Tzu-Ping Ko

The crystal structure of the DNase domain of colicin E7 in complex with its inhibitor Im7 protein

BACKGROUND Colicin E7 (ColE7) is one of the bacterial toxins classified as a DNase-type E-group colicin. The cytotoxic activity of a colicin in a colicin-producing cell can be counteracted by binding of the colicin to a highly specific immunity protein. This biological event is a good model system for the investigation of protein recognition. RESULTS The crystal structure of a one-to-one complex between the DNase domain of colicin E7 and its cognate immunity protein Im7 has been determined at 2.3 A resolution. Im7 in the complex is a varied four-helix bundle that is identical to the structure previously determined for uncomplexed Im7. The structure of the DNase domain of ColE7 displays a novel alpha/beta fold and contains a Zn2+ ion bound to three histidine residues and one water molecule in a distorted tetrahedron geometry. Im7 has a V-shaped structure, extending two arms to clamp the DNase domain of ColE7. One arm (alpha1(*)-loop12-alpha2(*); where * represents helices in Im7) is located in the region that displays the greatest sequence variation among members of the immunity proteins in the same subfamily. This arm mainly uses acidic sidechains to interact with the basic sidechains in the DNase domain of ColE7. The other arm (loop 23-alpha3(*)-loop 34) is more conserved and it interacts not only with the sidechain but also with the mainchain atoms of the DNase domain of ColE7. CONCLUSIONS The protein interfaces between the DNase domain of ColE7 and Im7 are charge-complementary and charge interactions contribute significantly to the tight and specific binding between the two proteins. The more variable arm in Im7 dominates the binding specificity of the immunity protein to its cognate colicin. Biological and structural data suggest that the DNase active site for ColE7 is probably near the metal-binding site.

The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin.

Significance Since 2009, an emergent shrimp disease, acute hepatopancreatic necrosis disease (AHPND), has been causing global losses to the shrimp farming industry. The causative agent of AHPND is a specific strain of Vibrio parahaemolyticus. We present evidence here that the opportunistic V. parahaemolyticus becomes highly virulent by acquiring a unique AHPND-associated plasmid. This virulence plasmid, which encodes a binary toxin [V. parahaemolyticus Photorhabdus insect-related toxins (PirAvp and PirBvp)] that induces cell death, is stably inherited via a postsegregational killing system and disseminated by conjugative transfer. The cytotoxicity of the PirAvp/PirBvp system is analogous to the structurally similar insecticidal pore-forming Cry toxin. These findings will significantly increase our understanding of this emerging disease, which is essential for developing anti-AHPND measures. Acute hepatopancreatic necrosis disease (AHPND) is a severe, newly emergent penaeid shrimp disease caused by Vibrio parahaemolyticus that has already led to tremendous losses in the cultured shrimp industry. Until now, its disease-causing mechanism has remained unclear. Here we show that an AHPND-causing strain of V. parahaemolyticus contains a 70-kbp plasmid (pVA1) with a postsegregational killing system, and that the ability to cause disease is abolished by the natural absence or experimental deletion of the plasmid-encoded homologs of the Photorhabdus insect-related (Pir) toxins PirA and PirB. We determined the crystal structure of the V. parahaemolyticus PirA and PirB (PirAvp and PirBvp) proteins and found that the overall structural topology of PirAvp/PirBvp is very similar to that of the Bacillus Cry insecticidal toxin-like proteins, despite the low sequence identity (<10%). This structural similarity suggests that the putative PirABvp heterodimer might emulate the functional domains of the Cry protein, and in particular its pore-forming activity. The gene organization of pVA1 further suggested that pirABvp may be lost or acquired by horizontal gene transfer via transposition or homologous recombination.

Bisphosphonates target multiple sites in both cis- and trans-prenyltransferases

Bisphosphonate drugs (e.g., Fosamax and Zometa) are thought to act primarily by inhibiting farnesyl diphosphate synthase (FPPS), resulting in decreased prenylation of small GTPases. Here, we show that some bisphosphonates can also inhibit geranylgeranyl diphosphate synthase (GGPPS), as well as undecaprenyl diphosphate synthase (UPPS), a cis-prenyltransferase of interest as a target for antibacterial therapy. Our results on GGPPS (10 structures) show that there are three bisphosphonate-binding sites, consisting of FPP or isopentenyl diphosphate substrate-binding sites together with a GGPP product- or inhibitor-binding site. In UPPS, there are a total of four binding sites (in five structures). These results are of general interest because they provide the first structures of GGPPS- and UPPS-inhibitor complexes, potentially important drug targets, in addition to revealing a remarkably broad spectrum of binding modes not seen in FPPS inhibition.

Structure and mechanism of Helicobacter pylori fucosyltransferase. A basis for lipopolysaccharide variation and inhibitor design.

