Hideaki Tsuge

Structure of the Human Cytomegalovirus Protease Catalytic Domain Reveals a Novel Serine Protease Fold and Catalytic Triad

Proteolytic processing of capsid assembly protein precursors by herpesvirus proteases is essential for virion maturation. A 2.5 A crystal structure of the human cytomegalovirus protease catalytic domain has been determined by X-ray diffraction. The structure defines a new class of serine protease with respect to global-fold topology and has a catalytic triad consisting of Ser-132, His-63, and His-157 in contrast with the Ser-His-Asp triads found in other serine proteases. However, catalytic machinery for activating the serine nucleophile and stabilizing a tetrahedral transition state is oriented similarly to that for members of the trypsin-like and subtilisin-like serine protease families. Formation of the active dimer is mediated primarily by burying a helix of one protomer into a deep cleft in the protein surface of the other.

Structure based development of novel specific inhibitors for cathepsin L and cathepsin S in vitro and in vivo

Specific inhibitors for cathepsin L and cathepsin S have been developed with the help of computer‐graphic modeling based on the stereo‐structure. The common fragment, N‐(L‐trans‐carbamoyloxyrane‐2‐carbonyl)‐phenylalanine‐dimethylamide, is required for specific inhibition of cathepsin L. Seven novel inhibitors of the cathepsin L inhibitor Katunuma (CLIK) specifically inhibited cathepsin L at a concentration of 10−7 M in vitro, while almost no inhibition of cathepsins B, C, S and K was observed. Four of the CLIKs are stable, and showed highly selective inhibition for hepatic cathepsin L in vivo. One of the CLIK inhibitors contains an aldehyde group, and specifically inhibits cathepsin S at 10−7 M in vitro.

Crystal structure and site-directed mutagenesis of enzymatic components from Clostridium perfringens iota-toxin.

Iota-toxin from Clostridium perfringens type E is an ADP-ribosylating toxin (ADPRT) that ADP-ribosylates actin, which is lethal and dermonecrotic in mammals. It is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib). Ia ADP-ribosylates G-actin at arginine 177, resulting in the depolymerization of the actin cytoskeleton. Here, we report on studies of the structure-function relationship by the crystal structures of Ia complexed with NADH and NADPH (at 1.8 A and 2.1 A resolution, respectively) and mutagenesis that map the active residues. The catalytic C-domain structure was similar to that of Bacillus cereus vegetative insecticidal protein (VIP2), which is an insect-targeted toxin, except for the EXE loop region. However, a significant structural difference could be seen in the N-domain, which interacts with Ib, suggesting an evolutionary difference between mammalian-targeted and insect-targeted ADPRT. The high resolution structure analysis revealed specific NAD conformation (a ring-like conformation of nicotinamide mononucleotide (NMN)) supported by Arg295, Arg296, Asn335, Arg352 and Glu380. Additionally, the mutagenesis study showed that the residues Tyr251, Arg295, Glu301, Ser338, Phe349, Arg352 and Glu380, including a newly identified one, are essential for NAD(+)-glycohydrolase (NADase) activity. At least one residue, Glu378, is an essential residue for ADP-ribosyltransferase (ARTase), but not for NADase. Consequently, the structural feature and these mutagenesis findings suggest that the catalytic mechanism of Ia proceeds via an Sn1-type reaction.

Structural basis of actin recognition and arginine ADP-ribosylation by Clostridium perfringens iota-toxin.

