Kei Wada

The Asymmetric Trimeric Architecture of [2Fe-2S] IscU : Implications for Its Scaffolding during Iron-Sulfur Cluster Biosynthesis

IscU is a key component of the ISC machinery and is involved in the biogenesis of iron-sulfur (Fe-S) proteins. IscU serves as a scaffold for assembly of a nascent Fe-S cluster prior to its delivery to an apo protein. Here, we report the first crystal structure of IscU with a bound [2Fe-2S] cluster from the hyperthermophilic bacterium Aquifex aeolicus, determined at a resolution of 2.3 A, using multiwavelength anomalous diffraction of the cluster. The holo IscU formed a novel asymmetric trimer that harbored only one [2Fe-2S] cluster. One iron atom of the cluster was coordinated by the S(gamma) atom of Cys36 and the N(epsilon) atom of His106, and the other was coordinated by the S(gamma) atoms of Cys63 and Cys107 on the surface of just one of the protomers. However, the cluster was buried inside the trimer between the neighboring protomers. The three protomers were conformationally distinct from one another and associated around a noncrystallographic pseudo-3-fold axis. The three flexible loop regions carrying the ligand-binding residues (Cys36, Cys63, His106 and Cys107) and the N-terminal alpha1 helices were positioned at the interfaces and underwent substantial conformational rearrangement, which stabilized the association of the asymmetric trimer. This unique trimeric A. aeolicus holo-IscU architecture was clearly distinct from other known monomeric apo-IscU/SufU structures, indicating that asymmetric trimer organization, as well as its association/dissociation, would be involved in the scaffolding function of IscU.

Crystal Structure of the γ-Glutamyltranspeptidase Precursor Protein from Escherichia coli STRUCTURAL CHANGES UPON AUTOCATALYTIC PROCESSING AND IMPLICATIONS FOR THE MATURATION MECHANISM

γ-Glutamyltranspeptidase (GGT) is an extracellular enzyme that plays a key role in glutathione metabolism. The mature GGT is a heterodimer consisting of L- and S-subunits that is generated by posttranslational cleavage of the peptide bond between Gln-390 and Thr-391 in the precursor protein. Thr-391, which becomes the N-terminal residue of the S-subunit, acts as the active residue in the catalytic reaction. The crystal structure of a mutant GGT, T391A, that is unable to undergo autocatalytic processing, has been determined at 2.55-Å resolution. Structural comparison of the precursor protein and mature GGT demonstrates that the structures of the core regions in the two proteins are unchanged, but marked differences are found near the active site. In particular, in the precursor, the segment corresponding to the C-terminal region of the L-subunit occupies the site where the loop (residues 438–449) forms the lid of the γ-glutamyl group-binding pocket in the mature GGT. This result demonstrates that, upon cleavage of the N-terminal peptide bond of Thr-391, the newly produced C terminus (residues 375–390) flips out, allowing the 438–449 segment to form the γ-glutamyl group-binding pocket. The electron density map for the T391A protein also identified a water molecule near the carbonyl carbon atom of Gln-390. The spatial arrangement around the water and Thr-391 relative to the scissile peptide bond appears suitable for the initiation of autocatalytic processing, as in other members of the N-terminal nucleophile hydrolase superfamily.

Crystal structure of the halotolerant γ-glutamyltranspeptidase from Bacillus subtilis in complex with glutamate reveals a unique architecture of the solvent-exposed catalytic pocket

γ‐Glutamyltranspeptidase (GGT; EC 2.3.2.2), an enzyme found in organisms from bacteria to mammals and plants, plays a central role in glutathione metabolism. Structural studies of GGTs from Escherichia coli and Helicobacter pylori have revealed detailed molecular mechanisms of catalysis and maturation. In these two GGTs, highly conserved residues form the catalytic pockets, conferring the ability of the loop segment to shield the bound γ‐glutamyl moiety from the solvent. Here, we have examined the Bacillus subtilis GGT, which apparently lacks the amino acids corresponding to the lid‐loop that are present in mammalian and plant GGTs as well as in most bacterial GGTs. Another remarkable feature of B. subtilis GGT is its salt tolerance; it retains 86% of its activity even in 3 m NaCl. To better understand these characteristics of B. subtilis GGT, we determined its crystal structure in complex with glutamate, a product of the enzymatic reaction, at 1.95 Å resolution. This structure revealed that, unlike the E. coli and H. pylori GGTs, the catalytic pocket of B. subtilis GGT has no segment that covers the bound glutamate; consequently, the glutamate is exposed to solvent. Furthermore, calculation of the electrostatic potential showed that strong acidic patches were distributed on the surface of the B. subtilis GGT, even under high‐salt conditions, and this may allow the protein to remain in the hydrated state and avoid self‐aggregation. The structural findings presented here have implications for the molecular mechanism of GGT.

