Monomer-collagen interactions studied by saturation transfer difference NMR.

Functional monomers in dentin adhesives are involved in wetting dental substrates, demineralization, and the formation of calcium salts. However, the interaction of these monomers with collagen is not understood at a molecular/atomic level. We performed saturation transfer difference (STD) NMR spectroscopy to investigate the binding interaction of 2 functional monomers, 4-methacryloyloxyethyl trimellitate anhydride (4-META) and 10-methacryloyloxydecyl dihydrogenphosphate (MDP), with atelocollagen as a triple-helical peptide model. High STD intensities were detected on the protons in the aliphatic region in MDP, whereas they were not detected for 4-META. The STD results imply that MDP has a relatively stable interaction with the collagen, because of the hydrophobic interactions between the hydrophobic MDP moieties and the hydrophobic collagen surface. This finding indicates that MDP-collagen complexation accounts for stable dentin bonding.

Characterization of acyl-CoA-binding protein (ACBP) in the pheromone gland of the silkworm, Bombyx mori.

Various fatty acyl-CoAs are involved as intermediates or precursors of sex pheromone components in the biosynthetic pathway of the pheromones in many lepidopteran insects. We have purified a 10-kDa protein from the cytosolic fraction of Bombyx mori pheromone glands by using affinity chromatography with a palmitoyl-CoA-agarose column and reversed-phase HPLC. Amino acid sequence analysis of the fragment peptides obtained from the purified protein, and a homology search, revealed that this protein was a member of acyl-CoA-binding proteins (ACBPs). MALDI-TOF mass spectral analysis of the purified protein and cloning of the gene from a pheromone gland cDNA library confirmed B. mori ACBP to be a 90 amino acid protein with 78.9% identity to that of Manduca sexta ACBP. The secondary structure of the recombinant B. mori ACBP was determined by NMR spectroscopy. Northern blot analysis demonstrated that B. mori ACBP was predominantly expressed in the pheromone gland and the corresponding transcript was expressed from the day before adult eclosion. Present results suggest that ACBP plays a significant role in the production of sex pheromones regulated by the neurohormone, pheromone biosynthesis activating neuropeptide (PBAN).

Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis.

Endocytic proteins such as epsin, AP180, and Hip1R (Sla2p) share a conserved modular region termed the epsin NH2-terminal homology (ENTH) domain, which plays a crucial role in clathrin-mediated endocytosis through an unknown target. Here, we demonstrate a strong affinity of the ENTH domain for phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. With nuclear magnetic resonance analysis of the epsin ENTH domain, we determined that a cleft formed with positively charged residues contributed to phosphoinositide binding. Overexpression of a mutant, epsin Lys76 --> Ala76, with an ENTH domain defective in phosphoinositide binding, blocked epidermal growth factor internalization in COS-7 cells. Thus, interaction between the ENTH domain and PtdIns(4,5)P2 is essential for endocytosis mediated by clathrin-coated pits.

Solution structure of the human parvulin-like peptidyl prolyl cis/trans isomerase, hPar14.

The hPar14 protein is a peptidyl prolyl cis/trans isomerase and is a human parvulin homologue. The hPar14 protein shows about 30 % sequence identity with the other human parvulin homologue, hPin1. Here, the solution structure of hPar14 was determined by nuclear magnetic resonance spectroscopy. The N-terminal 35 residues preceding the peptidyl prolyl isomerase domain of hPar14 are unstructured, whereas hPin1 possesses the WW domain at its N terminus. The fold of residues 36-131 of hPar14, which comprises a four-stranded beta-sheet and three alpha-helices, is superimposable onto that of the peptidyl prolyl isomerase domain of hPin1. To investigate the interaction of hPar14 with a substrate, the backbone chemical-shift changes of hPar14 were monitored during titration with a tetra peptide. Met90, Val91, and Phe94 around the N terminus of alpha3 showed large chemical-shift changes. These residues form a hydrophobic patch on the molecular surface of hPar14. Two of these residues are conserved and have been shown to interact with the proline residue of the substrate in hPin1. On the other hand, hPar14 lacks the hPin1 positively charged residues (Lys63, Arg68, and Arg69), which determine the substrate specificity of hPin1 by interacting with phosphorylated Ser or Thr preceding the substrate Pro, and exhibits a different structure in the corresponding region. Therefore, the mechanism determining the substrate specificity seems to be different between hPar14 and hPin1.

