Phospholipid transfer function of PTPIP51 at mitochondria-associated ER membranes.

In eukaryotic cells, mitochondria are closely tethered to the endoplasmic reticulum (ER) at sites called mitochondria-associated ER membranes (MAMs). Ca2+ ion and phospholipid transfer occurs at MAMs to support diverse cellular functions. Unlike those in yeast, the protein complexes involved in phospholipid transfer at MAMs in humans have not been identified. Here, we determine the crystal structure of the tetratricopeptide repeat domain of PTPIP51 (PTPIP51_TPR), a mitochondrial protein that interacts with the ER-anchored VAPB protein at MAMs. The structure of PTPIP51_TPR shows an archetypal TPR fold, and an electron density map corresponding to an unidentified lipid-like molecule probably derived from the protein expression host is found in the structure. We reveal functions of PTPIP51 in phospholipid binding/transfer, particularly of phosphatidic acid, in vitro. Depletion of PTPIP51 in cells reduces the mitochondrial cardiolipin level. Additionally, we confirm that the PTPIP51-VAPB interaction is mediated by the FFAT-like motif of PTPIP51 and the MSP domain of VAPB. Our findings suggest that PTPIP51 is a phospholipid transfer protein with a MAM-tethering function.

Structural basis of operator sites recognition and effector binding in the TetR family transcription regulator FadR.

FadR is a fatty acyl-CoA dependent transcription factor that regulates genes encoding proteins involved in fatty-acid degradation and synthesis pathways. In this study, the crystal structures of Bacillus halodurans FadR, which belong to the TetR family, have been determined in three different forms: ligand-bound, ligand-free and DNA-bound at resolutions of 1.75, 2.05 and 2.80 Å, respectively. Structural and functional data showed that B. halodurans FadR was bound to its operator site without fatty acyl-CoAs. Structural comparisons among the three different forms of B. halodurans FadR revealed that the movement of DNA binding domains toward the operator DNA was blocked upon binding of ligand molecules. These findings suggest that the TetR family FadR negatively regulates the genes involved in fatty acid metabolism by binding cooperatively to the operator DNA as a dimer of dimers.

Structural Analysis of Thymidylate Synthase from Kaposi's Sarcoma-Associated Herpesvirus with the Anticancer Drug Raltitrexed.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a highly infectious human herpesvirus that causes Kaposi's sarcoma. KSHV encodes functional thymidylate synthase, which is a target for anticancer drugs such as raltitrexed or 5-fluorouracil. Thymidylate synthase catalyzes the conversion of 2'-deoxyuridine-5'-monophosphate (dUMP) to thymidine-5'-monophosphate (dTMP) using 5,10-methylenetetrahydrofolate (mTHF) as a co-substrate. The crystal structures of thymidylate synthase from KSHV (apo), complexes with dUMP (binary), and complexes with both dUMP and raltitrexed (ternary) were determined at 1.7 Å, 2.0 Å, and 2.4 Å, respectively. While the ternary complex structures of human thymidylate synthase and E. coli thymidylate synthase had a closed conformation, the ternary complex structure of KSHV thymidylate synthase was observed in an open conformation, similar to that of rat thymidylate synthase. The complex structures of KSHV thymidylate synthase did not have a covalent bond between the sulfhydryl group of Cys219 and C6 atom of dUMP, unlike the human thymidylate synthase. The catalytic Cys residue demonstrated a dual conformation in the apo structure, and its sulfhydryl group was oriented toward the C6 atom of dUMP with no covalent bond upon ligand binding in the complex structures. These structural data provide the potential use of antifolates such as raltitrexed as a viral induced anticancer drug and structural basis to design drugs for targeting the thymidylate synthase of KSHV.

Structural Analysis and Insights into the Oligomeric State of an Arginine-Dependent Transcriptional Regulator from Bacillus halodurans.

The arginine repressor (ArgR) is an arginine-dependent transcription factor that regulates the expression of genes encoding proteins involved in the arginine biosynthesis and catabolic pathways. ArgR is a functional homolog of the arginine-dependent repressor/activator AhrC from Bacillus subtilis, and belongs to the ArgR/AhrC family of transcriptional regulators. In this research, we determined the structure of the ArgR (Bh2777) from Bacillus halodurans at 2.41 Å resolution by X-ray crystallography. The ArgR from B. halodurans appeared to be a trimer in a size exclusion column and in the crystal structure. However, it formed a hexamer in the presence of L-arginine in multi-angle light scattering (MALS) studies, indicating the oligomerization state was dependent on the presence of L-arginine. The trimeric structure showed that the C-terminal domains form the core, which was made by inter-subunit interactions mainly through hydrophobic contacts, while the N-terminal domains containing a winged helix-turn-helix DNA binding motif were arranged around the periphery. The arrangement of trimeric structure in the B. halodurans ArgR was different from those of other ArgR homologs previously reported. We finally showed that the B. halodurans ArgR has an arginine-dependent DNA binding property by an electrophoretic mobility shift assay.

Crystallization and preliminary X-ray diffraction analysis of the arginine repressor ArgR from Bacillus halodurans.

