Yoshinori Satow

Crystal structures of human MD-2 and its complex with antiendotoxic lipid IVa

Endotoxic lipopolysaccharide (LPS) with potent immunostimulatory activity is recognized by the receptor complex of MD-2 and Toll-like receptor 4. Crystal structures of human MD-2 and its complex with the antiendotoxic tetra-acylated lipid A core of LPS have been determined at 2.0 and 2.2 angstrom resolutions, respectively. MD-2 shows a deep hydrophobic cavity sandwiched by two β sheets, in which four acyl chains of the ligand are fully confined. The phosphorylated glucosamine moieties are located at the entrance to the cavity. These structures suggest that MD-2 plays a principal role in endotoxin recognition and provide a basis for antiseptic drug development.

A new type of X-ray area detector utilizing laser stimulated luminescence

Abstract A conceptually new integrating area (250 mm×200 mm or larger) detector system, originally developed for diagnostic radiography, has been used for synchrotron radiation experiments. In this detector system, an X-ray image is temporarily stored as a distribution of F-centers in a photostimulable phosphor (BaFBr:Eu 2+ ) screen. The stored image is read out by measuring the intensity of fluorescence ( λ ∼390 nm) stimulated by a HeNe laser beam scanned over the surface of the screen. The detector has 100% detective quantum efficiency for 8–17 keV X-rays, a spatial resolution better than 0.2 mm (fwhm) in both directions, a dynamic range of 1:10 5 and no counting rate limitation. Diffraction patterns from muscle and a protein crystal were recorded in several tens times less exposure than that of high-sensitivity X-ray films.

Crystal structure of a bacterial protein proteinase inhibitor (Streptomyces subtilisin inhibitor) at 2·6 Å resolution

The crystal structure of a bacterial protein proteinase inhibitor (Streptomyces subtilisin inhibitor) was solved at 2·6 A resolution. Each subunit of the dimeric inhibitor has a five-stranded antiparallel β-sheet and two short α-helices. The subunit-subunit interface formed by a stack of two β-sheets provided by the two subunits resembles the dimer-dimer interface of concanavalin A. Conformation of the reactive site around the scissible bond Met73-Val74 seems very rigid. Between bovine pancreatic trypsin inhibitor (Kunitz) and the Streptomyces inhibitor, the reactive site conformations are almost identical with each other from the P2 to P2′ residues, while between the soybean trypsin inhibitor (Kunitz) and the Streptomyces inhibitor they are similar from the P2 to P1′ residues. There are overall similarities in conformation extending from the P3 to P2′ residues between the Streptomyces inhibitor and a hypothetical substrate presumed (Robertus et al., 1972b) to be bound to subtilisin BPN′ in a productive binding mode. Apart from the reactive site, there seems to be no structural relationship among the Streptomyces, bovine pancreatic and soybean inhibitors, suggesting their convergent evolution from separate ancestral proteins.

Identification of Three Core Regions Essential for Protein Splicing of the Yeast Vma1 Protozyme A RANDOM MUTAGENESIS STUDY OF THE ENTIRE VMA1-DERIVED ENDONUCLEASE SEQUENCE

The translation product of the VMA1gene of Saccharomyces cerevisiae undergoes protein splicing, in which the intervening region is autocatalytically excised and the franking regions are ligated. The splicing reaction is catalyzed essentially by the in-frame insert, VMA1-derived endonuclease (VDE), which is a site-specific endonuclease to mediate gene homing. Previous mutational analysis of the splicing reaction has been concentrated extensively upon the splice junctions. However, it still remains unknown which amino acid residues are crucial for the splicing reaction within the entire region of VDE and its neighboring elements. In this work, a polymerase chain reaction-based random mutagenesis strategy was used to identify such residues throughout the overall intervening sequence of the VMA1 gene. Splicing-defective mutant proteins were initially screened using a bacterial expression system and then analyzed further in yeast cells. Mutations were mapped at the N- and C-terminal splice junctions and around the N-terminal one-third of VDE. We identified four potent mutants that yielded aberrant products with molecular masses of 200, 90, and 80 kDa. We suggest that the conserved His362, newly identified as the essential residue for the splicing reaction, contributes to the first cleavage at the N-terminal junction, whereas His736 assists the second cleavage by Asn cyclization at the C-terminal junction. Mutations in these regions did not appear to destroy the endonuclease activity of VDE.