Dosage effect of minor arginyl- and isoleucyl-tRNAs on protein synthesis in an Escherichia coli in vitro coupled transcription/translation system.

In Escherichia coli mRNA, the arginine codons AGA/AGG and the isoleucine codon AUA are rarely used with frequencies of about 0.35% and 0.41%, respectively. Six genes with a different number of these codons were expressed in an E. coli in vitro coupled transcription/translation system, which contained either tRNA prepared from E. coli cells carrying a plasmid with argU and ileX genes encoding rare tRNAs (tRNA(arg)(AGA/AGG) and tRNA(ile)(AUA)), designated codon-plus tRNA, or normal tRNA from cells lacking the plasmid. Genes having a low number of the rare codons, such as genes encoding chloramphenicol acetyltransferase and anti-gp120 single-chain Fv (artificially constructed to remove rare codons), were expressed at similar levels using with both tRNA preparations. On the other hand, the use of codon-plus tRNA increased the expression levels of genes having a relatively large number of the rare codons, including genes encoding archaeal proteins, green fluorescent protein of jelly fish origin, and a single-chain Fv of mouse origin, by about 20% higher than that using normal tRNA.

Structural genomics projects in Japan.

Two major structural genomics projects exist in Japan. The oldest, the RIKEN Structural Genomics Initiative, has two major goals: to determine bacterial, mammalian, and plant protein structures by X-ray crystallography and NMR spectroscopy and to perform functional analyses with the target proteins. The newest, the structural genomics project at the Biological Information Research Center, focuses on human membrane proteins.

Nuclear magnetic resonance and molecular dynamics studies on the interactions of the Ras-binding domain of Raf-1 with wild-type and mutant Ras proteins.

The Ras protein and its homolog, Rap1A, have an identical "effector region" (residues 32-40) preceded by Asp30-Glu31 and Glu30-Lys31, respectively. In the complex of the "Ras-like" E30D/K31E mutant Rap1A with the Ras-binding domain (RBD), residues 51-131 of Raf-1, Glu31 in Rap1A forms a tight salt bridge with Lys84 in Raf-1. However, we have recently found that Raf-1 RBD binding of Ras is indeed reduced by the E31K mutation, but is not affected by the E31A mutation. Here, the "Rap1A-like" D30E/E31K mutant of Ras was prepared and shown to bind the Raf-1 RBD less strongly than wild-type Ras, but slightly more tightly than the E31K mutant. The backbone 1H, 13C, and 15N magnetic resonances of the Raf-1 RBD were assigned in complexes with the wild-type and D30E/E31K mutant Ras proteins in the guanosine 5'-O-(beta,gamma-imidotriphosphate)-bound form. The Lys84 residue in the Raf-1 RBD exhibited a large change in chemical shift upon binding wild-type Ras, suggesting that Lys84 interacts with wild-type Ras. The D30E/E31K mutant of Ras caused nearly the same perturbations in Raf-1 chemical shifts, including that of Lys84. We hypothesized that Glu31 in Ras may not be the major salt bridge partner of Lys84 in Raf-1. A molecular dynamics simulation of a model structure of the Raf-1 RBD.Ras.GTP complex suggested that Lys84 in Raf-1 might instead form a tight salt bridge with Asp33 in Ras. Consistent with this, the D33A mutation in Ras greatly reduced its Raf-I RBD binding activity. We conclude that the major salt bridge partner of Lys84 in Raf-1 may be Asp33 in Ras.

Solution structure of the Eps15 homology domain of a human POB1 (partner of RalBP1).

