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To develop artificial cell models that mimic living cells, cell-sized lipid vesicles encapsulating cell-free protein synthesis (CFPS) systems are useful for protein expressions or artificial gene circuits for vesicle-vesicle communications. Therefore, investigating the transcriptional and translational properties of CFPS systems in lipid vesicles is important for maximizing the synthesis and functions of proteins. Although transcription and translation using CFPS systems inside lipid vesicles are more important than that outside lipid vesicles, the former processes are not investigated by changing the lipid composition of lipid vesicles. Herein, we investigated changes in transcription and translation using CFPS systems inside giant lipid vesicles (approximately 5-20 μm in diameter) caused by changing the lipid composition of lipid vesicles containing neutral, positively, and negatively charged lipids. After incubating for 30 min, 1 h, 2 h, and 4 h, the transcriptional and translational activities in these lipid vesicles were determined by detecting the fluorescence intensities of the fluorogenic RNA aptamer on the 3'-untranslated region of mRNA (transcription) and the fluorescent protein sfCherry (translation), respectively. The results revealed that transcriptional and translational activities in a lipid vesicle containing positively charged lipids were high when the protein was synthesized using the CFPS system inside the lipid vesicle. Thus, the present study provides an experimental basis for constructing complex artificial cell models using bottom-up approaches., (© 2024. The Author(s).)

The inside of a cell is highly crowded with proteins and other biomolecules. How proteins express their specific functions together with many off-target proteins in crowded cellular environments is largely unknown. Here, we investigate an inhibitor binding with c-Src kinase using atomistic molecular dynamics (MD) simulations in dilute as well as crowded protein solution. The populations of the inhibitor, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), in bulk solution and on the surface of c-Src kinase are reduced as the concentration of crowder bovine serum albumins (BSAs) increases. This observation is consistent with the reduced PP1 inhibitor efficacy in experimental c-Src kinase assays in addition with BSAs. The crowded environment changes the major binding pathway of PP1 toward c-Src kinase compared to that in dilute solution. This change is explained based on the population shift mechanism of local conformations near the inhibitor binding site in c-Src kinase.

Epilepsy is a common brain disorder throughout history. Epilepsy-related ligand-receptor complex, LGI1-ADAM22, regulates synaptic transmission and has emerged as a determinant of brain excitability, as their mutations and acquired LGI1 autoantibodies cause epileptic disorders in human. Here, we report the crystal structure of human LGI1-ADAM22 complex, revealing a 2:2 heterotetrameric assembly. The hydrophobic pocket of the C-terminal epitempin-repeat (EPTP) domain of LGI1 binds to the metalloprotease-like domain of ADAM22. The N-terminal leucine-rich repeat and EPTP domains of LGI1 mediate the intermolecular LGI1-LGI1 interaction. A pathogenic R474Q mutation of LGI1, which does not exceptionally affect either the secretion or the ADAM22 binding, is located in the LGI1-LGI1 interface and disrupts the higher-order assembly of the LGI1-ADAM22 complex in vitro and in a mouse model for familial epilepsy. These studies support the notion that the LGI1-ADAM22 complex functions as the trans-synaptic machinery for precise synaptic transmission.

Cell-free protein synthesis is used to produce proteins with various structural traits. Recent bioinformatics analyses indicate that more than half of eukaryotic proteins possess long intrinsically disordered regions. However, no systematic study concerning the connection between intrinsic disorder and expression success of cell-free protein synthesis has been presented until now. To address this issue, we examined correlations of the experimentally observed cell-free protein expression yields with the contents of intrinsic disorder bioinformatically predicted in the expressed sequences. This analysis revealed strong relationships between intrinsic disorder and protein amenability to heterologous cell-free expression. On the one hand, elevated disorder content was associated with the increased ratio of soluble expression. On the other hand, overall propensity for detectable protein expression decreased with disorder content. We further demonstrated that these tendencies are rooted in some distinct features of intrinsically disordered regions, such as low hydrophobicity, elevated surface accessibility and high abundance of sequence motifs for proteolytic degradation, including sites of ubiquitination and PEST sequences. Our findings suggest that identification of intrinsically disordered regions in the expressed amino acid sequences can be of practical use for predicting expression success and optimizing cell-free protein synthesis.

A new carbon-carbon bond has been regioselectively introduced into a target position (position 32 or 174) of the Ras protein by two types of organopalladium reactions (Mizoroki-Heck and Sonogashira reactions). Reaction conditions were screened by using a model peptide, and the stability of the Ras protein under the reaction conditions was examined by using the wild-type Ras protein. Finally, the iF-Ras proteins containing a 4-iodo-L-phenylalanine residue were subjected to organopalladium reactions with vinylated or propargylated biotin. Site-specific biotinylations of the Ras protein were confirmed by Western blot and LC-MS/MS.

Palladium-catalyzed reactions have contributed to the advancement of many areas of organic chemistry, in particular, the synthesis of organic compounds such as natural products and polymeric materials. In this study, we have used a Mizoroki-Heck reaction for site-specific carbon-carbon bond formation in the Ras protein. This was performed by the following two steps: 1) the His6-fused Ras protein containing 4-iodo-L-phenylalanine at position 32 (iF32-Ras-His) was prepared by genetic engineering and 2) the aryl iodide group on the iF32-Ras-His was coupled with vinylated biotin in the presence of a palladium catalyst. The biotinylation was confirmed by Western blotting and liquid chromatography-mass spectrometry (LC-MS). The regioselectivity of the Mizoroki-Heck reaction was furthermore confirmed by LC-MS/MS analysis. However, in addition to the biotinylated product (bF32-Ras-His), a dehalogenated product (F32-Ras-His) was detected by LC-MS/MS. This dehalogenation resulted from the undesired termination of the Mizoroki-Heck reaction due to steric and electrostatic hindrance around residue 32. The biotinylated Ras showed binding activity for the Ras-binding domain as its downstream target, Raf-1, with no sign of decomposition. This study is the first report of an application of organometallic chemistry in protein chemistry.