Helicobacter pylori α1,3-fucosyltransferase (FucT) is involved in catalysis to produce the Lewis x trisaccharide, the major component of the bacterias lipopolysaccharides, which has been suggested to mimic the surface sugars in gastric epithelium to escape host immune surveillance. We report here three x-ray crystal structures of FucT, including the FucT·GDP-fucose and FucT·GDP complexes. The protein structure is typical of the glycosyltransferase-B family despite little sequence homology. We identified a number of catalytically important residues, including Glu-95, which serves as the general base, and Glu-249, which stabilizes the developing oxonium ion during catalysis. The residues Arg-195, Tyr-246, Glu-249, and Lys-250 serve to interact with the donor substrate, GDP-fucose. Variations in the protein and ligand conformations, as well as a possible FucT dimer, were also observed. We propose a catalytic mechanism and a model of polysaccharide binding not only to explain the observed variations in H. pylori lipopolysaccharides, but also to facilitate the development of potent inhibitors.

Structures of three crystal forms of the sweet protein thaumatin.

Three crystal forms of the sweet-tasting protein thaumatin from the African berry Thaumatococcus daniellii have been grown. These include two naturally occurring isoforms, A and B, that differ by a single amino acid, and a recombinant form of isoform B expressed in yeast. The crystals are of space groups C2 with a = 117.7, b = 44.9, c = 38.0 A, and beta = 94.0 degrees, P2(1)2(1)2(1) with a = 44.3, b = 63.7 and c = 72.7 A, and a tetragonal form P4(1)2(1)2 with a = b = 58.6 and c = 151.8 A. The structures of all three crystals have been solved by molecular replacement and subsequently refined to R factors of 0.184 for the monoclinic at 2.6 A, 0.165 for the orthorhombic at 1.75 A, and 0.181 for the tetragonal, also at 1.75 A resolution. No solvent was included in the monoclinic crystal while 123 and 105 water molecules were included in the higher resolution orthorhombic and tetragonal structures, respectively. A bound tartrate molecule was also clearly visible in the tetragonal structure. The r.m.s. deviations between molecular structures in the three crystals range from 0.6 to 0.7 A for Calpha atoms, and 1.1 to 1.3 A for all atoms. This is comparable to the r.m.s. deviation between the three structures and the starting model. Nevertheless, several peptide loops show particularly large variations from the initial model.

Crystal Structure of Type-III Geranylgeranyl Pyrophosphate Synthase from Saccharomyces cerevisiae and the Mechanism of Product Chain Length Determination

Geranylgeranyl pyrophosphate synthase (GGPPs) catalyzes a condensation reaction of farnesyl pyrophosphate with isopentenyl pyrophosphate to generate C20 geranylgeranyl pyrophosphate, which is a precursor for carotenoids, chlorophylls, geranylgeranylated proteins, and archaeal ether-linked lipid. For short-chain trans-prenyltransferases that synthesize C10-C25 products, bulky amino acid residues generally occupy the fourth or fifth position upstream from the first DDXXD motif to block further elongation of the final products. However, the short-chain type-III GGPPs in eukaryotes lack any large amino acid at these positions. In this study, the first structure of type-III GGPPs from Saccharomyces cerevisiae has been determined to 1.98 Å resolution. The structure is composed entirely of 15 α-helices joined by connecting loops and is arranged with α-helices around a large central cavity. Distinct from other known structures of trans-prenyltransferases, the N-terminal 17 amino acids (9-amino acid helix A and the following loop) of this GGPPs protrude from the helix core into the other subunit and contribute to the tight dimer formation. Deletion of the first 9 or 17 amino acids caused the dissociation of dimer into monomer, and the Δ(1-17) mutant showed abolished enzyme activity. In each subunit, an elongated hydrophobic crevice surrounded by D, F, G, H, and I α-helices contains two DDXXD motifs at the top for substrate binding with one Mg2+ coordinated by Asp75, Asp79, and four water molecules. It is sealed at the bottom with three large residues of Tyr107, Phe108, and His139. Compared with the major product C30 synthesized by mutant H139A, the products generated by mutant Y107A and F108A are predominantly C40 and C30, respectively, suggesting the most important role of Tyr107 in determining the product chain length.

Structure of a heterotetrameric geranyl pyrophosphate synthase from mint (Mentha piperita) reveals intersubunit regulation