The ADP-ribosylating toxins (ADPRTs) produced by pathogenic bacteria modify intracellular protein and affect eukaryotic cell function. Actin-specific ADPRTs (including Clostridium perfringens ι-toxin and Clostridium botulinum C2 toxin) ADP-ribosylate G-actin at Arg-177, leading to disorganization of the cytoskeleton and cell death. Although the structures of many actin-specific ADPRTs are available, the mechanisms underlying actin recognition and selective ADP-ribosylation of Arg-177 remain unknown. Here we report the crystal structure of actin-Ia in complex with the nonhydrolyzable NAD analog βTAD at 2.8 Å resolution. The structure indicates that Ia recognizes actin via five loops around NAD: loop I (Tyr-60–Tyr-62 in the N domain), loop II (active-site loop), loop III, loop IV (PN loop), and loop V (ADP-ribosylating turn–turn loop). We used site-directed mutagenesis to confirm that loop I on the N domain and loop II are essential for the ADP-ribosyltransferase activity. Furthermore, we revealed that Glu-378 on the EXE loop is in close proximity to Arg-177 in actin, and we proposed that the ADP-ribosylation of Arg-177 proceeds by an SN1 reaction via first an oxocarbenium ion intermediate and second a cationic intermediate by alleviating the strained conformation of the first oxocarbenium ion. Our results suggest a common reaction mechanism for ADPRTs. Moreover, the structure might be of use in rational drug design to block toxin-substrate recognition.

Structural Basis of the Influenza A Virus RNA Polymerase PB2 RNA-binding Domain Containing the Pathogenicity-determinant Lysine 627 Residue

Because the influenza A virus has an RNA genome, its RNA-dependent RNA polymerase, comprising the PA, PB1, and PB2 subunits, is essential for viral transcription and replication. The binding of RNA primers/promoters to the polymerases is an initiation step in viral transcription. In our current study, we reveal the 2.7 Å tertiary structure of the C-terminal RNA-binding domain of PB2 by x-ray crystallography. This domain incorporates lysine 627 of PB2, and this residue is associated with the high pathogenicity and host range restriction of influenza A virus. We found from our current analyses that this lysine is located in a unique “ϕ”-shaped structure consisting of a helix and an encircled loop within the PB2 domain. By electrostatic analysis, we identified a highly basic groove along with this ϕ loop and found that lysine 627 is located in the ϕ loop. A PB2 domain mutant in which glutamic acid is substituted at position 627 shows significantly lower RNA binding activity. This is the first report to show a relationship between RNA binding activity and the pathogenicity-determinant lysine 627. Using the Matras program for protein three-dimensional structural comparisons, we further found that the helix bundles in the PB2 domain are similar to that of activator 1, the 40-kDa subunit of DNA replication clamp loader (replication factor C), which is also an RNA-binding protein. This suggests a functional and structural relationship between the RNA-binding mechanisms underlying both influenza A viral transcription and cellular DNA replication. Our present results thus provide important new information for developing novel drugs that target the primer/promoter RNA binding of viral RNA polymerases.

Crystal structure of human D-amino acid oxidase : Context-dependent variability of the backbone conformation of the VAAGL hydrophobic stretch located at the si-face of the flavin ring

In the brain, the extensively studied FAD‐dependent enzyme D‐amino acid oxidase (DAO) degrades the gliotransmitter D‐serine, a potent activator of N‐methyl‐D‐aspartate type glutamate receptors, and evidence suggests that DAO, together with its activator G72 protein, may play a key role in the pathophysiology of schizophrenia. Indeed, its potential clinical importance highlights the need for structural and functional analyses of human DAO. We recently succeeded in purifying human DAO, and found that it weakly binds FAD and shows a significant slower rate of flavin reduction compared with porcine DAO. However, the molecular basis for the different kinetic features remains unclear because the active site of human DAO was considered to be virtually identical to that of porcine DAO, as would be expected from the 85% sequence identity. To address this issue, we determined the crystal structure of human DAO in complex with a competitive inhibitor benzoate, at a resolution of 2.5 Å. The overall dimeric structure of human DAO is similar to porcine DAO, and the catalytic residues are fully conserved at the re‐face of the flavin ring. However, at the si‐face of the flavin ring, despite the strict sequence identity, a hydrophobic stretch (residues 47–51, VAAGL) exists in a significantly different conformation compared with both of the independently determined porcine DAO–benzoate structures. This suggests that a context‐dependent conformational variability of the hydrophobic stretch accounts for the low affinity for FAD as well as the slower rate of flavin reduction, thus highlighting the unique features of the human enzyme.