Human CA125, encoded by the MUC16 gene, is an ovarian cancer antigen widely used for a serum assay. Its extracellular region consists of tandem repeats of SEA domains. In this study we determined the three-dimensional structure of the SEA domain from the murine MUC16 homologue using multidimensional NMR spectroscopy. The domain forms a unique alpha/beta sandwich fold composed of two alpha helices and four antiparallel beta strands and has a characteristic turn named the TY-turn between alpha1 and alpha2. The internal mobility of the main chain is low throughout the domain. The residues that form the hydrophobic core and the TY-turn are fully conserved in all SEA domain sequences, indicating that the fold is common in the family. Interestingly, no other residues are conserved throughout the family. Thus, the sequence alignment of the SEA domain family was refined on the basis of the three-dimensional structure, which allowed us to classify the SEA domains into several subfamilies. The residues on the surface differ between these subfamilies, suggesting that each subfamily has a different function. In the MUC16 SEA domains, the conserved surface residues, Asn-10, Thr-12, Arg-63, Asp-75, Asp-112, Ser-115, and Phe-117, are clustered on the beta sheet surface, which may be functionally important. The putative epitope (residues 58-77) for anti-MUC16 antibodies is located around the beta2 and beta3 strands. On the other hand the tissue tumor marker MUC1 has a SEA domain belonging to another subfamily, and its GSVVV motif for proteolytic cleavage is located in the short loop connecting beta2 and beta3.

The BolA-like proteins are widely conserved from prokaryotes to eukaryotes. The BolA-like proteins seem to be involved in cell proliferation or cell-cycle regulation, but the molecular function is still unknown. Here we determined the structure of a mouse BolA-like protein. The overall topology is alphabetabetaalphaalphabetaalpha, in which beta(1) and beta(2) are antiparallel, and beta(3) is parallel to beta(2). This fold is similar to the class II KH fold, except for the absence of the GXXG loop, which is well conserved in the KH fold. The conserved residues in the BolA-like proteins are assembled on the one side of the protein.

We studied the interactions between the SH2 domain of growth factor receptor binding protein 2 (Grb2) and ErbB receptor-derived phosphotyrosyl peptides using molecular dynamics, free energy calculations, and surface plasmon resonance (SPR) analysis. Binding free energies for nine phosphotyrosyl peptides were calculated using the MM-PBSA continuum solvent method, and excellent qualitative agreement with the SPR experimental data, with a correlation coefficient of 0.92, was obtained. Consistent with previous experimental findings, phosphotyrosyl peptides with the consensus sequence pYXNX showed favorable binding affinity for the Grb2. Unexpectedly, phosphotyrosyl peptides with the consensus sequence pYQQD, which had not shown any specific binding affinity for the Grb2 in earlier studies, also showed favorable binding affinity for the Grb2 in our experimental and computational analyses. Component analysis of the calculated binding free energies revealed that van der Waals interaction between the Grb2 and the phosphotyrosyl peptide was the dominant factor for specificity and binding affinity. These results indicate that current methods of estimating binding free energies are efficient for obtaining important information about protein-protein interactions, which are essential for the transmission of signals in cellular signaling pathways.

A suppressor tRNA(Tyr) and mutant tyrosyl-tRNA synthetase (TyrRS) pair was developed to incorporate 3-iodo-L-tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNA(Tyr) gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E.coli TyrRS and Bacillus stearothermophilus suppressor tRNA(Tyr), which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3-iodo-L-tyrosine, its specific E.coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B.stearothermophilus suppressor tRNA(Tyr), while 3-iodo-L-tyrosine was supplied in the growth medium. 3-Iodo-L-tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of >95%. Finally, we demonstrated conditional 3-iodo-L-tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline-regulated promoter.

Expression of constitutively active Src protein tyrosine kinase in Xenopus oocytes has been shown to accelerate oocyte maturation suggesting that Src may be involved in meiotic progression. However, meiotic regulation of endogenous Src kinase in oocytes has not been investigated in detail. To address this problem, we measured the activity, expression level, and phosphorylation state of the endogenous Xenopus Src (xSrc) and overexpressed xSrc mutants in the process of progesterone-induced oocyte maturation. We found that the enzyme is first transiently activated in the plasma membrane-containing fraction of oocytes within 3 min of progesterone administration. This event represents one of the earliest responses of oocytes to the hormone and should be related to triggering some early signaling pathways of maturation. Thereafter, xSrc activity increases again at the time of germinal vesicle breakdown (GVBD) and remains elevated till the completion of maturation. This elevation of xSrc activity is associated with a 2-fold increase of xSrc protein content in the absence of change in its specific activity and xSrc mRNA content. No significant changes in the phosphorylation state of C-terminal regulatory phosphotyrosine can be registered either in endogenous xSrc or in overexpressed kinase-negative and wild-type xSrc proteins during maturation. Altogether, these results indicate that upregulation of xSrc in the meiotic metaphase occurs at the translation level. We also demonstrate here that the expression of constitutively active xSrc in Xenopus oocytes is accompanied by the activation of mitogen-activated protein kinase (MAPK). Our data suggest that the Src kinase acts through the MAPK pathway to accelerate oocyte maturation. Keywords: Src kinase; Xenopus; Oocyte; Maturation; Meiosis