Crystal structure of Escherichia coli SufA involved in biosynthesis of iron-sulfur clusters : Implications for a functional dimer

IscA and SufA are paralogous proteins that play crucial roles in the biosynthesis of Fe–S clusters, perhaps through a mechanism involving transient Fe–S cluster formation. We have determined the crystal structure of E. coli SufA at 2.7 Å resolution. SufA exists as a homodimer, in contrast to the tetrameric organization of IscA. Furthermore, a C‐terminal segment containing two essential cysteine residues (Cys‐Gly‐Cys), which is disordered in the IscA structure, is clearly visible in one molecule (the α1 subunit) of the SufA homodimer. Although this segment is disordered in the other molecule (the α2 subunit), computer modeling of this segment based on the well‐defined conformation of α1 subunit suggests that the four cysteine residues (Cys114 and Cys116 in each subunit) in the Cys‐Gly‐Cys motif are positioned in close proximity at the dimer interface. The arrangement of these cysteines together with the nearby Glu118 in SufA dimer may allow coordination of an Fe–S cluster and/or an Fe atom.

Crystallization and preliminary X-ray diffraction studies of catalase-peroxidase from Synechococcus PCC 7942

The recombinant catalase-peroxidase of Synechococcus PCC 7942 overexpressed in Escherichia coli was purified and crystallized by the hanging-drop vapour-diffusion method using sodium formate as a precipitant. The crystals belonged to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 109.3, c = 202.0 A. The calculated V(M) value based on a dimer in the asymmetric unit was 1.9 A(3) Da(-1). A native data set was collected to 2.3 A resolution from a frozen crystal using synchrotron radiation at SPring-8.

Crystal Structures of Escherichia coli γ-Glutamyltranspeptidase in Complex with Azaserine and Acivicin: Novel Mechanistic Implication for Inhibition by Glutamine Antagonists

gamma-Glutamyltranspeptidase (GGT) catalyzes the cleavage of such gamma-glutamyl compounds as glutathione, and the transfer of their gamma-glutamyl group to water or to other amino acids and peptides. GGT is involved in a number of biological phenomena such as drug resistance and metastasis of cancer cells by detoxification of xenobiotics. Azaserine and acivicin are classical and irreversible inhibitors of GGT, but their binding sites and the inhibition mechanisms remain to be defined. We have determined the crystal structures of GGT from Escherichia coli in complex with azaserine and acivicin at 1.65 A resolution. Both inhibitors are bound to GGT at its substrate-binding pocket in a manner similar to that observed previously with the gamma-glutamyl-enzyme intermediate. They form a covalent bond with the O(gamma) atom of Thr391, the catalytic residue of GGT. Their alpha-carboxy and alpha-amino groups are recognized by extensive hydrogen bonding and charge interactions with the residues that are conserved among GGT orthologs. The two amido nitrogen atoms of Gly483 and Gly484, which form the oxyanion hole, interact with the inhibitors directly or via a water molecule. Notably, in the azaserine complex the carbon atom that forms a covalent bond with Thr391 is sp(3)-hybridized, suggesting that the carbonyl of azaserine is attacked by Thr391 to form a tetrahedral intermediate, which is stabilized by the oxyanion hole. Furthermore, when acivicin is bound to GGT, a migration of the single and double bonds occurs in its dihydroisoxazole ring. The structural characteristics presented here imply that the unprecedented binding modes of azaserine and acivicin are conserved in all GGTs from bacteria to mammals and give a new insight into the inhibition mechanism of glutamine amidotransferases by these glutamine antagonists.

Crystal structure of Escherichia coli SufC, an ABC‐type ATPase component of the SUF iron–sulfur cluster assembly machinery

SufC is an ATPase component of the SUF machinery, which is involved in the biosynthesis of Fe–S clusters. To gain insight into the function of this protein, we have determined the crystal structure of Escherichia coli SufC at 2.5 Å resolution. Despite the similarity of the overall structure with ABC‐ATPases (nucleotide‐binding domains of ABC transporters), some key differences were observed. Glu171, an invariant residue involved in ATP hydrolysis, is rotated away from the nucleotide‐binding pocket to form a SufC‐specific salt bridge with Lys152. Due to this salt bridge, D‐loop that follows Glu171 is flipped out to the molecular surface, which may sterically inhibit the formation of an active dimer. Thus, the salt bridge may play a critical role in regulating ATPase activity and preventing wasteful ATP hydrolysis. Furthermore, SufC has a unique Q‐loop structure on its surface, which may form a binding site for its partner proteins, SufB and/or SufD.

