant and bacterial enzymes (epimerase, germin). The protein forms homodimers in the crystal, confirming measurements of its molecular mass in solution. Two monomers have their beta-sheet packed together to form the dimer. The presence of a hydrophobic ligand in a well conserved pocket inside the barrel and local sequence similarity with bacterial epimerases may suggest a biochemical function for this protein.
Amino Acid Sequence↗
Crystal structure and functional characterization of yeast YLR011wp, an enzyme with NAD(P)H-FMN and ferric iron reductase activities.
Flavodoxins are involved in a variety of electron transfer reactions that are essential for life. Although FMN-binding proteins are well characterized in prokaryotic organisms, information is scarce for eukaryotic flavodoxins. We describe the 2.0-A resolution crystal structure of the Saccharomyces cerevisiae YLR011w gene product, a predicted flavoprotein. YLR011wp indeed adopts a flavodoxin fold, binds the FMN cofactor, and self-associates as a homodimer. Despite the absence of the flavodoxin key fingerprint motif involved in FMN binding, YLR011wp binds this cofactor in a manner very analogous to classical flavodoxins. YLR011wp closest structural homologue is the homodimeric Bacillus subtilis Yhda protein (25% sequence identity) whose homodimer perfectly superimposes onto the YLR011wp one. Yhda, whose function is not documented, has 53% sequence identity with the Bacillus sp. OY1-2 azoreductase. We show that YLR011wp has an NAD(P)H-dependent FMN reductase and a strong ferricyanide reductase activity. We further demonstrate a weak but specific reductive activity on azo dyes and nitrocompounds.
Amino Acid Sequence↗
Crystal structure of the YGR205w protein from Saccharomyces cerevisiae: close structural resemblance to E. coli pantothenate kinase.
The protein product of the YGR205w gene of Saccharomyces cerevisiae was targeted as part of our yeast structural genomics project. YGR205w codes for a small (290 amino acids) protein with unknown structure and function. The only recognizable sequence feature is the presence of a Walker A motif (P loop) indicating a possible nucleotide binding/converting function. We determined the three-dimensional crystal structure of Se-methionine substituted protein using multiple anomalous diffraction. The structure revealed a well known mononucleotide fold and strong resemblance to the structure of small metabolite phosphorylating enzymes such as pantothenate and phosphoribulo kinase. Biochemical experiments show that YGR205w binds specifically ATP and, less tightly, ADP. The structure also revealed the presence of two bound sulphate ions, occupying opposite niches in a canyon that corresponds to the active site of the protein. One sulphate is bound to the P-loop in a position that corresponds to the position of beta-phosphate in mononucleotide protein ATP complex, suggesting the protein is indeed a kinase. The nature of the phosphate accepting substrate remains to be determined.
Adenosine Diphosphate↗
Crystal structure of the YDR533c S. cerevisiae protein, a class II member of the Hsp31 family.
The ORF YDR533c from Saccharomyces cerevisiae codes for a 25.5 kDa protein of unknown biochemical function. Transcriptome analysis of yeast has shown that this gene is activated in response to various stress conditions together with proteins belonging to the heat shock family. In order to clarify its biochemical function, we determined the crystal structure of YDR533c to 1.85 A resolution by the single anomalous diffraction method. The protein possesses an alpha/beta hydrolase fold and a putative Cys-His-Glu catalytic triad common to a large enzyme family containing proteases, amidotransferases, lipases, and esterases. The protein has strong structural resemblance with the E. coli Hsp31 protein and the intracellular protease I from Pyrococcus horikoshii, which are considered class I and class III members of the Hsp31 family, respectively. Detailed structural analysis strongly suggests that the YDR533c protein crystal structure is the first one of a class II member of the Hsp31 family.
Amino Acid Sequence↗
Refolding strategies from inclusion bodies in a structural genomics project.
The South-Paris Yeast Structural Genomics Project aims at systematically expressing, purifying and determining the structure of S. cerevisiae proteins with no detectable homology to proteins of known structure. We brought 250 yeast ORFs to expression in E. coli, but 37% of them form inclusion bodies. This important fraction of proteins that are well expressed but lost for structural studies prompted us to test methodologies to recover these proteins. Three different strategies were explored in parallel on a set of 20 proteins: (1) refolding from solubilized inclusion bodies using an original and fast 96-well plates screening test, (2) co-expression of the targets in E. coli with DnaK-DnaJ-GrpE and GroEL-GroES chaperones, and (3) use of the cell-free expression system. Most of the tested proteins (17/20) could be resolubilized at least by one approach, but the subsequent purification proved to be difficult for most of them.
Genome, Fungal↗
Crystal structure of the yeast Phox homology (PX) domain protein Grd19p complexed to phosphatidylinositol-3-phosphate.
Phox homology (PX) domains have been recently identified in a number of different proteins and are involved in various cellular functions such as vacuolar targeting and membrane protein trafficking. It was shown that these modules of about 130 amino acids specifically binding to phosphoinositides and that this interaction is crucial for their cellular function. The yeast genome contains 17 PX domain proteins. One of these, Grd19p, is involved in the localization of the late Golgi membrane proteins DPAP A and Kex2p. Grd19p consists of the PX domain with 30 extra residues at the N-terminal and is homologous to the functionally characterized human sorting nexin protein SNX3. We determined the 2.0 A crystal structure of Grd19p in the free form and in complex with d-myo-phosphatidylinositol 3-phosphate (diC4PtdIns(3)P), representing the first case of both free and ligand-bound conformations of the same PX module. The ligand occupies a well defined positively charged binding pocket at the interface between the beta-sheet and alpha-helical parts of the molecule. The structure of the free and bound protein are globally similar but show some significant differences in a region containing a polyproline peptide and a putative membrane attachment site.
Amino Acid Sequence↗
The 62-kb upstream region of Bombyx mori fibroin heavy chain gene is clustered of repetitive elements and candidate matrix association regions.
We sequenced an 80 kb DNA region containing the complete sequence of the silkworm Bombyx mori fibroin gene and its flanking, especially the upstream, regions (-62 kb). About 30% of the 62 kb upstream region is composed of repetitive elements including short interspersed elements Bm1, long interspersed elements L1Bm and mariner-like elements Bmmar1 which are widespread over the silkworm genome. This 62 kb region is also enriched of commonly considered matrix association region (MAR) motifs. A total of 25 individual MAR recognition signatures (MRSs) were identified, with 24 at the upstream and one at the downstream region. Combining two newly developed MAR prediction programs (MAR-finder and Chrclass), ten candidate MARs were predicted, with five containing MRS and seven related to the repetitive elements. The wide distribution of nested repetitive elements, candidate MARs, DNase I hypersensitive sites and other potential regulatory factors recognition sites indicates this region is probably a unique huge cis-acting element contributing to the regulation of the spatial and temporal specificity and efficiency of fibroin gene expression.
5' Flanking Region↗
The Paris-Sud yeast structural genomics pilot-project: from structure to function.
We present here the outlines and results from our yeast structural genomics (YSG) pilot-project. A lab-scale platform for the systematic production and structure determination is presented. In order to validate this approach, 250 non-membrane proteins of unknown structure were targeted. Strategies and final statistics are evaluated. We finally discuss the opportunity of structural genomics programs to contribute to functional biochemical annotation.
Genomics↗