Members of the leucine-rich repeat-containing 8 (LRRC8) protein family, composed of the five LRRC8A-E isoforms, are pore-forming components of the volume-regulated anion channel (VRAC). LRRC8A and at least one of the other LRRC8 isoforms assemble into heteromers to generate VRAC transport activities. Despite the availability of the LRRC8A structures, the structural basis of how LRRC8 isoforms other than LRRC8A contribute to the functional diversity of VRAC has remained elusive. Here, we present the structure of the human LRRC8D isoform, which enables the permeation of organic substrates through VRAC. The LRRC8D homo-hexamer structure displays a two-fold symmetric arrangement, and together with a structure-based electrophysiological analysis, revealed two key features. The pore constriction on the extracellular side is wider than that in the LRRC8A structures, which may explain the increased permeability of organic substrates. Furthermore, an N-terminal helix protrudes into the pore from the intracellular side and may be critical for gating. Ryoki Nakamura et al. report the cryo-EM structure of the volume-regulated anion channel LRRC8D isoform, which enables permeation of organic compounds into cells. Compared to the LRRC8A isoform, the LRRC8D isoform has a wider extracellular pore and an intracellular N-terminal helix that may function in gating. [ABSTRACT FROM AUTHOR]

Growing numbers of therapeutic antibodies offer excellent treatment strategies for many diseases. Elucidation of the interaction between a potential therapeutic antibody and its target protein by structural analysis reveals the mechanism of action and offers useful information for developing rational antibody designs for improved affinity. Here, we developed a rapid, high-yield cell-free system using dialysis mode to synthesize antibody fragments for the structural analysis of antibody–antigen complexes. Optimal synthesis conditions of fragments (Fv and Fab) of the anti-EGFR antibody 059–152 were rapidly determined in a day by using a 30-μl-scale unit. The concentration of supplemented disulfide isomerase, DsbC, was critical to obtaining soluble antibody fragments. The optimal conditions were directly applicable to a 9-ml-scale reaction, with linear scalable yields of more than 1 mg/ml. Analyses of purified 059-152-Fv and Fab showed that the cell-free synthesized antibody fragments were disulfide-bridged, with antigen binding activity comparable to that of clinical antibodies. Examination of the crystal structure of cell-free synthesized 059-152-Fv in complex with the extracellular domain of human EGFR revealed that the epitope of 059-152-Fv broadly covers the EGF binding surface on domain III, including residues that formed critical hydrogen bonds with EGF (Asp355EGFR, Gln384EGFR, H409EGFR, and Lys465EGFR), so that the antibody inhibited EGFR activation. We further demonstrated the application of the cell-free system to site-specific integration of non-natural amino acids for antibody engineering, which would expand the availability of therapeutic antibodies based on structural information and rational design. This cell-free system could be an ideal antibody-fragment production platform for functional and structural analysis of potential therapeutic antibodies and for engineered antibody development. [ABSTRACT FROM AUTHOR]

Protein Nε-acylation is emerging as a ubiquitous post-translational modification. In Corynebacterium glutamicum, which is utilized for industrial production of l-glutamate, the levels of protein acetylation and succinylation change drastically under the conditions that induce glutamate overproduction. Here, the acylation of phosphoenolpyruvate carboxylase (PEPC), an anaplerotic enzyme that supplies oxaloacetate for glutamate overproduction was characterized. It was shown that acetylation of PEPC at lysine 653 decreased enzymatic activity, leading to reduced glutamate production. An acetylation-mimic (KQ) mutant of K653 showed severely reduced glutamate production, while the corresponding KR mutant showed normal production levels. Using an acetyllysine-incorporated PEPC protein, we verified that K653-acetylation negatively regulates PEPC activity. In addition, NCgl0616, a sirtuin-type deacetylase, deacetylated K653-acetylated PEPC in vitro. Interestingly, the specific activity of PEPC was increased during glutamate overproduction, which was blocked by the K653R mutation or deletion of sirtuin-type deacetylase homologues. These findings suggested that deacetylation of K653 by NCgl0616 likely plays a role in the activation of PEPC, which maintains carbon flux under glutamate-producing conditions. PEPC deletion increased protein acetylation levels in cells under glutamate-producing conditions, supporting the hypothesis that PEPC is responsible for a large carbon flux change under glutamate-producing conditions. [ABSTRACT FROM AUTHOR]

Peripheral stalk subunits of eukaryotic or mammalian vacuolar ATPases (V-ATPases) play key roles in regulating its assembly and disassembly. In a previous study, we purified several subunits and their isoforms of the peripheral stalk region of Homo sapiens (human) V-ATPase; such as C1, E1G1, H, and the N-terminal cytoplasmic region of Vo, a1. Here, we investigated the in vitro binding interactions of the subunits at the stalk region and measured their specific affinities. Surface plasmon resonance experiments revealed that the subunit C1 binds the E1G1 heterodimer with both high and low affinities (2.8 nM and 1.9 µM, respectively). In addition, an E1G1-H complex can be formed with high affinity (48 nM), whereas affinities of other subunit pairs appeared to be low (∼0.21−3.0 µM). The putative ternary complex of C1-H-E1G1 was not much strong on co-incubation of these subunits, indicating that the two strong complexes of C1-E1G1 and H-E1G1 in cooperation with many other weak interactions may be sufficiently strong enough to withstand the torque of rotation during catalysis. We observed a partially stable quaternary complex (consisting of E1G1, C1, a1NT, and H subunits) resulting from discrete peripheral subunit interactions stabilizing the complex through their intrinsic affinities. No binding was observed in the absence of E1G1 (using only H, C1, and a1NT); therefore, it is likely that, in vivo, the E1G1 heterodimer has a significant role in the initiation of subunit assembly. Multiple interactions of variable affinity in the stalk region may be important to the mechanism of reversible dissociation of the intact V-ATPase. [ABSTRACT FROM AUTHOR]

