Yan-Bin Teng

Structural basis for the allosteric control of the global transcription factor NtcA by the nitrogen starvation signal 2-oxoglutarate

2-oxogluatarate (2-OG), a metabolite of the highly conserved Krebs cycle, not only plays a critical role in metabolism, but also constitutes a signaling molecule in a variety of organisms ranging from bacteria to plants and animals. In cyanobacteria, the accumulation of 2-OG constitutes the signal of nitrogen starvation and NtcA, a global transcription factor, has been proposed as a putative receptor for 2-OG. Here we present three crystal structures of NtcA from the cyanobacterium Anabaena: the apoform, and two ligand-bound forms in complex with either 2-OG or its analogue 2,2-difluoropentanedioic acid. All structures assemble as homodimers, with each subunit composed of an N-terminal effector-binding domain and a C-terminal DNA-binding domain connected by a long helix (C-helix). The 2-OG binds to the effector-binding domain at a pocket similar to that used by cAMP in catabolite activator protein, but with a different pattern. Comparative structural analysis reveals a putative signal transmission route upon 2-OG binding. A tighter coiled-coil conformation of the two C-helices induced by 2-OG is crucial to maintain the proper distance between the two F-helices for DNA recognition. Whereas catabolite activator protein adopts a transition from off-to-on state upon cAMP binding, our structural analysis explains well why NtcA can bind to DNA even in its apoform, and how 2-OG just enhances the DNA-binding activity of NtcA. These findings provided the structural insights into the function of a global transcription factor regulated by 2-OG, a metabolite standing at a crossroad between carbon and nitrogen metabolisms.

Structure of autophagy-related protein Atg8 from the silkworm Bombyx mori.

Autophagy-related protein Atg8 is ubiquitous in all eukaryotes. It is involved in the Atg8-PE ubiquitin-like conjugation system, which is essential for autophagosome formation. The structures of Atg8 from different species are very similar and share a ubiquitin-fold domain at the C-terminus. In the 2.40 A crystal structure of Atg8 from the silkworm Bombyx mori reported here, the ubiquitin fold at the C-terminus is preceded by two additional helices at the N-terminus.

Structural insights into the substrate tunnel of Saccharomyces cerevisiae carbonic anhydrase Nce103

BackgroundThe carbonic anhydrases (CAs) are involved in inorganic carbon utilization. They have been classified into six evolutionary and structural families: α-, β-, γ-, δ-, ε-, ζ- CAs, with β-CAs present in higher plants, algae and prokaryotes. The yeast Saccharomyces cerevisiae encodes a single copy of β-CA Nce103/YNL036W.ResultsWe determined the crystal structure of Nce103 in complex with a substrate analog at 2.04 Å resolution. It assembles as a homodimer, with the active site located at the interface between two monomers. At the bottom of the substrate pocket, a zinc ion is coordinated by the three highly conserved residues Cys57, His112 and Cys115 in addition to a water molecule. Residues Asp59, Arg61, Gly111, Leu102, Val80, Phe75 and Phe97 form a tunnel to the bottom of the active site which is occupied by a molecule of the substrate analog acetate. Activity assays of full length and two truncated versions of Nce103 indicated that the N-terminal arm is indispensable.ConclusionThe quaternary structure of Nce103 resembles the typical plant type β-CAs of known structure, with an N-terminal arm indispensable for the enzymatic activity. Comparative structure analysis enables us to draw a possible tunnel for the substrate to access the active site which is located at the bottom of a funnel-shaped substrate pocket.

Structural basis for the different activities of yeast Grx1 and Grx2

Yeast glutaredoxins Grx1 and Grx2 catalyze the reduction of both inter- and intra-molecular disulfide bonds using glutathione (GSH) as the electron donor. Although sharing the same dithiolic CPYC active site and a sequence identity of 64%, they have been proved to play different roles during oxidative stress and to possess different glutathione-disulfide reductase activities. To address the structural basis of these differences, we solved the crystal structures of Grx2 in oxidized and reduced forms, at 2.10 A and 1.50 A, respectively. With the Grx1 structures we previously reported, comparative structural analyses revealed that Grx1 and Grx2 share a similar GSH binding site, except for a single residue substitution from Asp89 in Grx1 to Ser123 in Grx2. Site-directed mutagenesis in combination with activity assays further proved this single residue variation is critical for the different activities of yeast Grx1 and Grx2.

Structural insights into the catalytic mechanism of the yeast pyridoxal 5-phosphate synthase Snz1

In most eubacteria, fungi, apicomplexa, plants and some metazoans, the active form of vitamin B6, PLP (pyridoxal 5-phosphate), is de novo synthesized from three substrates, R5P (ribose 5-phosphate), DHAP (dihydroxyacetone phosphate) and ammonia hydrolysed from glutamine by a complexed glutaminase. Of the three active sites of DXP (deoxyxylulose 5-phosphate)independent PLP synthase (Pdx1), the R5P isomerization site has been assigned, but the sites for DHAP isomerization and PLP formation remain unknown. In the present study, we present the crystal structures of yeast Pdx1/Snz1, in apo-, G3P (glyceraldehyde 3-phosphate)- and PLP-bound forms, at 2.3, 1.8 and 2.2 Å (1 Å=0.1 nm) respectively. Structural and biochemical analysis enabled us to assign the PLP-formation site, a G3P-binding site and a G3P-transfer site. We propose a putative catalytic mechanism for Pdx1/Snz1 in which R5P and DHAP are isomerized at two distinct sites and transferred along well-defined routes to a final destination for PLP synthesis.