The solution structure of the Eps15 homology (EH) domain of a human POB1 (partner of RaIBP1) has been determined by uniform 13C/15N labeling and heteronuclear multidimensional nuclear magnetic resonance spectroscopy. The POB1 EH domain consists of two EF-hand structures, and the second one binds a calcium ion. In the calcium-bound state, the orientation of the fourth alpha-helix relative to the other helices of the POB1 EH domain is slightly different from that of calbindin, and much more different from those of calmodulin and troponin C, on the basis of their atomic coordinates.

Cell-free production and stable-isotope labeling of milligram quantities of proteins.

We have improved the productivity of an Escherichia coli cell-free protein synthesis system. First, creatine phosphate and creatine kinase were used as the energy source regeneration system, and the other components of the reaction mixture were optimized. Second, the E. coli S30 cell extract was condensed by dialysis against a polyethylene glycol solution to increase the rate of synthesis. Third, during the protein synthesis, the reaction mixture was dialyzed against a low-molecular-weight substrate solution to prolong the reaction. Thus, the yield of chloramphenicol acetyltransferase was raised to 6 mg/ml of reaction mixture. Stable-isotope labeling of a protein with 13C/15N-labeled amino acids for NMR spectroscopy was achieved by this method.

Dual amino acid-selective and site-directed stable-isotope labeling of the human c-Ha-Ras protein by cell-free synthesis.

We developed two methods for stable-isotope labeling of proteins by cell-free synthesis. Firstly, we applied cell-free synthesis to the dual amino acid-selective 13C-15N labeling method, originally developed for in vivo systems by Kainosho and co-workers. For this purpose, we took one of the advantages of a cell-free protein synthesis system; the amino acid-selective stable-isotope labeling is free of the isotope scrambling problem. The targets of selective observation were Thr35 and Ser39 in the effector region (residues 32-40) of the Ras protein complexed with the Ras-binding domain of c-Raf-1 (Raf RBD) (the total molecular mass is about 30 kDa). Using a 15-mL Escherichia coli cell-free system, which was optimized to produce about 0.4 mg of Ras protein per 1-mL reaction, with 2 mg each of DL-[13C']proline and L-[15N]threonine, we obtained about 6 mg of Ras protein. As the Pro-Thr sequence is unique in the Ras protein, the Thr35 cross peak of the Ras.Raf RBD complex was unambiguously identified by the 2D 1H-15N HNCO experiment. The Ser-39 cross peak was similarly identified with the [13C']Asp/[15N]Ser-selectively labeled Ras protein. There were no isotope scrambling problems in this study. Secondly, we have established a method for producing a milligram quantity of site-specifically stable-isotope labeled protein by a cell-free system involving amber suppression. The E. coli amber suppressor tRNATyrCUA (25 mg) was prepared by in vitro transcription with T7 RNA polymerase. We aminoacylated the tRNATyrCUA transcript with purified E. coli tyrosyl-tRNA synthetase, using 2 mg of L-[15N]tyrosine. In the gene encoding the Ras protein, the codon for Tyr32 was changed to an amber codon (TAG). This template DNA and the [15N]Tyr-tRNATyrCUA were reacted for 30 min in 30 mL of E. coli cell-free system. The subsequent purification yielded 2.2 mg of [15N]Tyr32-Ras protein. In the 1H-15N HSQC spectrum of the labeled Ras protein, only one cross peak was observed, which was unambiguously assigned to Tyr32.

A helix-turn-helix structure unit in human centromere protein B (CENP-B).