This work presents the crystal structure of mint heteromeric prenyltransferase, which is responsible for the biosynthesis of geranyl pyrophosphate, a precursor of the monoterpene menthol. By combining biochemical and genetic complementation approaches, the authors show that the molecular mechanism regulating specific product formation is mediated by protein–protein interactions. Terpenes (isoprenoids), derived from isoprenyl pyrophosphates, are versatile natural compounds that act as metabolism mediators, plant volatiles, and ecological communicators. Divergent evolution of homomeric prenyltransferases (PTSs) has allowed PTSs to optimize their active-site pockets to achieve catalytic fidelity and diversity. Little is known about heteromeric PTSs, particularly the mechanisms regulating formation of specific products. Here, we report the crystal structure of the (LSU · SSU)2-type (LSU/SSU = large/small subunit) heterotetrameric geranyl pyrophosphate synthase (GPPS) from mint (Mentha piperita). The LSU and SSU of mint GPPS are responsible for catalysis and regulation, respectively, and this SSU lacks the essential catalytic amino acid residues found in LSU and other PTSs. Whereas no activity was detected for individually expressed LSU or SSU, the intact (LSU · SSU)2 tetramer produced not only C10-GPP at the beginning of the reaction but also C20-GGPP (geranylgeranyl pyrophosphate) at longer reaction times. The activity for synthesizing C10-GPP and C20-GGPP, but not C15-farnesyl pyrophosphate, reflects a conserved active-site structure of the LSU and the closely related mustard (Sinapis alba) homodimeric GGPPS. Furthermore, using a genetic complementation system, we showed that no C20-GGPP is produced by the mint GPPS in vivo. Presumably through protein–protein interactions, the SSU remodels the active-site cavity of LSU for synthesizing C10-GPP, the precursor of volatile C10-monoterpenes.

White spot syndrome virus protein ICP11: A histone-binding DNA mimic that disrupts nucleosome assembly.

White spot syndrome virus (WSSV) is a large (≈300 kbp), double-stranded DNA eukaryotic virus that has caused serious disease in crustaceans worldwide. ICP11 is the most highly expressed WSSV nonstructural gene/protein, which strongly suggests its importance in WSSV infection; but until now, its function has remained obscure. We show here that ICP11 acts as a DNA mimic. In crystal, ICP11 formed a polymer of dimers with 2 rows of negatively charged spots that approximated the duplex arrangement of the phosphate groups in DNA. Functionally, ICP11 prevented DNA from binding to histone proteins H2A, H2B, H3, and H2A.x, and in hemocytes from WSSV-infected shrimp, ICP11 colocalized with histone H3 and activated-H2A.x. These observations together suggest that ICP11 might interfere with nucleosome assembly and prevent H2A.x from fulfilling its critical function of repairing DNA double strand breaks. Therefore, ICP11 possesses a functionality that is unique among the handful of presently known DNA mimic proteins.

Structure and properties of recombinant human pyridoxine 5'-phosphate oxidase

Pyridoxine 5′‐phosphate oxidase catalyzes the terminal step in the synthesis of pyridoxal 5′‐phosphate. The cDNA for the human enzyme has been cloned and expressed in Escherichia coli. The purified human enzyme is a homodimer that exhibits a low catalytic rate constant of ∼0.2 sec−1 and Km values in the low micromolar range for both pyridoxine 5′phosphate and pyridoxamine 5′‐phosphate. Pyridoxal 5′‐phosphate is an effective product inhibitor. The three‐dimensional fold of the human enzyme is very similar to those of the E. coli and yeast enzymes. The human and E. coli enzymes share 39% sequence identity, but the binding sites for the tightly bound FMN and substrate are highly conserved. As observed with the E. coli enzyme, the human enzyme binds one molecule of pyridoxal 5′‐phosphate tightly on each subunit.

Crystal Structures of the BlaI Repressor from Staphylococcus aureus and Its Complex with DNA: Insights into Transcriptional Regulation of the bla and mec Operons

The 14-kDa BlaI protein represses the transcription of blaZ, the gene encoding beta-lactamase. It is homologous to MecI, which regulates the expression of mecA, the gene encoding the penicillin binding protein PBP2a. These genes mediate resistance to beta-lactam antibiotics in staphylococci. Both repressors can bind either bla or mec DNA promoter-operator sequences. Regulated resistance genes are activated via receptor-mediated cleavage of the repressors. Cleavage is induced when beta-lactam antibiotics bind the extramembrane sensor of the sensor-transducer signaling molecules, BlaR1 or MecR1. The crystal structures of BlaI from Staphylococcus aureus, both in free form and in complex with 32 bp of DNA of the mec operator, have been determined to 2.0- and 2.7-A resolutions, respectively. The structure of MecI, also in free form and in complex with the bla operator, has been previously reported. Both repressors form homodimers, with each monomer composed of an N-terminal DNA binding domain of winged helix-turn-helix topology and a C-terminal dimerization domain. The structure of BlaI in complex with the mec operator shows a protein-DNA interface that is conserved between both mec and bla targets. The recognition helix alpha3 interacts specifically with the conserved TACA/TGTA DNA binding motif. BlaI and, probably, MecI dimers bind to opposite faces of the mec DNA double helix in an up-and-down arrangement, whereas MecI and, probably, BlaI dimers bind to the same DNA face of bla promoter-operator DNA. This is due to the different spacing of mec and bla DNA binding sites. Furthermore, the flexibility of the dimeric proteins may make the C-terminal proteolytic cleavage site more accessible when the repressors are bound to DNA than when they are in solution, suggesting that the induction cascade involves bound rather than free repressor.