Nobumasa Hino

Adding l-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases

We report a method for site-specifically incorporating l-lysine derivatives into proteins in mammalian cells, based on the expression of the pyrrolysyl-tRNA synthetase (PylRS)-tRNA(Pyl) pair from Methanosarcina mazei. Different types of external promoters were tested for the expression of tRNA(Pyl) in Chinese hamster ovary cells. When tRNA(Pyl) was expressed from a gene cluster under the control of the U6 promoter, the wild-type PylRS-tRNA(Pyl) pair facilitated the most efficient incorporation of a pyrrolysine analog, N(epsilon)-tert-butyloxycarbonyl-l-lysine (Boc-lysine), into proteins at the amber position. This PylRS-tRNA(Pyl) system yielded the Boc-lysine-containing protein in an amount accounting for 1% of the total protein in human embryonic kidney (HEK) 293 cells. We also created a PylRS variant specific to N(epsilon)-benzyloxycarbonyl-l-lysine, to incorporate this long, bulky, non-natural lysine derivative into proteins in HEK293. The recently reported variant specific to N(epsilon)-acetyllysine was also expressed, resulting in the genetic encoding of this naturally-occurring lysine modification in mammalian cells.

Protein photo-cross-linking in mammalian cells by site-specific incorporation of a photoreactive amino acid

We report a method of photo-cross-linking proteins in mammalian cells, which is based on site-specific incorporation of a photoreactive amino acid, p-benzoyl-L-phenylalanine (pBpa), through the use of an expanded genetic code. To analyze the cell signaling interactions involving the adaptor protein Grb2, pBpa was incorporated in its Src homology 2 (SH2) domain. The human GRB2 gene with an amber codon was introduced into Chinese hamster ovary (CHO) cells, together with the genes for the Bacillus stearothermophilus suppressor tRNATyr and a pBpa-specific variant of Escherichia coli tyrosyl-tRNA synthetase (TyrRS). The Grb2 variant with pBpa in the amber position was synthesized when pBpa was included in the growth medium. Upon exposure of cells to 365-nm light, protein variants containing pBpa in the positions proximal to the ligand-binding pocket were cross-linked with the transiently expressed epidermal growth factor (EGF) receptor in the presence of an EGF stimulus. Cross-linked complexes with endogenous proteins were also detected. In vivo photo-cross-linking with pBpa incorporated in proteins will be useful for studying protein-protein interactions in mammalian cells.

Crystal structure of bacterial RNA polymerase bound with a transcription inhibitor protein

The multi-subunit DNA-dependent RNA polymerase (RNAP) is the principal enzyme of transcription for gene expression. Transcription is regulated by various transcription factors. Gre factor homologue 1 (Gfh1), found in the Thermus genus, is a close homologue of the well-conserved bacterial transcription factor GreA, and inhibits transcription initiation and elongation by binding directly to RNAP. The structural basis of transcription inhibition by Gfh1 has remained elusive, although the crystal structures of RNAP and Gfh1 have been determined separately. Here we report the crystal structure of Thermus thermophilus RNAP complexed with Gfh1. The amino-terminal coiled-coil domain of Gfh1 fully occludes the channel formed between the two central modules of RNAP; this channel would normally be used for nucleotide triphosphate (NTP) entry into the catalytic site. Furthermore, the tip of the coiled-coil domain occupies the NTP β-γ phosphate-binding site. The NTP-entry channel is expanded, because the central modules are ‘ratcheted’ relative to each other by ∼7°, as compared with the previously reported elongation complexes. This ‘ratcheted state’ is an alternative structural state, defined by a newly acquired contact between the central modules. Therefore, the shape of Gfh1 is appropriate to maintain RNAP in the ratcheted state. Simultaneously, the ratcheting expands the nucleic-acid-binding channel, and kinks the bridge helix, which connects the central modules. Taken together, the present results reveal that Gfh1 inhibits transcription by preventing NTP binding and freezing RNAP in the alternative structural state. The ratcheted state might also be associated with other aspects of transcription, such as RNAP translocation and transcription termination.