The N-Terminal β-Sheet of Peroxiredoxin 4 in the Large Yellow Croaker Pseudosciaena crocea Is Involved in Its Biological Functions

Peroxiredoxins (Prxs) are thiol-specific antioxidant proteins that exhibit peroxidase and peroxynitrite reductase activities involved in the reduction of reactive oxygen species. The peroxiredoxin Prx4 from the large yellow croaker Pseudosciaena crocea is a typical 2-Cys Prx with an N-terminal signal peptide. We solved the crystal structure of Prx4 at 1.90 Å and revealed an N-terminal antiparallel β-sheet that contributes to the dimer interface. Deletion of this β-sheet decreased the in vitro peroxidase activity to about 50% of the wild-type. In vivo assays further demonstrated that removal of this β-sheet led to some impairment in the ability of Prx4 to negatively regulate nuclear factor-κB (NF-κB) activity and to perform its role in anti-bacterial immunity. These results provide new insights into the structure and function relationship of a peroxiredoxin from bony fish.

Crystal structures and putative interface of Saccharomyces cerevisiae mitochondrial matrix proteins Mmf1 and Mam33

The yeast Saccharomyces cerevisiae mitochondrial matrix factor Mmf1, a member in the YER057c/Yigf/Uk114 family, participates in isoleucine biosynthesis and mitochondria maintenance. Mmf1 physically interacts with another mitochondrial matrix protein Mam33, which is involved in the sorting of cytochrome b₂ to the intermembrane space as well as mitochondrial ribosomal protein synthesis. To elucidate the structural basis for their interaction, we determined the crystal structures of Mmf1 and Mam33 at 1.74 and 2.10 Å, respectively. Both Mmf1 and Mam33 adopt a trimeric structure: each subunit of Mmf1 displays a chorismate mutase fold with a six-stranded β-sheet flanked by two α-helices on one side, whereas a subunit of Mam33 consists of a twisted six-stranded β-sheet surrounded by five α-helices. Biochemical assays combined with structure-based computational simulation enable us to model a putative complex of Mmf1-Mam33, which consists of one Mam33 trimer and two tandem Mmf1 trimers in a head-to-tail manner. The two interfaces between the ring-like trimers are mainly composed of electrostatic interactions mediated by complementary negatively and positively charged patches. These results provided the structural insights into the putative function of Mmf1 during mitochondrial protein synthesis via Mam33, a protein binding to mitochondrial ribosomal proteins.

Crystal structures of holo and Cu-deficient Cu/Zn-SOD from the silkworm Bombyx mori and the implications in amyotrophic lateral sclerosis.

Cu/Zn superoxide dismutases (Cu/Zn-SODs) are a large family of cytosolic antioxidant proteins involved in responses to oxidative stress.1 They catalyze the disproportionation of superoxide radicals into less toxic hydrogen peroxide and dioxygen via cyclic reduction and reoxidation of its bound copper ion as shown below.2 SOD CuðIIÞ þ O2 ! SOD CuðIÞ þ O2 SOD CuðIÞ þO2 þ 2Hþ ! SOD CuðIIÞ þH2O2

Crystal structure of Arabidopsis translation initiation factor eIF‐5A2

The protein eukaryotic translation initiation factor 5A (eIF-5A) is a highly conserved eukaryotic translation initiation factor (eIF) found in eukaryotes and archaea.1–3 Biochemical and molecular studies revealed that eIF-5A is the sole protein that contains a modified amino acid residue hypusine (Ne-(4-amino-2-hydroxybutyl)lysine).4 The hypusination modification is made by two sequential reactions catalyzed by deoxyhypusine synthase (EC 1.1.1.249) and deoxyhypusine hydroxylase (EC 1.14.99.29).5–7 eIF-5A was originally purified and identified from immature red blood cells.8 However, unlike the traditional translation initiation factors, eIF-5A is not essential for global protein synthesis8,9 but might be involved in mRNA translocation across the nuclear envelope.10,11 The hypusinated yeast eIF-5A was recently found to promote translation elongation.12 Moreover, hypusine of the yeast eIF-5A has been found to be required for the sequence-specific interaction with RNA.13 To help clarify these diverse and even somewhat controversial functions, seven structures of eIF-5A from various organisms have been solved (Methanococcus jannaschii, PDB codes: 1eif and 2eif14; Pyrobaculum aerophilum, PDB code: 1bkb15; Pyrococcus horikoshii, PDB code: 1iz616; Leishmania braziliensis, PDB code: 1 3 6o; Leishmania mexicana, PDB code:1 3 td; Homo sapiens, PDB code: 3cpf; Saccharomyces cerevisiae, PDB code: 3er0). They all share an overall structure of two domains, both of which resemble the nucleic acid binding fold.14 The plant Arabidopsis thaliana encodes three isoforms of eIF-5A: AteIF-5A1, 2, and 3 (GenBank Accession Numbers AF296082, BE039424, and AV526594). As the best investigated one, eIF-5A2 has been found to play a crucial role in plant growth and development by controlling cell proliferation and senescence.17 Here, we report the crystal structure of eIF-5A2 at 2.3 Å resolution, which represents a novel dimerization pattern specifically conserved in all plants.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of hypothetical protein SCO4226 from Streptomyces coelicolor A3(2).

A non-Pfam hypothetical protein SCO4226 of molecular weight 9 kDa from Streptomyces coelicolor A3(2) was overexpressed in Escherichia coli and the purified recombinant protein was crystallized using the sitting-drop vapour-diffusion method. An X-ray diffraction data set was collected to 2.0 A resolution. The crystal belonged to space group P2(1), with unit-cell parameters a = 29.67, b = 67.00, c = 34.43 A, alpha = gamma = 90.00, beta = 94.26 degrees .