High-throughput cell-free protein synthesis is being used increasingly in structural/functional genomics projects. However, the factors determining expression success are poorly understood. Here, we evaluated the expression of 3066 human proteins and their domains in a bacterial cell-free system and analyzed the correlation of protein expression with 39 physicochemical and structural properties of proteins. As a result of the bioinformatics analysis performed, we determined the 18 most influential features that affect protein amenability to cell-free expression. They include protein length; hydrophobicity; pI; content of charged, nonpolar, and aromatic residues;, cysteine content; solvent accessibility; presence of coiled coil; content of intrinsically disordered and structured (α-helix and β-sheet) sequence; number of disulfide bonds and functional domains; presence of transmembrane regions; PEST motifs; and signaling sequences. This study represents the first comprehensive bioinformatics analysis of heterologous protein synthesis in a cell-free system. The rules and correlations revealed here provide a plethora of important insights into rationalization of cell-free protein production and can be of practical use for protein engineering with the aim of increasing expression success. [ABSTRACT FROM AUTHOR]

TTHA1846 is a conserved hypothetical protein from Thermus thermophilus HB8 with a molecular mass of 15.1 kDa that belongs to the thioesterase superfamily (Pfam 03061). Here, the 1.9 Å resolution crystal structure of TTHA1846 from T. thermophilus is reported. The crystal structure is a dimer of dimers. Each subunit adopts the so-called hot-dog fold composed of five antiparallel β-strands flanked on one side by a rather long α-helix and shares structural similarity to a number of thioesterases. Unexpectedly, TTHA1846 binds one metal ion and one ligand per subunit. The ligand density was modelled as coenzyme A (CoA). Its structure was confirmed by MALDI-TOF mass spectrometry and electron-density mapping. X-ray absorption fine-structure (XAFS) measurement of the crystal unambiguously characterized the metal ion as zinc. The zinc ion is tetrahedrally coordinated by the side chains of Asp18, His22 and Glu50 and the CoA thiol group. This is the first structural report of the interaction of CoA with a zinc ion. From structural and database analyses, it was speculated that the zinc ion may play an inhibitory role in the enzymatic activity. [ABSTRACT FROM AUTHOR]

Ligand-induced homo- and hetero-dimer formation of ErbB receptors results in different biological outcomes irrespective of recruitment and activation of similar effector proteins. Earlier experimental research indicated that cells expressing both EGFR (epidermal growth factor receptor) and the ErbB4 receptor (E1/4 cells) induced E1/4 cell-specific B-Raf activation and higher extracellular signal-regulated kinase (ERK) activation, followed by cellular transformation, than cells solely expressing EGFR (E1 cells) in Chinese hamster ovary (CHO) cells. Since our experimental data revealed the presence of positive feedback by ERK on upstream pathways, it was estimated that the cross-talk/feedback pathway structure of the Raf-MEK-ERK cascade might affect ERK activation dynamics in our cell system. To uncover the regulatory mechanism concerning the ERK dynamics, we used topological models and performed parameter estimation for all candidate structures that possessed ERK-mediated positive feedback regulation of Raf. The structure that reliably reproduced a series of experimental data regarding signal amplitude and duration of the signaling molecules was selected as a solution. We found that the pathway structure is characterized by ERK-mediated positive feedback regulation of B-Raf and B-Raf-mediated negative regulation of Raf-1. Steady-state analysis of the estimated structure indicated that the amplitude of Ras activity might critically affect ERK activity through ERK-B-Raf positive feedback coordination with sustained B-Raf activation in E1/4 cells. However, Rap1 that positively regulates B-Raf activity might be less effective concerning ERK and B-Raf activity. Furthermore, we investigated how such Ras activity in E1/4 cells can be regulated by EGFR/ErbB4 heterodimer-mediated signaling. From a sensitivity analysis of the detailed upstream model for Ras activation, we concluded that Ras activation dynamics is dominated by heterodimer-mediated signaling coordination with a large initial speed of dimerization when the concentration of the ErbB4 receptor is considerably high. Such characteristics of the signaling cause the preferential binding of the Grb2-SOS complex to heterodimer-mediated signaling molecules. [ABSTRACT FROM AUTHOR]

The gene encoding TTHA1544 is a singleton found in the Thermus thermophilus HB8 genome and encodes a 131-amino-acid protein. The crystal structure of TTHA1544 has been determined at 2.0 Å resolution by the single-wavelength anomalous dispersion method in order to elucidate its function. There are two molecules in the asymmetric unit. Each molecule consists of four α-helices and six β-strands, with the β-strands composing a central β-sheet. A structural homology search revealed that the overall structure of TTHA1544 resembles the α/β-hydrolase fold, although TTHA1544 lacks the catalytic residues of a hydrolase. These results suggest that TTHA1544 represents the minimized α/β-hydrolase fold and that an additional component would be required for its activity. [ABSTRACT FROM AUTHOR]

The transcription termination factor NusA from Aeropyrum pernix was crystallized using a counter-diffusion technique in both terrestrial and microgravity environments. Crystallization under microgravity conditions significantly reduced the twinning content (1.0%) compared with terrestrially grown crystals (18.3%) and improved the maximum resolution from 3.0 to 2.29 Å, with similar unit-cell parameters. Based on a comparison of the crystal parameters, the effect of microgravity on protein crystallization is discussed. [ABSTRACT FROM AUTHOR]