CENP-B has been suggested to organize arrays of centromere satellite DNA into a higher order structure which then directs centromere formation and kinetochore assembly in mammalian chromosomes. The N-terminal portion of CENP-B is a 15 kDa DNA binding domain (DBD) consisting of two repeating units, RP1 and RP2. The DBD specifically binds to the CENP-B box sequence (17 bp) in centromere DNA. We determined the solution structure of human CENP-B DBD RP1 by multi-dimensional 1H, 13C and 15N NMR methods. The CENP-B DBD RP1 structure consists of four helices and has a helix-turn-helix structure. The overall folding is similar to those of some other eukaryotic DBDs, although significant sequence homology with these proteins was not found. The DBD of yeast RAP1, a telomere binding protein, is most similar to CENP-B DBD RP1. We studied the interaction between CENP-B DBD RP1 and the CENP-B box by the use of NMR chemical shift perturbation. The results suggest that CENP-B DBD RP1 interacts with one of the essential regions of the CENP-B box DNA, mainly at the N-terminal basic region, the N-terminal portion of helix 2 and helix 3.

Solution structure of the Ras-binding domain of RGL.

The RGL protein, a homolog of the Ral GDP dissociation stimulator (RalGDS), has been identified as a downstream effector of Ras. In the present study, the solution structure of the Ras-binding domain of RGL (RGL-RBD) was determined by NMR spectroscopy. The overall fold of RGL-RBD consists of a five-stranded beta-sheet and two alpha-helices, which is the same topology as that of RalGDS-RBD. The backbone chemical shift perturbation of RGL-RBD upon interaction with the GTP analog-bound Ras was also examined. The solution structure of RGL-RBD, especially around some of the Ras-interacting residues, is appreciably different from that of RalGDS-RBD.

A characteristic arrangement of aromatic amino acid residues in the solution structure of the amino-terminal RNA-binding domain of Drosophila sex-lethal.

The Sex-lethal (Sxl) protein from Drosophila melanogaster has two RNA-binding domains (RBDs). As the amino-terminal RBD (RBD1) of the Sxl protein exhibits low sequence homology to the typical RBDs, particularly at the putative functional residues, it was difficult to unambiguously locate the RNP1 and RNP2 motifs. Therefore, in the present study, we defined the amino and carboxy-terminal borders of the first RNA-binding domain (RBD1) of the Sxl protein by limited tryptic digestion. By replacement of Phe166 by Tyr, we constructed a highly soluble mutant, which exhibits the same RNA-binding properties as those of the wild-type. Using this mutant protein, we performed NMR measurements, and elucidated the secondary and tertiary structures of the Sxl RBD1 in solution. The betaalphabetabetaalphabeta folding pattern is conserved in the solution structure of the Sxl RBD1, as in other reported RBD structures. This allowed us to identify both the RNP1 and RNP2 motifs of the Sxl RBD1 unambiguously. Intriguingly, the RNP2 motif of the Sxl RBD1 has an Ile residue at the second position, which is generally occupied by an aromatic amino acid residue in RBDs and has been suggested to be involved in their RNA binding. Furthermore, the loop region between beta2 and beta3 of the Sxl RBD1 has an exceptional cluster of aromatic amino acid residues, in place of the normal basic amino acid cluster. In contrast, the second RBD of Sxl does not exhibit these characteristic features.

The solution structure of the pleckstrin homology domain of mouse Son-of-sevenless 1 (mSos1).

The solution structure of the pleckstrin homology (PH) domain of mouse Son-of-sevenless 1 (mSos1), a guanine nucleotide exchange factor for Ras, was determined by multidimensional NMR spectroscopy. The structure of the mSos1 PH domain involves the fundamental PH fold, consisting of seven beta-strands and one alpha-helix at the C terminus, as determined for the PH domains of other proteins. By contrast, the mSos1 PH domain showed two major characteristic features. First, the N-terminal region, whose amino acid sequence is highly conserved among Sos proteins, was found to form an alpha-helix, which interacts with the beta-sheet structure of the fundamental PH fold. Second, there is a long unstructured loop between beta3 and beta4. Furthermore, the mSos1 PH domain was found to bind phosphatidylinositol-4,5-bisphosphate by a centrifugation assay. The addition of inositol-1,4,5-trisphosphate to the mSos1 PH domain induced backbone amide chemical shift changes mainly in the beta1/beta2 loop and the N- and C-terminal parts of the long beta3/beta4 loop. This inositol-1,4,5-trisphosphate-binding mode of the mSos1 PH domain is somewhat similar to those of the PH domains of pleckstrin and phospholipase Cdelta1, and is clearly different from those of other PH domains.