Structural basis of the sphingomyelin phosphodiesterase activity in neutral sphingomyelinase from Bacillus cereus

Sphingomyelinase (SMase) from Bacillus cereus (Bc-SMase) hydrolyzes sphingomyelin to phosphocholine and ceramide in a divalent metal ion-dependent manner. Bc-SMase is a homologue of mammalian neutral SMase (nSMase) and mimics the actions of the endogenous mammalian nSMase in causing differentiation, development, aging, and apoptosis. Thus Bc-SMase may be a good model for the poorly characterized mammalian nSMase. The metal ion activation of sphingomyelinase activity of Bc-SMase was in the order Co2+ ≥ Mn2+ ≥ Mg2+ » Ca2+ ≥ Sr2+. The first crystal structures of Bc-SMase bound to Co2+, Mg2+, or Ca2+ were determined. The water-bridged double divalent metal ions at the center of the cleft in both the Co2+- and Mg2+-bound forms were concluded to be the catalytic architecture required for sphingomyelinase activity. In contrast, the architecture of Ca2+ binding at the site showed only one binding site. A further single metal-binding site exists at one side edge of the cleft. Based on the highly conserved nature of the residues of the binding sites, the crystal structure of Bc-SMase with bound Mg2+ or Co2+ may provide a common structural framework applicable to phosphohydrolases belonging to the DNase I-like folding superfamily. In addition, the structural features and site-directed mutagenesis suggest that the specific β-hairpin with the aromatic amino acid residues participates in binding to the membrane-bound sphingomyelin substrate.

The calcium-binding property of equine lysozyme

It was found that equine lysozyme binds one Ca2+. It was eluted with equimolar Ca2+ from a Bio‐Gel P‐4 column. Human lysozyme did not behave similarly. Equine lysozyme is concluded to be a calcium metalloprotein like α‐lactalbumin, which is a homologue of hen egg white lysozyme.

Clostridium perfringens Iota-Toxin: Structure and Function

Clostridium perfringens iota-toxin is composed of the enzyme component (Ia) and the binding component (Ib). Ib binds to receptor on targeted cells and translocates Ia into the cytosol of the cells. Ia ADP-ribosylates actin, resulting in cell rounding and death. Comparisons of the deduced amino acid sequence from the gene and three-dimensional structure of Ia with those of ADP-ribosylating toxins (ARTs) suggests that there is striking structural similarity among these toxins. Our objectives are to review the recent advances in the character, structure-function, and the mode of action of iota-toxin by consideration of the findings about ARTs.

Sequential Aldol Condensation Catalyzed by Hyperthermophilic 2-Deoxy-d-Ribose-5-Phosphate Aldolase

ABSTRACT Genes encoding 2-deoxy-d-ribose-5-phosphate aldolase (DERA) homologues from two hyperthermophiles, the archaeon Pyrobaculum aerophilum and the bacterium Thermotoga maritima, were expressed individually in Escherichia coli, after which the structures and activities of the enzymes produced were characterized and compared with those of E. coli DERA. To our surprise, the two hyperthermophilic DERAs showed much greater catalysis of sequential aldol condensation using three acetaldehydes as substrates than the E. coli enzyme, even at a low temperature (25°C), although both enzymes showed much less 2-deoxy-d-ribose-5-phosphate synthetic activity. Both the enzymes were highly resistant to high concentrations of acetaldehyde and retained about 50% of their initial activities after a 20-h exposure to 300 mM acetaldehyde at 25°C, whereas the E. coli DERA was almost completely inactivated after a 2-h exposure under the same conditions. The structure of the P. aerophilum DERA was determined by X-ray crystallography to a resolution of 2.0 Å. The main chain coordinate of the P. aerophilum enzyme monomer was quite similar to those of the T. maritima and E. coli enzymes, whose crystal structures have already been solved. However, the quaternary structure of the hyperthermophilic enzymes was totally different from that of the E. coli DERA. The areas of the subunit-subunit interface in the dimer of the hyperthermophilic enzymes are much larger than that of the E. coli enzyme. This promotes the formation of the unique dimeric structure and strengthens the hydrophobic intersubunit interactions. These structural features are considered responsible for the extremely high stability of the hyperthermophilic DERAs.