The Thermus thermophilus hypothetical protein TTHA1280 belongs to a family of predicted S-adenosyl-l-methionine (AdoMet) dependent RNA methyltransferases (MTases) present in many bacterial and archaeal species. Inspection of amino-acid sequence motifs common to class I Rossmann-fold-like MTases suggested a specific role as an RNA 5-methyluridine MTase. Selenomethionine (SeMet) labelled and native versions of the protein were expressed, purified and crystallized. Two crystal forms of the SeMet-labelled apoprotein were obtained: SeMet-ApoI and SeMet-ApoII. Cocrystallization of the native protein with S-adenosyl-L-homocysteine (AdoHcy) yielded a third crystal form, Native-AdoHcy. The SeMet-ApoI structure was solved by the multiple anomalous dispersion method and refined at 2.55 Å resolution. The SeMet-ApoII and Native-AdoHcy structures were solved by molecular replacement and refined at 1.80 and 2.60 Å , respectively. TTHA1280 formed a homodimer in the crystals and in solution. Each subunit folds into a three-domain structure composed of a small N-terminal PUA domain, a central α/β-domain and a C-terminal Rossmann-fold-like MTase domain. The three domains form an overall clamp-like shape, with the putative active site facing a deep cleft. The architecture of the active site is consistent with specific recognition of uridine and catalysis of methyl transfer to the 5-carbon position. The cleft is suitable in size and charge distribution for binding single-stranded RNA. [ABSTRACT FROM AUTHOR]

Describes the solution structure of the mouse enhancer of rudimentary (ER) protein determined by using heteronuclear nuclear magnetic resonance spectroscopy. Role of the regulatory or enzymatic activity of ER in pyrimidine biosynthesis and the cell cycle; Dimer or trimer behavior of ER in solution.

Examines how the structures of both the human feline sarcoma oncogene (Fes) protein and Src SH2 domains operate differently in the context of the full length Fes protein. Approach in which the Fes SH2 domain acts in a negative regulatory way; Description on the structures of Fes and SrC SH2 domains; Involvement of Fes in the growth and differentiation of myeloid hematopoietic cells.

Describes an engineered Escherichia coli tyrosyl-transfer RNA (tRNA) synthetase for site-specific incorporation of unnatural amino acids into proteins in eukaryotic translation. Efforts to engineer the translation system of amino acids incorporated into proteins; Synthesis of proteins containing amino acids; Method of attaching unnatural amino acids to its adapter tRNA.

Knots in polypeptide chains have been found in very few proteins. Only two proteins are considered to have a shallow 'trefoil' knot, which tucks a few residues at one end of the chain through a loop exposed on the protein surface. Recently, another protein was found by a mathematical algorithm to have a deep 'figure-of-eight' knot which had not been visually identified. In the present study, the crystal structure of a hypothetical RNA 2'-O-ribose methyltransferase from Thermus thermophilus (RrmA) was determined at 2.4 Å resolution and a deep trefoil knot was found for the first time. The present knot is formed by the threading of a 44-residue polypeptide chain through a 41-residue loop and is better defined than the previously reported knots. Two of the three catalytic residues conserved in the 2'-O-ribose methyltransferase family are located in the knotting loop and in the knotted carboxy-terminal chain, which is the first observation that the enzyme active site is constructed right on the knot. On the other hand, the amino-terminal domain exhibits a geometrical similarity to the ribosomal proteins which recognize an internal loop of RNA. [ABSTRACT FROM AUTHOR]

Eukaryotic ribosomal protein L10 is an essential component of the large ribosomal subunit, which organizes the architecture of the aminoacyl-tRNA binding site. The human L10 protein is also called the QM protein and consists of 214 amino-acid residues. For crystallization, the L10 core domain (L10CD, Phe34–Glu182) was recombinantly expressed in Escherichia coli and purified to homogeneity. A hexagonal crystal of L10CD was obtained by the sitting-drop vapour-diffusion method. The L10CD crystal diffracted to 2.5 Å resolution and belongs to space group P3121 or P3221. [ABSTRACT FROM AUTHOR]

Ribosomal proteins are subjected to a variety of post-transational modifications, of which methylation is the most frequently found in all three kingdoms of life. PrmA is the only bacterial enzyme identified to date that catalyzes the methy1ation of a ribosomal protein. It is responsible for the introduction of nice methyl groups into the N-terminal domain of ribosomal protein L11. The PrmA protein from Thermus thermophilus HB8 was crystallized and a preliminary X-ray diffraction analysis was performed. A cryocooled crystal diffracted X-rays beyond 1.9 Å using synchrotron radiation. [ABSTRACT FROM AUTHOR]

The gene encoding LysX, an essential component of the lysine-biosynthesis pathway in Thermus thermophilus (molecular weight ≃31 000 Da), was cloned and expressed and the purified protein was crystallized by the hanging-drop vapour-diffusion technique in two different space groups, C2 (unit-cell parameters a = 124.7, b = 51.4, c = 103.6 Å β = 122.8°) and R3 (a = b = 122.6, c = 97.6Å). Crystals improved by macroseeding diffracted to beyond 2.3 and 3Å resolution for the C2 and R3 crystal forms, respectively. Complete diffraction data sets were collected for the C2 and R3 crystal forms at 2.5 and 3.1Å resolution, respectively. Crystals of selenomethionine-containing LysX protein were obtained by cross-microseeding, using the native microcrystals as a seed. Structure determination is now in progress. [ABSTRACT FROM AUTHOR]