Structural basis for the electron transfer from an open form of NADPH-cytochrome P450 oxidoreductase to heme oxygenase

Significance Heme oxygenase (HO) is a key enzyme for heme degradation that is deeply involved in iron homeostasis, defensive reaction against oxidative stress, and signal transduction mediated by carbon monoxide. To complete a single HO reaction, seven electrons supplied from NADPH-cytochrome P450 reductase (CPR) are required. Based on crystallography, X-ray scattering, and NMR analyses of CPR, it has been proposed that CPR has a dynamic equilibrium of the “closed-open transition.” The crystal structure of the transient complex of CPR with heme-bound HO clearly demonstrated that it is the open form of CPR that can interact with and transfer electrons to heme-bound HO. Moreover, the complex structure provides a scaffold to research the protein–protein interactions between CPR and other redox partners. NADPH-cytochrome P450 oxidoreductase (CPR) supplies electrons to various heme proteins including heme oxygenase (HO), which is a key enzyme for heme degradation. Electrons from NADPH flow first to flavin adenine dinucleotide, then to flavin mononucleotide (FMN), and finally to heme in the redox partner. For electron transfer from CPR to its redox partner, the ‘‘closed-open transition’’ of CPR is indispensable. Here, we demonstrate that a hinge-shortened CPR variant, which favors an open conformation, makes a stable complex with heme–HO-1 and can support the HO reaction, although its efficiency is extremely limited. Furthermore, we determined the crystal structure of the CPR variant in complex with heme–HO-1 at 4.3-Å resolution. The crystal structure of a complex of CPR and its redox partner was previously unidentified. The distance between heme and FMN in this complex (6 Å) implies direct electron transfer from FMN to heme.

Molecular Dynamism of Fe–S Cluster Biosynthesis Implicated by the Structure of the SufC2–SufD2 Complex

Maturation of iron-sulfur (Fe-S) proteins is achieved by the SUF machinery in a wide number of eubacteria and archaea, as well as eukaryotic chloroplasts. This machinery is encoded in Escherichia coli by the sufABCDSE operon, where three Suf components, SufB, SufC, and SufD, form a complex and appear to provide an intermediary site for the Fe-S cluster assembly. Here, we report the quaternary structure of the SufC(2)-SufD(2) complex in which SufC is bound to the C-terminal domain of SufD. Comparison with the monomeric structure of SufC revealed conformational change of the active-site residues: SufC becomes competent for ATP binding and hydrolysis upon association with SufD. The two SufC subunits were spatially separated in the SufC(2)-SufD(2) complex, whereas cross-linking experiments in solution have indicated that two SufC molecules associate with each other in the presence of Mg(2+) and ATP. Such dimer formation of SufC may lead to a gross structural change of the SufC(2)-SufD(2) complex. Furthermore, genetic analysis of SufD revealed an essential histidine residue buried inside the dimer interface, suggesting that conformational change may expose this crucial residue. These findings, together with biochemical characterization of the SufB-SufC-SufD complex, have led us to propose a model for the Fe-S cluster biosynthesis in the complex.

Functional Dynamics Revealed by the Structure of the SufBCD Complex, a Novel ATP-binding Cassette (ABC) Protein That Serves as a Scaffold for Iron-Sulfur Cluster Biogenesis

ATP-binding cassette (ABC)-type ATPases are chemomechanical engines involved in diverse biological pathways. Recent genomic information reveals that ABC ATPase domains/subunits act not only in ABC transporters and structural maintenance of chromosome proteins, but also in iron-sulfur (Fe-S) cluster biogenesis. A novel type of ABC protein, the SufBCD complex, functions in the biosynthesis of nascent Fe-S clusters in almost all Eubacteria and Archaea, as well as eukaryotic chloroplasts. In this study, we determined the first crystal structure of the Escherichia coli SufBCD complex, which exhibits the common architecture of ABC proteins: two ABC ATPase components (SufC) with function-specific components (SufB-SufD protomers). Biochemical and physiological analyses based on this structure provided critical insights into Fe-S cluster assembly and revealed a dynamic conformational change driven by ABC ATPase activity. We propose a molecular mechanism for the biogenesis of the Fe-S cluster in the SufBCD complex.