Rational Strategy for Designing Peptidomimetic Small Molecules Based on Cyclic Peptides Targeting Protein-Protein Interaction between CTLA-4 and B7-1.

Currently, various pharmaceutical modalities are being developed rapidly. Targeting protein-protein interactions (PPIs) is an important objective in such development. Cyclic peptides, because they have good specificity and activity, have been attracting much attention as an alternative to antibody drugs. However, cyclic peptides involve some difficulties, such as oral availability and cell permeability. Therefore, while small-molecule drugs still present many benefits, the screening of functional small-molecule compounds targeting PPIs requires a great deal of time and effort, including structural analysis of targets and hits. In this study, we investigated a rational two-step strategy to design small-molecule compounds targeting PPIs. First, we obtained inhibitory cyclic peptides that bind to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) by ribosomal display using PUREfrex® (PUREfrex®RD) to get structure-activity relation (SAR) information. Based on that information, we converted cyclic peptides to small molecules using PepMetics® scaffolds that can mimic the α-helix or ß-turn of the peptide. Finally, we succeeded in generating small-molecule compounds with good IC50 (single-digit µM values) against CTLA-4. This strategy is expected to be a useful approach for small-molecule design targeting PPIs, even without having structural information such as that associated with X-ray crystal structures.

Adverse birth outcomes associated with maternal smoking and polymorphisms in the N-Nitrosamine-metabolizing enzyme genes NQO1 and CYP2E1.

Maternal smoking during pregnancy can result in both pregnancy complications and reduced size of the fetus and neonate. Among women who smoke, genetic susceptibility to tobacco smoke also is a likely causative factor in adverse pregnancy outcomes. A prospective cohort study was conducted among 460 pregnant women who delivered live singletons in Sapporo, Japan, from 2002 to 2005. Multiple linear regression models were used to estimate associations of maternal smoking and polymorphisms in two genes encoding N-nitrosamine-metabolizing enzymes-NAD(P)H: quinone oxidoreductase 1 (NQO1) and cytochrome P-450 2E1 (CYP2E1)-with birth size. Among infants born to smokers with the NQO1 homozygous wild-type allele, birth weight, birth length, and birth head circumference were significantly reduced (p < 0.01 for each factor). For the homozygous wild-type CYP2E1 allele, birth weight was lower by an estimated 195 g (standard error, 55; p < 0.001) among smokers. These genotypes did not confer adverse effects among women who had never smoked or who quit smoking during the first trimester. The adverse effects of maternal smoking on infant birth size may be modified by maternal genetic polymorphisms in N-nitrosamine-metabolizing enzymes among Japanese subjects. These results may help in directing smoking cessation interventions during pregnancy, especially among susceptible women.

Thermodynamic basis for the stabilities of three CutA1s from Pyrococcus horikoshii,Thermus thermophilus, and Oryza sativa, with unusually high denaturation temperatures.

In order to elucidate the stabilization mechanism of CutA1 from Pyrococcus horikoshii (PhCutA1) with a denaturation temperature of nearly 150 degrees C, GuHCl denaturation and heat denaturation were examined at neutral and acidic pHs. As a comparison, CutA1 proteins from Thermus thermophilus (TtCutA1) and Oryza sativa (OsCutA1) were also examined, which have lower optimum growth temperatures of 75 and 28 degrees C, respectively, than that (98 degrees C) of P. horikoshii. GuHCl-induced unfolding and refolding curves of the three proteins showed hysteresis effects due to an unusually slow unfolding rate. The midpoints of refolding for PhCutA1, TtCutA1 and OsCutA1 were 5.7 M, 3.3 M, and 2.3 M GuHCl, respectively, at pH 8.0 and 37 degrees C. DSC experiments with TtCutA1 and OsCutA1 showed that the denaturation temperatures were remarkably high, 112.8 and 97.3 degrees C, respectively, at pH 7.0 and that the good heat reversibility was amenable to thermodynamic analyses. At acidic pH, TtCutA1 showed higher stability to both heat and denaturant than PhCutA1. Combined with the data for DSC and denaturant denaturation, the unfolding Gibbs energy of PhCutA1 could be depicted as a function of temperature. It was experimentally revealed that (1) the unusually high stability of PhCutA1 basically originates from a common trimer structure of the three proteins, (2) the stability of PhCutA1 is superior to those of the other two CutA1s over all temperatures above 0 degrees C at neutral pH, due to the decrease in both enthalpy and entropy, and (3) ion pairs of PhCutA1 contribute to the unusually high stability at neutral pH.