In the final step of tRNA splicing, the 2′-phosphotransferase catalyzes the transfer of the extra 2′-phosphate from the precursor-ligated tRNA to NAD. We have determined the crystal structure of the 2′-phosphotransferase protein from Aeropyrum pernix K1 at 2.8Å resolution. The structure of the 2′-phosphotransferase contains two globular domains (N and C-domains), which form a cleft in the center. The N-domain has the winged helix motif, a subfamily of the helix-turn-helix family, which is shared by many DNA-binding proteins. The C-domain of the 2′-phosphotransferase superimposes well on the NAD-binding fold of bacterial (diphtheria) toxins, which catalyze the transfer of ADP ribose from NAD to target proteins, indicating that the mode of NAD binding by the 2′-phosphotransferase could be similar to that of the bacterial toxins. The conserved basic residues are assembled at the periphery of the cleft and could participate in the enzyme contact with the sugar-phosphate backbones of tRNA. The modes by which the two functional domains recognize the two different substrates are clarified by the present crystal structure of the 2′-phosphotransferase. [Copyright &y& Elsevier]

Ribosomal protein L16 is an essential component of the bacterial ribosome. It organizes the architecture of aminoacyl tRNA binding site in the ribosome 50S subunit. The three-dimensional structure of L16 from Thermus thermophilus HB8 was determined by NMR. In solution, L16 forms an α+β sandwich structure combined with two additional β sheets located at the loop regions connecting the two layers. The terminal regions and a central loop region did not show any specific secondary structure. The structured part of L16 could be superimposed well on the Cα model of L16 determined in the crystal structure of the ribosome 50S subunit. By overlaying the L16 solution structure onto the coordinates of the ribosome crystal structure, we constructed the combined model that represents the ribosome-bound state of L16 in the detailed structure. The model showed that L16 possesses residues in contact with helices 38, 39, 42, 43 and 89 of 23S rRNA and helix 4 of 5S rRNA. This suggests its broad effect on the ribosome architecture. Comparison of L16 with the L10e protein, which is the archaeal counterpart, showed that they share a common fold, but differ in some regions of functional importance, especially in the N-terminal region. All known mutation sites in L16 that confer resistance to avilamycin and evernimicin were positioned so that their side-chains were exposed to solvent in the internal cavity of the ribosome. This suggests the direct participation of L16 as a part of the binding site for antibiotics. [Copyright &y& Elsevier]

Summary: ADP-ATP exchange by the molecular chaperone Hsp70 is enhanced by several cochaperones. BAG5 consists of five BAG domains and associates with the nucleotide-binding domain (NBD) of Hsp70. The overexpression of BAG5 in the cytosol reportedly disturbs Hsp70-mediated protein refolding and induces Parkinson''s disease. In the present study, we found that the fifth BAG domain (BD5) of BAG5 is responsible for the interaction between Hsp70 and BAG5. We also determined the crystal structures of the BD5•NBD complex. BD5 binding caused two different types of NBD conformational changes, which both disrupted the nucleotide-binding groove. In fact, BD5 reduced the affinity of the NBD for ADP. Moreover, BD5, as well as the full-length BAG5, accelerated Hsp70-mediated refolding in an in vitro assay. Therefore, BAG5 can function as the nucleotide exchange factor of Hsp70 for the enhancement of protein refolding. [Copyright &y& Elsevier]

Summary: We developed an Escherichia coli cell-based system to generate proteins containing 3-iodo-l-tyrosine at desired sites, and we used this system for structure determination by single-wavelength anomalous dispersion (SAD) phasing with the strong iodine signal. Tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii was engineered to specifically recognize 3-iodo-l-tyrosine. The 1.7 Å crystal structure of the engineered variant, iodoTyrRS-mj, bound with 3-iodo-l-tyrosine revealed the structural basis underlying the strict specificity for this nonnatural substrate; the iodine moiety makes van der Waals contacts with 5 residues at the binding pocket. E. coli cells expressing iodoTyrRS-mj and the suppressor tRNA were used to incorporate 3-iodo-l-tyrosine site specifically into the ribosomal protein N-acetyltransferase from Thermus thermophilus. The crystal structure of this enzyme with iodotyrosine was determined at 1.8 and 2.2 Å resolutions by SAD phasing at CuKα and CrKα wavelengths, respectively. The native structure, determined by molecular replacement, revealed no significant structural distortion caused by iodotyrosine incorporation. [Copyright &y& Elsevier]

Abstract: Leukocyte cell-surface antigen CD38 is a single-transmembrane protein. CD38 ligation by anti-CD38 antibodies triggers the growth or apoptosis of immune cells. Although the extracellular domain of CD38 has multifunctional catalytic activities including NAD+ glycohydrolase and cyclase, the CD38-mediated cell survival or death appears to be independent of its catalytic activity. It is proposed that a conformational change of CD38 triggers the signalling. The conformational change of CD38 could influence its catalytic activity. However, the agonistic anti-CD38 antibody that alters the catalytic activity of CD38 has not been reported so far. In the present study, we demonstrated that two agonistic anti-mouse CD38 mAbs (CS/2 and clone 90) change the catalytic activities of CD38. CS/2 was clearly more potent than clone 90 in prolonging B cell survival and activation. CS/2 inhibited the NAD+ glycohydrolase activity of both the isolated extracellular domain of CD38 (FLAG-CD38) and cell-surface CD38. Kinetic analysis suggested a non-competitive inhibition. On the other hand, clone 90 stimulated the NAD+ glycohydrolase activity of FLAG-CD38 and had little effect on the NAD+ glycohydrolase activity of cell-surface CD38. CS/2 and clone 90 had no effect on the cyclase activity of FLAG-CD38 and inhibited the cyclase activity of cell-surface CD38. Accordingly, these agonistic antibodies probably induce the conformational changes of CD38 that are evident in the distinct alterations of the catalytic site. The antibodies will be useful tools to analyze the conformational change of CD38 in the process of triggering B cell survival and the activation signal. [Copyright &y& Elsevier]