Impaired α-TTP-PIPs Interaction Underlies Familial Vitamin E Deficiency

Vitamin E Out Familial vitamin E deficiency is caused by mutations in the α-tocopherol transfer protein (α-TTP) gene. Kono et al. (p. 1106, published online 18 April; see the Perspective by Mesmin and Antonny) studied natural mutations in α-TTP. α-TTP bound phosphatidylinositol polyphosphates (PIPs), especially PI(4,5)P2, and a disease-related missense mutation abolished PIP binding but not α-tocopherol binding. The x-ray crystal structure of the α-TTP–PIP complex suggested that PIP binding opens the lid of the α-tocopherol–binding pocket to facilitate the release of α-tocopherol. Thus, PIP binding to α-TTP at the target membrane may facilitate the release of α-tocopherol in the hydrophobic pocket of α-TTP to the lipid bilayer of the target membrane, providing a mechanism for the transfer of lipids from the lipid-transfer protein to the target membrane. Phosphatidylinositol phosphates may play a role in lipid-transfer protein–mediated vitamin E efflux from hepatocytes. [Also see Perspective by Mesmin and Antonny] α-Tocopherol (vitamin E) transfer protein (α-TTP) regulates the secretion of α-tocopherol from liver cells. Missense mutations of some arginine residues at the surface of α-TTP cause severe vitamin E deficiency in humans, but the role of these residues is unclear. Here, we found that wild-type α-TTP bound phosphatidylinositol phosphates (PIPs), whereas the arginine mutants did not. In addition, PIPs in the target membrane promoted the intermembrane transfer of α-tocopherol by α-TTP. The crystal structure of the α-TTP–PIPs complex revealed that the disease-related arginine residues interacted with phosphate groups of the PIPs and that the PIPs binding caused the lid of the α-tocopherol–binding pocket to open. Thus, PIPs have a role in promoting the release of a ligand from a lipid-transfer protein.

Crystal Structure of Human β-Galactosidase STRUCTURAL BASIS OF GM1 GANGLIOSIDOSIS AND MORQUIO B DISEASES

Background: Deficiencies in β-d-galactosidase cause lysosomal storage diseases. Results: This is the first report to describe the crystal structure of human β-Gal. Human β-Gal is composed of a TIM barrel domain and two β-domains. Conclusion: The mutations were classified as mutations directly affecting the ligand recognition, mutations inside the protein core, or mutations located in the protein surface. Significance: Structural insights into lysosomal storage diseases mutations can be demonstrated. GM1 gangliosidosis and Morquio B are autosomal recessive lysosomal storage diseases associated with a neurodegenerative disorder or dwarfism and skeletal abnormalities, respectively. These diseases are caused by deficiencies in the lysosomal enzyme β-d-galactosidase (β-Gal), which lead to accumulations of the β-Gal substrates, GM1 ganglioside, and keratan sulfate. β-Gal is an exoglycosidase that catalyzes the hydrolysis of terminal β-linked galactose residues. This study shows the crystal structures of human β-Gal in complex with its catalytic product galactose or with its inhibitor 1-deoxygalactonojirimycin. Human β-Gal is composed of a catalytic TIM barrel domain followed by β-domain 1 and β-domain 2. To gain structural insight into the molecular defects of β-Gal in the above diseases, the disease-causing mutations were mapped onto the three-dimensional structure. Finally, the possible causes of the diseases are discussed.