EL5 is involved in root development as an anti-cell death ubiquitin ligase.

Ubiquitin ligase (E3) plays a central role in substrate recognition during ubiquitination, a post-translational modification of proteins. Rice EL5 is an E3 with a RING-H2 finger domain (RFD) and its transcript is upregulated by a chitin elicitor. The EL5-RFD has been intensively studied and demonstrated to exhibit E3 activity. Its three-dimensional structure was determined for the first time in plant E3, and the amino acid residues required for the interaction with the ubiquitin-conjugating enzyme (E2) were identified. Recent analyses revealed that EL5 plays a crucial role as an E3 in the maintenance of cell viability during root development in rice. In this addendum, we report that the EL5-RFD catalyzes polyubiquitination via the Lys48 residue of ubiquitin. We also discuss the possible role of EL5 as an anti-cell death enzyme. We hypothesize that EL5 might be responsible for mediating the degradation of cytotoxic proteins produced in root cells after the actions of phytohormones.

RING-H2 type ubiquitin ligase EL5 is involved in root development through the maintenance of cell viability in rice.

Rice EL5 is an ATL family gene characterized by a transmembrane domain at the N-terminal and a RING-H2 finger domain (RFD), which exhibits ubiquitin ligase (E3) activity. To elucidate the physiological roles of EL5, we analyzed transgenic rice plants overexpressing mutant EL5 proteins that are impaired in E3 activity to various degrees. Plants expressing EL5C153A and EL5W165A, which encode an inactive E3, showed a rootless phenotype accompanied by cell death in root primordia, and those expressing EL5V162A, with moderately impaired E3 activity, formed short crown roots with necrotic lateral roots. The dominant-negative phenotype was specifically observed in root meristems where EL5 is expressed, and not recovered by exogenous auxin. When wild-type EL5 was transcriptionally overexpressed, the EL5 protein was barely detected by Western blotting. Neither treatment with a proteasome inhibitor nor mutation of the sole lysine residue, a potential target of ubiquitination, resulted in increased EL5 accumulation, whereas mutations in the RFD led to increased EL5 accumulation. The stabilized EL5 without the RFD was localized in the plasma membrane. Deletion of the transmembrane domain prevented the EL5 from localizing in the membrane and from exerting an inhibitory effect on root formation. Deletion of the C-terminal region also neutralized the negative effect. We concluded that EL5 plays a major role as a membrane-anchored E3 for the maintenance of cell viability after the initiation of root primordial formation. In addition, we propose that EL5 is an unstable protein, of which degradation is regulated by the RFD in a proteasome-independent manner.

The cooperative role of OsCnfU-1A domain I and domain II in the iron sulphur cluster transfer process as revealed by NMR.

OsCnfU-1A is a chloroplast-type Nfu-like protein that consists of tandem repeats sharing high sequence homology. Domain I of this protein, but not domain II, has a C-X-X-C motif that is thought to assemble an iron-sulphur cluster. Herein we report the solution structure of OsCnfU-1A domain I (73-153). Although OsCnfU-1A domain I is structurally similar to OsCnfU-1A domain II (154-226), the electrostatic surface potential of the 2 domains differs. Domain I has an acidic surface, whereas that of domain II is predominantly basic. Chemical shift perturbation studies on OsCnfU-1A domain I and domain II with ferredoxin revealed negligible chemical shift changes in domain I, whereas much larger chemical shift changes were observed in domain II. The residues with larger chemical shift changes were located on the basic surface of domain II. Considering that ferredoxin is predominantly negatively charged, we propose the following hypothesis: First, an iron-sulphur cluster is assembled on domain I. Next, domain II interacts with the ferredoxin, thus tethering domain I close to the ferredoxin. Finally, domain I transfers the iron-sulphur cluster to the ferredoxin. Thus, domain II facilitates the efficient transfer of the iron-sulphur cluster from domain I to the ferredoxin.

The NMR structure of the domain II of a chloroplastic NifU-like protein OsNifU1A.

NifU-like proteins are a highly conserved protein that serves as the scaffold for assembly of Fe-S clusters. Chloroplastic NifU-like proteins have tandem NifU like domains, named domain I and domain II. Although the amino acid sequences of these domains are very similar to each other, the predicted functional region for the Fe-S cluster assembly, the CXXC motif, exists only in domain I. The structure of the domain II of chloroplastic NifU-like protein OsNifU1A has an alpha-beta sandwich structure containing two alpha helices located on one side of the beta-sheet. The electrostatic surface potential of OsNifU1A domain II is predominantly positively charged. Chloroplastic NifU-like proteins are targeted to ferredoxin for transferring the Fe-S cluster. The ferredoxin presents an overall negatively charged surface, which may evoke an electrostatic association with OsNifU1A domain II.