Abstract: SWIRM is a conserved domain found in several chromatin-associated proteins. Based on their sequences, the SWIRM family members can be classified into three subfamilies, which are represented by Swi3, LSD1, and Ada2. Here we report the SWIRM structure of human MYb-like, Swirm and Mpn domain-containing protein-1 (MYSM1). The MYSM1 SWIRM structure forms a compact HTH-related fold comprising five α-helices, which best resembles the Swi3 SWIRM structure, among the known SWIRM structures. The MYSM1 and Swi3 SWIRM structures are more similar to the LSD1 structure than the Ada2α structure. The SWIRM domains of MYSM1 and LSD1 lacked DNA binding activity, while those of Ada2α and the human Swi3 counterpart, SMARCC2, bound DNA. The dissimilarity in the DNA-binding ability of the MYSM1 and SMARCC2 SWIRM domains might be due to a couple of amino acid differences in the last helix. These results indicate that the SWIRM family has indeed diverged into three structural subfamilies (Swi3/MYSM1, LSD1, and Ada2 types), and that the Swi3/MYSM1-type subfamily has further diverged into two functionally distinct groups. We also solved the structure of the SANT domain of MYSM1, and demonstrated that it bound DNA with a similar mode to that of the c-Myb DNA-binding domain. [Copyright &y& Elsevier]

Summary: Pombe Cdc15 homology (PCH) proteins play an important role in a variety of actin-based processes, including clathrin-mediated endocytosis (CME). The defining feature of the PCH proteins is an evolutionarily conserved EFC/F-BAR domain for membrane association and tubulation. In the present study, we solved the crystal structures of the EFC domains of human FBP17 and CIP4. The structures revealed a gently curved helical-bundle dimer of ∼220 Å in length, which forms filaments through end-to-end interactions in the crystals. The curved EFC dimer fits a tubular membrane with an ∼600 Å diameter. We subsequently proposed a model in which the curved EFC filament drives tubulation. In fact, striation of tubular membranes was observed by phase-contrast cryo-transmission electron microscopy, and mutations that impaired filament formation also impaired membrane tubulation and cell membrane invagination. Furthermore, FBP17 is recruited to clathrin-coated pits in the late stage of CME, indicating its physiological role. [Copyright &y& Elsevier]

The BET (bromodomains and extra terminal domain) family proteins recognize acetylated chromatin through their bromodomain and act as transcriptional activators. One of the BET proteins, BRD2, associates with the transcription factor E2F, the mediator components CDK8 and TRAP220, and RNA polymerase II, as well as with acetylated chromatin during mitosis. BRD2 contains two bromodomains (BD1 and BD2), which are considered to be responsible for binding to acetylated chromatin. The BRD2 protein specifically recognizes the histone H4 tail acetylated at Lys12. Here, we report the crystal structure of the N-terminal bromodomain (BD1, residues 74–194) of human BRD2. Strikingly, the BRD2 BD1 protein forms an intact dimer in the crystal. This is the first observation of a homodimer among the known bromodomain structures, through the buried hydrophobic core region at the interface. Biochemical studies also demonstrated BRD2 BD1 dimer formation in solution. The two acetyllysine-binding pockets and a negatively charged secondary binding pocket, produced at the dimer interface in BRD2 BD1, may be the unique features that allow BRD2 BD1 to selectively bind to the acetylated H4 tail. [ABSTRACT FROM AUTHOR]

The concave surface of the crescent-shaped Bin-amphiphy-sin-Rvs (BAR) domain is postulated to bind to the cell membrane to induce membrane deformation of a specific curvature. The Rac binding (RCB) domain/IRSp53-MIM homology domain (IMD) has a dimeric structure that is similar to the structure of the BAR domain; however, the RCB domain/IMD has a ‘zeppelin-shaped’ dimer. Interestingly, the RCB domain/IMD of IRSp53 possesses Rac binding, membrane binding, and actin filament binding abilities. Here we report that the RCB domain/IMD of IRSp53 induces membrane deformation independent of the actin filaments in a Rac-dependent manner. In contrast to the BAR domain, the RCB domain/IMD did not cause long tubulation of the artificial liposomes; however, the Rac binding domain caused the formation of small buds on the liposomal surface. When expressed in cells, the Rac binding domain induced outward protrusion of the plasma membrane in a direction opposite to that induced by the BAR domain, Mapping of the amino acids responsible for membrane deformation suggests that the convex surface of the Rac binding domain binds to the membrane in a Rac-dependent manner, which may explain the mechanism of the membrane deformation induced by the RCB domain/IMD. [ABSTRACT FROM AUTHOR]

Rap2-interacting protein x (RPIPx) is a homolog of RPIP8, a specific effector of Rap2 GTPase. The N-terminal region of RPIP8, which contains the RUN domain, interacts with Rap2. Using cell-free synthesis and NMR, we determined that the region encompassing residues 83-255 of mouse RPIPx, which is 40-residues larger than the predicted RUN domain (residues 113-245), is the minimum fragment that forms a correctly folded protein. This fragment, the RPIPx RUN domain, interacted specifically with Rap2B in vitro in a nucleotide-dependent manner. The crystal structure of the RPIPx RUN domain was determined at 2.0 Å of resolution by the multiwavelength anomalous dispersion (MAD) method. The RPIPx RUN domain comprises eight anti-parallel α-helices, which form an extensive hydrophobic core, followed by an extended segment. The residues in the core region are highly conserved, suggesting the conservation of the RUN domain-fold among the RUN domain-containing proteins. The residues forming a positively charged surface are conserved between RPIP8 and its homologs, suggesting that this surface is important for Rap2 binding. In the crystal the putative Rap2 binding site of the RPIPx RUN domain interacts with the extended segment in a segment-swapping manner. [ABSTRACT FROM AUTHOR]