Genetic-code evolution for protein synthesis with non-natural amino acids.

The genetic encoding of synthetic or non-natural amino acids promises to diversify the functions and structures of proteins. We applied rapid codon-reassignment for creating Escherichia coli strains unable to terminate translation at the UAG stop triplet, but efficiently decoding it as various tyrosine and lysine derivatives. This complete change in the UAG meaning enabled protein synthesis with these non-natural molecules at multiple defined sites, in addition to the 20 canonical amino acids. UAG was also redefined in the E. coli BL21 strain, suitable for the large-scale production of recombinant proteins, and its cell extract served the cell-free synthesis of an epigenetic protein, histone H4, fully acetylated at four specific lysine sites.

Structural basis for extracellular interactions between calcitonin receptor-like receptor and receptor activity-modifying protein 2 for adrenomedullin-specific binding

The calcitonin receptor‐like receptor (CRLR), a class B GPCR, forms a heterodimer with receptor activity‐modifying protein 2 (RAMP2), and serves as the adrenomedullin (AM) receptor to control neovascularization, while CRLR and RAMP1 form the calcitonin gene‐related peptide (CGRP) receptor. Here, we report the crystal structures of the RAMP2 extracellular domain alone and in the complex with the CRLR extracellular domain. The CRLR–RAMP2 complex exhibits several intermolecular interactions that were not observed in the previously reported CRLR–RAMP1 complex, and thus the shape of the putative ligand‐binding pocket of CRLR–RAMP2 is distinct from that of CRLR–RAMP1. The CRLR–RAMP2 interactions were confirmed for the full‐length proteins on the cell surface by site‐specific photo‐crosslinking. Mutagenesis revealed that AM binding requires RAMP2 residues that are not conserved in RAMP1. Therefore, the differences in both the shapes and the key residues of the binding pocket are essential for the ligand specificity.

Genetic Incorporation of a Photo-Crosslinkable Amino Acid Reveals Novel Protein Complexes with GRB2 in Mammalian Cells

Cell signaling pathways are essentially organized through the distribution of various types of binding domains in signaling proteins, with each domain binding to specific target molecules. Although identification of these targets is crucial for mapping the pathways, affinity-based or copurification methods are insufficient to distinguish between direct and indirect interactions in a cellular context. In the present study, we developed another approach involving the genetic encoding of a photo-crosslinkable amino acid. p-Trifluoromethyl-diazirinyl-l-phenylalanine was thus incorporated at a defined site in the Src homology 2 (SH2) domain of the adaptor protein GRB2 in human embryonic kidney cells. These cells were exposed to 365-nm light after an epidermal growth factor stimulus, and the crosslinkable GRB2-SH2 domain exclusively formed covalent bonds with directly interacting proteins. Proteomic mass spectrometry analysis identified these direct binders of GRB2-SH2 separately from the proteins noncovalently bound to the Src homology 3 domains of GRB2. In addition to two signaling-associated proteins (GIT1 and AF6), the heterogeneous nuclear ribonucleoproteins F, H1, and H2 were thus identified as novel direct binders. The results revealed a connection between the cell signaling protein and the nuclear machinery involved in mRNA processing, and demonstrated the usefulness of genetically encoded photo-crosslinkers for mapping protein-protein interactions in cells.

Structural basis of interleukin-5 dimer recognition by its α receptor

Interleukin‐5 (IL‐5), a major hematopoietin, stimulates eosinophil proliferation, migration, and activation, which have been implicated in the pathogenesis of allergic inflammatory diseases, such as asthma. The specific IL‐5 receptor (IL‐5R) consists of the IL‐5 receptor α subunit (IL‐5RA) and the common receptor β subunit (βc). IL‐5 binding to IL‐5R on target cells induces rapid tyrosine phosphorylation and activation of various cellular proteins, including JAK1/JAK2 and STAT1/STAT5. Here, we report the crystal structure of dimeric IL‐5 in complex with the IL‐5RA extracellular domains. The structure revealed that IL‐5RA sandwiches the IL‐5 homodimer by three tandem domains, arranged in a “wrench‐like” architecture. This association mode was confirmed for human cells expressing IL‐5 and the full‐length IL‐5RA by applying expanded genetic code technology: protein photo‐cross‐linking experiments revealed that the two proteins interact with each other in vivo in the same manner as that in the crystal structure. Furthermore, a comparison with the previously reported, partial GM‐CSF•GM‐CSFRA•βc structure enabled us to propose complete structural models for the IL‐5 and GM‐CSF receptor complexes, and to identify the residues conferring the cytokine‐specificities of IL‐5RA and GM‐CSFRA.