The arginine repressor (ArgR) is a transcriptional regulator which regulates genes encoding proteins involved in arginine biosynthesis and the arginine catabolic pathway. ArgR from the alkaliphilic bacterium Bacillus halodurans was cloned and overexpressed in Escherichia coli. ArgR (Bh2777) from B. halodurans is composed of 149 amino-acid residues with a molecular mass of 16 836 Da. ArgR was crystallized at 296 K using 1,2-propanediol as a precipitant. Crystals of N-terminally His-tagged ArgR were obtained by the sitting-drop vapour-diffusion method. Dehydrated crystals showed a dramatic improvement in diffraction quality and diffracted to 2.35 Šresolution. The crystals belonged to the cubic space group I23, with unit-cell parameters a = b = c = 104.68 Å. The asymmetric unit contained one monomer of ArgR, which generates a trimer by the threefold axis of the space group, giving a crystal volume per mass (VM) of 2.98 Å(3) Da(-1) and a solvent content of 56.8%.

Structural analysis and insight into metal-ion activation of the iron-dependent regulator from Thermoplasma acidophilum.

The iron-dependent regulator (IdeR) is a metal ion-activated transcriptional repressor that regulates the expression of genes encoding proteins involved in iron uptake to maintain metal-ion homeostasis. IdeR is a functional homologue of the diphtheria toxin repressor (DtxR), and both belong to the DtxR/MntR family of metalloregulators. The structure of Fe(2+)-bound IdeR (TA0872) from Themoplasma acidophilum was determined at 2.1 Å resolution by X-ray crystallography using single-wavelength anomalous diffraction. The presence of Fe(2+), which is the true biological activator of IdeR, in the metal-binding site was ascertained by the use of anomalous difference electron-density maps using diffraction data collected at the Fe absorption edge. Each DtxR/IdeR subunit contains two metal ion-binding sites separated by 9 Å, labelled the primary and ancillary sites, whereas the crystal structures of IdeR from T. acidophilum show a binuclear iron cluster separated by 3.2 Å, which is novel to T. acidophilum IdeR. The metal-binding site analogous to the primary site in DtxR was unoccupied, and the ancillary site was occupied by binuclear clustered ions. This difference suggests that T. acidophilum IdeR and its closely related homologues are regulated by a mechanism distinct from that of either DtxR or MntR. T. acidophilum IdeR was also shown to have a metal-dependent DNA-binding property by electrophoretic mobility shift assay.

Crystallization and preliminary X-ray diffraction analysis of the TetR-family transcriptional repressor YhgD from Bacillus halodurans.

YhgD is a member of the TetR-family transcription factors, which regulate genes encoding proteins involved in multidrug resistance, virulence, osmotic stress and pathogenicity. YhgD from the alkaliphilic bacterium Bacillus halodurans was cloned and overexpressed in Escherichia coli. YhgD (Bh2145) from B. halodurans is composed of 193 amino-acid residues with a molecular mass of 21 853 Da. YhgD was crystallized at 296 K using ethylene glycol as a precipitant by the sitting-drop vapour-diffusion method. The crystal diffracted to 1.9 Šresolution and belonged to the apparent triclinic space group P1, with unit-cell parameters a = 37.22, b = 47.85, c = 54.15 Å, α = 92.75, ß = 107.9, γ = 90.27°. The asymmetric unit is likely to contain two molecules of monomeric YhgD, giving a crystal volume per mass (VM) of 2.05 Å(3) Da(-1) and a solvent content of 40.2%.

Crystallization and preliminary X-ray diffraction analysis of a fatty-acid metabolism regulatory protein, FadR, from Bacillus halodurans.

FadR is an acyl-CoA-dependent transcription factor which regulates genes encoding proteins involved in fatty-acid degradation and synthesis in order to maintain lipid homeostasis. FadR from the alkaliphilic bacterium Bacillus halodurans was cloned and overexpressed in Escherichia coli. The FadR (Bh3102) protein from B. halodurans is composed of 195 amino-acid residues with a molecular mass of 22 378 Da. Crystals were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.05 Šresolution. FadR was crystallized at 296 K using polyethylene glycol 3350 as a precipitant. The crystal belonged to the apparent trigonal space group P3(2)21, with unit-cell parameters a = b = 56.34, c = 199.73 Å. The Matthews coefficient and solvent content were estimated to be 2.0 Å(3) Da(-1) and 39.8%, respectively, assuming that the asymmetric unit contained two molecules of FadR, which was subsequently confirmed by molecular-replacement calculations.

Crystallization and preliminary X-ray diffraction analysis of the metalloregulatory protein DtxR from Thermoplasma acidophilum.

The diphtheria toxin repressor (DtxR) is a metal-ion-dependent transcriptional regulator which regulates genes encoding proteins involved in metal-ion uptake to maintain metal-ion homeostasis. DtxR from Thermoplasma acidophilum was cloned and overexpressed in Escherichia coli. Crystals of N-terminally His-tagged DtxR were obtained by hanging-drop vapour diffusion and diffracted to 1.8 Å resolution. DtxR was crystallized at 296 K using polyethylene glycol 4000 as a precipitant. The crystals belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 61.14, b = 84.61, c = 46.91 Å, α = ß = γ = 90°. The asymmetric unit contained approximately one monomer of DtxR, giving a crystal volume per mass (V(M)) of 2.22 Å(3) Da(-1) and a solvent content of 44.6%.