Identification of glycosylation genes and glycosylated amino acids of flagellin in Pseudomonas syringae pv. tabaci.

A glycosylation island is a genetic region required for glycosylation. The glycosylation island of flagellin in Pseudomonas syringae pv. tabaci 6605 consists of three orfs: orf1, orf2 and orf3. Orf1 and orf2 encode putative glycosyltransferases, and their deletion mutants, Deltaorf1 and Deltaorf2, exhibit deficient flagellin glycosylation or produce partially glycosylated flagellin respectively. Digestion of glycosylated flagellin from wild-type bacteria and non-glycosylated flagellin from Deltaorf1 mutant using aspartic N-peptidase and subsequent HPLC analysis revealed candidate glycosylated amino acids. By generation of site-directed Ser/Ala-substituted mutants, all glycosylated amino acid residues were identified at positions 143, 164, 176, 183, 193 and 201. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) analysis revealed that each glycan was about 540 Da. While all glycosylation-defective mutants retained swimming ability, swarming ability was reduced in the Deltaorf1, Deltaorf2 and Ser/Ala-substituted mutants. All glycosylation mutants were also found to be impaired in the ability to adhere to a polystyrene surface and in the ability to cause disease in tobacco. Based on the predicted tertiary structure of flagellin, S176 and S183 are expected to be located on most external surface of the flagellum. Thus the effect of Ala-substitution of these serines is stronger than that of other serines. These results suggest that glycosylation of flagellin in P. syringae pv. tabaci 6605 is required for bacterial virulence. It is also possible that glycosylation of flagellin may mask elicitor function of flagellin molecule.

Active site residues and amino acid specificity of the ubiquitin carrier protein-binding RING-H2 finger domain.

EL5 is a rice ubiquitin-protein isopeptide ligase (E3) containing a RING-H2 finger domain that interacts with Oryza sativa (Os) UBC5b, a rice ubiquitin carrier protein. We introduced point mutations into the EL5 RING-H2 finger so that residues that functionally interact with OsUBC5b could be identified when assayed for ubiquitination activity in vitro. The residue positions were selected based on the results of an EL5 RING-H2 finger/OsUBC5b NMR titration experiment. These RING-H2 finger residues form or are adjacent to a shallow groove that is recognized by OsUBC5b. The E3 activity of EL5 is shown to be dependent on a Trp located at the center of the groove. We classified rice RING fingers according to the type of metal-chelating motif, i.e. RING-H2 or RING-HC, and according to the presence or absence of a conserved EL5-like Trp. We discuss the probable relationship between E3 activity and the conserved Trp.

Refolding and purification of recombinant OsNifU1A domain II that was expressed by Escherichia coli.

OsNifU1A is a NifU-like rice (Oryza sativa) protein, discovered recently. Its amino acid sequence is very homologous to the sequence of cyanobacterial CnfU and to the sequences of NifU C-terminal domains. Based on its sequence, OsNifU1A is probably a modular structure consisting of two CnfU-like domains, with domain I (formed by residues Leu73 to Gly153) and domain II (formed by residues Leu154 to Ser226). Domain I have a conserved Cys-X-X-Cys motif, which may function as an iron-sulfur cluster assembly scaffold. Domain II lacks a Cys-X-X-Cys motif and therefore, cannot function analogously. Other NifU-like proteins, with sequences homologous to OsNifU1A domain II, have been identified during plant genomic projects; however, the biological roles of these domains remain unknown. We successfully constructed an Escherichia coli expression system for OsNifU1A domain II that enabled us to synthesize and purify milligram quantities of protein for use in structural and functional studies. Using the Gateway system, we built DNA sequences corresponding to two OsNifU1A domain II fusion proteins. One construct has a (His)6 sequence upstream of the OsNifU1A domain II sequence; the other has an upstream thioredoxin-(His)6 sequence. Recombinant OsNifU1A domain II fusion proteins were extracted from E. coli inclusion bodies by dissolving them in 6 M guanidine-HCl. About 36% of the total (His)6/OsNifU1A domain II fusion protein initially present remained soluble after guanidine-HCl was completely removed by step-wise dialysis; whereas, recovery of soluble Trx-(His)6 fusion protein was about 60% of the total cell lysate. About 2 mg of 15N-labeled OsNifU1A domain II was purified for NMR spectral studies. Examination of the OsNifU1A domain II 1H-15N HSQC NMR spectrum indicated that the purified protein was monomeric and correctly folded. Therefore, we established an efficient procedure for synthesis and purification of 15N-labeled OsNifU1A domain II in quantities sufficient for heteronuclear NMR solution structure studies.