Site-specific incorporation of non-natural amino acids into proteins in mammalian cells with an expanded genetic code

We describe a detailed protocol for incorporating non-natural amino acids, 3-iodo-L-tyrosine (IY) and p-benzoyl-L-phenylalanine (pBpa), into proteins in response to the amber codon (the UAG stop codon) in mammalian cells. These amino acids, IY and pBpa, are applicable for structure determination and the analysis of a network of protein–protein interactions, respectively. This method involves (i) the mutagenesis of the gene encoding the protein of interest to create an amber codon at the desired site, (ii) the expression in mammalian cells of the bacterial pair of an amber suppressor tRNA and an aminoacyl-tRNA synthetase specific to IY or pBpa and (iii) the supplementation of the growth medium with these amino acids. The amber mutant gene, together with these bacterial tRNA and synthetase genes, is introduced into mammalian cells. Culturing these cells for 16–40 h allows the expression of the full-length product from the mutant gene, which contains the non-natural amino acid at the introduced amber position. This method is implemented using the conventional tools for molecular biology and treating cultured mammalian cells. This protocol takes 5–6 d for plasmid construction and 3–4 d for incorporating the non-natural amino acids into proteins.

Efficient Decoding of the UAG Triplet as a Full-Fledged Sense Codon Enhances the Growth of a prfA-Deficient Strain of Escherichia coli

We previously reassigned the amber UAG stop triplet as a sense codon in Escherichia coli by expressing a UAG-decoding tRNA and knocking out the prfA gene, encoding release factor 1. UAG triplets were left at the ends of about 300 genes in the genome. In the present study, we showed that the detrimental effect of UAG reassignment could be alleviated by increasing the efficiency of UAG translation instead of reducing the number of UAGs in the genome. We isolated an amber suppressor tRNA(Gln) variant displaying enhanced suppression activity, and we introduced it into the prfA knockout strain, RFzero-q, in place of the original suppressor tRNA(Gln). The resulting strain, RFzero-q3, translated UAG to glutamine almost as efficiently as the glutamine codons, and it proliferated faster than the parent RFzero-q strain. We identified two major factors in this growth enhancement. First, the sucB gene, which is involved in energy regeneration and has two successive UAG triplets at the end, was expressed at a higher level in RFzero-q3 than RFzero-q. Second, the ribosome stalling that occurred at UAG in RFzero-q was resolved in RFzero-q3. The results revealed the importance of backup stop triplets, UAA or UGA downstream of UAG, to avoid the deleterious impact of UAG reassignment on the proteome.

Tetrameric Interaction of the Ectoenzyme CD38 on the Cell Surface Enables Its Catalytic and Raft-Association Activities

The leukocyte cell-surface antigen CD38 is the major nicotinamide adenide dinucleotide glycohydrolase in mammals, and its ectoenzyme activity is involved in calcium mobilization. CD38 is also a raft-dependent signaling molecule. CD38 forms a tetramer on the cell surface, but the structural basis and the functional significance of tetramerization have remained unexplored. We identified the interfaces contributing to the homophilic interaction of mouse CD38 by site-specific crosslinking on the cell surface with an expanded genetic code, based on a crystallographic analysis. A combination of the three interfaces enables CD38 to tetramerize: one interface involving the juxtamembrane α-helix is responsible for the formation of the core dimer, which is further dimerized via the other two interfaces. This dimerization of dimers is required for the catalytic activity and the localization of CD38 in membrane rafts. The glycosylation prevents further self-association of the tetramer. Accordingly, the tetrameric interaction underlies the multifaceted actions of CD38.