2-Thioribothymidine (s²T) is a modified nucleoside of U, specifically found at position 54 of tRNAs from extreme thermophilic microorganisms. The function of the 2-thiocarbonyl group of s²T54 is thermostabilization of the three-dimensional structure of tRNA; however, its biosynthesis has not been clarified until now. Using an in vivo tRNA labeling experiment, we demonstrate that the sulfur atom of s²T in tRNA is derived from cysteine or sulfate. We attempted to reconstitute 2-thiolation of s2T in vitro, using a cell extract of Thermus thermophilus. Specific 2-thiolation of ribothymidine, at position 54, was observed in vitro, in the presence of ATP. Using this assay, we found a strong temperature dependence of the 2-thiolation reaction in vitro as well as expression of 2-thiolation enzymes in vivo. These results suggest that the variable content of s2T in vivo at different temperatures may be explained by the above characteristics of the enzymes responsible for the 2-thiolation reaction. Furthermore, we found that another posttranscriptionally modified nucleoside, 1-methyladenosine at position 58, is required for the efficient 2-thiolation of ribothymidine 54 both in vivo and in vitro. [ABSTRACT FROM AUTHOR]

Abstract: TTHA1554 was found as a hypothetical protein composed of 95 amino acids in the genome of the extremely thermophilic bacterium, Thermus thermophilus HB8. Proteins homologous to TTHA1554 are conserved in several bacteria and archaea, although their functions are unknown. To investigate the function of TTHA1554, we identified interacting proteins by using a pull-down assay and mass spectrometry. TTHA1329, which is glutamine synthetase, and TTHA1620, a putative aminotransferase, were identified as TTHA1554 binding proteins. The interactions with TTHA1329 and TTHA1620 were validated using in vitro pull-down assays and surface plasmon resonance biosensor assays with recombinant proteins. Since sequence homology analyses suggested that TTHA1620 was a pyridoxal 5′-phosphate-dependent enzyme, such as an aminotransferase, a cystathionine β-lyase or a cystalysin, putative substrates were investigated. When cystathionine, cystine and S-methylcysteine were used as substrates, pyruvate was produced by TTHA1620. The data revealed that TTHA1620 has cystathionine β-lyase enzymatic activity. When TTHA1554 was added to the reaction mixtures, the glutamine synthetase and cystathionine β-lyase enzymatic activities both increased by approximately two-fold. These results indicated that TTHA1554 is a novel protein (we named it GCBP: glutamine synthetase and cystathionine β-lyase binding protein) that binds to glutamine synthetase and cystathionine β-lyase. [Copyright &y& Elsevier]

Ethylene-insensitive3 (EIN3) and EIN3-like (EIL) proteins are essential transcription factors in the ethylene signaling of higher plants. The EIN3/EIL proteins bind to the promoter regions of the downstream genes and regulate their expression. The location of the DNA-binding domain (DBD) in the primary structure was unclear, since the proteins show no sequence similarity to other known DBDs. Here, we identify the major DBD of an EIN3/EIL protein, Arabidopsis thaliana EIL3, containing a key mutational site for DNA binding and signaling (ein3-3 site), and determine its solution structure by NMR spectroscopy. The structure consists of five α-helices, possessing a novel fold dissimilar to known DBD structures. By a chemical-shift perturbation analysis, a region including the ein3-3 site is suggested to be involved in DNA binding. [Copyright &y& Elsevier]

Ligand-activated and tyrosine-phosphorylated ErbB3 receptor binds to the SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase and initiates intracellular signaling. Here, we studied the interactions between the N- (N-SH2) and C- (C-SH2) terminal SH2 domains of the p85 subunit of the phosphatidylinositol 3-kinase and eight ErbB3 receptor-derived phosphotyrosyl peptides (P-peptides) by using molecular dynamics, free energy, and surface plasmon resonance (SPR) analyses. In SPR analysis, these P-peptides showed no binding to the CSH2 domain, but P-peptides containing a phospho-YXXM or a non-phospho-YXXM motif did bind to the N-SH2 domain. The N-SH2 domain has two phosphotyrosine binding sites in its N- (N1) and C- (N2) terminal regions. Interestingly, we found that P-peptides of pY1180 and pY1241 favored to bind to the N2 site, although all other P-peptides showed favorable binding to the N1 site. Remarkably, two phosphotyrosines, pY1178 and pY1243, which are just 63 amino acids apart from the pY1241 and pY1180, respectively, showed favorable binding to the N1 site. These findings indicate a possibility that the pair of phosphotyrosines, pY1178-pY1241 or pY1243-pY1180, will fold into an appropriate configuration for binding to the N1 and N2 sites simultaneously. Our model structures of the cytoplasmic C-terminal domain of ErbB3 receptor also strongly supported the speculation. The calculated binding free energies between the N-SH2 domain and P-peptides showed excellent qualitative agreement with SPR data with a correlation coefficient of 0.91. The total electrostatic solvation energy between the N-SH2 domain and P-peptide was the dominant factor for its binding affinity. [ABSTRACT FROM AUTHOR]