A highly efficient cell-free protein synthesis system from Escherichia coli.

We modified a cell-free coupled transcription/translation system from Escherichia coli with the T7 phage RNA polymerase, and achieved a productivity as high as 0.4 mg protein/ml reaction mixture. First, we found that the optimal concentrations of phosphoenolpyruvate and poly(ethylene glycol) are interdependent; higher concentrations of the former should be used at higher concentrations of the latter. Second, the use of a condensed 30000 x g cell extract, in place of the conventional one, significantly increased the initial rate of protein synthesis. This phenomenon was demonstrated to be due to a reason other than elimination of inhibitory molecule(s) from the extract. For this system with the condensed extract, the phosphoenolpyruvate and poly(ethylene glycol) concentrations were again co-optimized, resulting in production of chloramphenicol acetyltransferase at a productivity of 0.3 mg/ml. Finally, the productivity was further increased up to 0.4 mg/ml, by supplementation of the pool of amino acids. This improved cell-free protein synthesis system is superior in productivity to any other cell-free systems reported so far, including the continuous-flow cell-free system.

Synthesis of N-acetylglucosaminyl asparagine-substituted puromycin analogues.

As part of our project aimed to introduce specifically glycosylated amino acids into proteins, new glycosylated puromycin analogues were chemically synthesized. Introduction of a free N-acetylglucosaminyl asparaginyl side chain abolished the activity of puromycin completely, but when the sugar OH groups were rendered increasingly hydrophobic by acetylation or benzylation, up to 8% of the activity was recovered. The results of our preliminary inhibition tests suggest that the interaction of puromycin analogues and therefore also of glycosylated aminoacyl tRNA, with the ribosomal A site increase with hydrophobicity of the modifying protecting groups.

Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis.

For the application of multidimensional NMR spectroscopy to larger proteins, it would be useful to perform selective labeling of one of the 20 amino acids. For some amino acids, however, amino acid metabolism drastically reduces the efficiency and selectivity of labeling in in vivo expression systems. In the present study, a cell-free protein synthesis system was optimized, so that highly efficient and selective stable isotope labeling of proteins can be achieved in the absence of amino acid metabolism. The productivity of the E. coli cell-free coupled transcription-translation system was first improved, by about fivefold, by using the T7 RNA polymerase for transcription and also by improving the translation conditions. Thus, about 0.1 mg protein per 1 ml reaction mixture was synthesized. Then, this improved cell-free system was used for Asp- or Ser-selective 15N-labeling of the human c-Ha-Ras protein. With a 15 ml cell-free reaction, using less than 1 mg of 15N-labeled amino acid, 1 mg of the Ras protein was obtained. 1H-15N HSQC experiments confirmed that the Ras protein was efficiently labeled with high selectivity. These results indicate that this cell-free protein synthesis system is useful for NMR studies.

Architectures of class-defining and specific domains of glutamyl-tRNA synthetase.

The crystal structure of a class I aminoacyl-transfer RNA synthetase, glutamyl-tRNA synthetase (GluRS) from Thermus thermophilus, was solved and refined at 2.5 A resolution. The amino-terminal half of GluRS shows a geometrical similarity with that of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) of the same subclass in class I, comprising the class I-specific Rossmann fold domain and the intervening subclass-specific alpha/beta domain. These domains were found to have two GluRS-specific, secondary-structure insertions, which then participated in the specific recognition of the D and acceptor stems of tRNA(Glu) as indicated by mutagenesis analyses based on the docking properties of GluRS and tRNA. In striking contrast to the beta-barrel structure of the GlnRS carboxyl-terminal half, the GluRS carboxyl-terminal half displayed an all-alpha-helix architecture, an alpha-helix cage, and mutagenesis analyses indicated that it had a role in the anticodon recognition.