Horizontal‐type four‐circle diffractometer station of the vertical wiggler beamline at the Photon Factory

An experimental station with a four‐circle diffractometer combined with tunable and focusing optics was designed and constructed for x‐ray crystallography using synchrotron radiation from the vertical wiggler at the Photon Factory. The optics implemented into beamline 14A comprises a double‐crystal monochromator and a double‐focusing mirror so as to yield high photon flux x rays tunable over wide wavelength regions. The diffractometer with its equatorial plane in the horizontal was specially fabricated for rapid and accurate measurements of x‐ray diffraction intensities from macromolecular crystals, and was provided with an alignment carriage as well as computer control and measuring systems. The station has proved to be particularly useful for such applications as anomalous scattering studies of protein crystals and also EXAFS studies at high photon energies.

Crystal Structure of Human Renal Dipeptidase Involved in β-Lactam Hydrolysis

Human renal dipeptidase is a membrane-bound glycoprotein hydrolyzing dipeptides and is involved in hydrolytic metabolism of penem and carbapenem beta-lactam antibiotics. The crystal structures of the saccharide-trimmed enzyme are determined as unliganded and inhibitor-liganded forms. They are informative for designing new antibiotics that are not hydrolyzed by this enzyme. The active site in each of the (alpha/beta)(8) barrel subunits of the homodimeric molecule is composed of binuclear zinc ions bridged by the Glu125 side-chain located at the bottom of the barrel, and it faces toward the microvillar membrane of a kidney tubule. A dipeptidyl moiety of the therapeutically used cilastatin inhibitor is fully accommodated in the active-site pocket, which is small enough for precise recognition of dipeptide substrates. The barrel and active-site architectures utilizing catalytic metal ions exhibit unexpected similarities to those of the murine adenosine deaminase and the catalytic domain of the bacterial urease.

Protein splicing: its discovery and structural insight into novel chemical mechanisms.

Protein splicing is a posttranslational cellular process, in which an intervening protein sequence (intein) is self‐catalytically excised out from a nascent protein precursor and the two flanking sequences (N‐ and C‐exteins) are ligated to produce two mature enzymes. This unique reaction was first discovered from studies of the structure and expression of the VMA1 gene in Saccharomyces cerevisiae. VMA1 consists of a single open reading frame and yet comprises two independent genetic information for Vma1p (a catalytic 70‐kDa subunit of the vacuolar H + ‐ATPase) and VDE (a 50‐kDa DNA endonuclease) as an in‐frame spliced insert in the gene. Subsequent studies have demonstrated that protein splicing is not unique for the VMA1 precursor and there are many operons in nature, which implement genetic information editing at protein level. To elucidate its precise reaction mechanisms from a viewpoint of structure‐directed chemistry, a series of crystal structural studies has been carried out with the use of splicing‐inactive and slowly spliceable precursors of VMA1 recombinants. One precursor structure revealed that the N‐terminal junction of the introduced extein polypeptide forms an intermediate containing a five‐membered thiazolidine ring. The other precursor structures showed spliced products with a linkage between the N‐ and C‐extein segments. This article summarizes biochemical and structural studies on a self‐catalytic mechanism for protein splicing that is triggered and terminated solely via thiazolidine intermediates with tetrahedral configurations formed within the splicing sites where proton ingress and egress are driven by balanced protonation and deprotonation. IUBMB Life, 57: 563‐574, 2005

Imaging plate for time‐resolved x‐ray measurements (invited)

Two apparatus have been developed for time‐resolved measurements of x‐ray diffraction patterns using an imaging plate detector. The first one is based on a cinema method which permits up to 40 exposures of a two‐dimensional x‐ray pattern (100×108 mm2) with a 0.3‐s time resolution. The second one works as a 200‐mm‐long linear detector which enables a time resolution of 23 μs for a duration of up to 46 ms, based on a streak‐camera method. These apparatus have no count rate limitation, a high detective quantum efficiency (more than 80% for 8–20 keV), a 1:105 dynamic range in x‐ray intensity and a spatial resolution of 150 μm (FWHM) due to the performance of the imaging plate.