Improving expression and solubility of rice proteins produced as fusion proteins in Escherichia coli.

For proteins of higher eukaryotes, such as plants, which have large genomes, recombinant protein expression and purification are often difficult. Expression levels tend to be low and the expressed proteins tend to misfold and aggregate. We tested seven different expression vectors in Escherichia coli for rapid subcloning of rice genes and for protein expression and solubility levels. Each expressed gene product has an N-terminal fusion protein and/or tag, and an engineered protease site upstream of the mature rice protein. Several different fusion proteins/tags and protease sites were tested. We found that the fusion proteins and the protease sites have significant and varying effects on expression and solubility levels. The expression vector with the most favorable characteristics is pDEST-trx. The vector, which is a modified version of the commercially available expression vector, pET-32a, contains an N-terminal thioredoxin fusion protein and a hexahistidine tag, and is adapted to the Gateway expression system. However, addition of an engineered protease site could drastically change the expression and solubility properties. We selected 135 genes corresponding to potentially interesting rice proteins, transferred the genes from cDNAs to expression vectors, and engineered in suitable protease sites N-terminal to the mature proteins. Of 135 genes, 131 (97.0%) could be expressed and 72 (53.3%) were soluble when the fusion proteins/tags were present. Thirty-eight mature-length rice proteins and domains (28.1%) are suitable for NMR solution structure studies and/or X-ray crystallography. Our expression systems are useful for the production of soluble plant proteins in E. coli to be used for structural genomics studies.

Improved expression, purification and crystallization of a putative N-acetyl-gamma-glutamyl-phosphate reductase from rice (Oryza sativa).

N-Acetyl-gamma-glutamyl-phosphate reductase (AGPR) catalyzes the third step in an eight-step arginine-biosynthetic pathway that starts with glutamate. This enzyme converts N-acetyl-gamma-glutamyl phosphate to N-acetylglutamate-gamma-semialdehyde by an NADPH-dependent reductive dephosphorylation. AGPR from Oryza sativa (OsAGPR) was expressed in Escherichia coli at 291 K as a soluble fusion protein with an upstream thioredoxin-hexahistidine [Trx-(His)6] extension. OsAGPR(Ala50-Pro366) was purified and crystals were obtained using the sitting-drop vapour-diffusion method at 293 K and diffract X-rays to at least 1.8 A resolution. They belong to the hexagonal space group P6(1), with unit-cell parameters a = 86.11, c = 316.3 A.

High precision NMR structure and function of the RING-H2 finger domain of EL5, a rice protein whose expression is increased upon exposure to pathogen-derived oligosaccharides.

EL5, a RING-H2 finger protein, is rapidly induced by N-acetylchitooligosaccharides in rice cell. We expressed the EL5 RING-H2 finger domain in Escherichia coli and determined its structure in solution by NMR spectroscopy. The EL5 RING-H2 finger domain consists of two-stranded beta-sheets (beta1, Ala(147)-Phe(149); beta2, Gly(156)-His(158)), one alpha-helix (Cys(161)-Leu(166)), and two large N- and C-terminal loops. It is stabilized by two tetrahedrally coordinated zinc ions. This structure is similar to that of other RING finger domains of proteins of known function. From structural analogies, we inferred that the EL5 RING-H2 finger is a binding domain for ubiquitin-conjugating enzyme (E2). The binding site is probably formed by solvent-exposed hydrophobic residues of the N- and C-terminal loops and the alpha-helix. We demonstrated that the fusion protein with EL5-(96-181) and maltose-binding protein (MBP) was polyubiquitinated by incubation with ubiquitin, ubiquitin-activating enzyme (E1), and a rice E2 protein, OsUBC5b. This supported the idea that the EL5 RING finger domain is essential for ubiquitin-ligase activity of EL5. By NMR titration experiments, we identified residues that are critical for the interaction between the EL5 RING-H2 finger and OsUBC5b. We conclude that the RING-H2 finger domain of EL5 is the E2 binding site of EL5.