Intron locations and functional deletions in relation to the design and evolution of a subgroup of class I tRNA synthetases

Schimmel, Paul; Shepard, Alyssa; Shiba, Kiyotaka

doi:10.1002/pro.5560011018

Protein Science

1992

DOI: 10.1002/pro.5560011018

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Intron locations and functional deletions in relation to the design and evolution of a subgroup of class I tRNA synthetases

Paul Schimmel

Alyssa Shepard

Kiyotaka Shiba

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Cited by 19 publications

(14 citation statements)

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“…In addition, the carbohydrates on the ␣ subunits are not the same in all the gonadotropin dimers (7-9) and the receptor contact sites on the ␣ subunit differ among the hormones (10, 39, 40). Thus, a priori one cannot predict based on the CG model that other members of the glycoprotein hormone family can be converted to single chains.Tethering a variety of multisubunit complexes into single chains has been performed by several laboratories to increase protein stability or activity (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). In such studies, a linker sequence was designed to give optimal alignment of determinants.…”

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confidence: 99%

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Expression of Biologically Active Fusion Genes Encoding the Common α Subunit and the Follicle-stimulating Hormone β Subunit

Sato

Kudo³

et al. 1996

Journal of Biological Chemistry

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The gonadotropin/thyrotropin hormone family is characterized by a heterodimeric structure composed of a common ␣ subunit noncovalently linked to a hormonespecific ␤ subunit. The conformation of the heterodimer is essential for controlling secretion, hormone-specific post-translational modifications, and signal transduction. Structure-function studies of follicle-stimulating hormone (FSH) and the other glycoprotein hormones are often hampered by mutagenesis-induced defects in subunit combination. Thus, the ability to overcome the limitation of subunit assembly would expand the range of structure-activity relationships that can be performed on these hormones. Here we converted the FSH heterodimer to a single chain by genetically fusing the carboxyl end of the FSH ␤ subunit to the amino end of the ␣ subunit in the presence or absence of a linker sequence. In the absence of the CTP linker, the secretion rate was decreased over 3-fold. Unexpectedly, however, receptor binding/signal transduction was unaffected by the absence of the linker. These data show that the single-chain FSH was secreted efficiently and is biologically active and that the conformation determinants required for secretion and biologic activity are not the same.One of the hallmarks of the gonadotropin and thyrotropin hormone family is their heterodimeric structure, consisting of a common ␣ subunit and a unique ␤ subunit (1). Subunit assembly is vital to the function of these hormones: (i) only the dimers are bioactive, (ii) maturation of the hormone-specific oligosaccharides is dependent on the formation of the heterodimer complex, and (iii) the secretion efficiency of the dimer is determined by the ␤ subunit. Previously, we constructed a chimera composed of the human chorionic gonadotropin (hCG) 1 ␤ subunit genetically fused to the ␣ subunit, and the resulting single polypeptide chain was efficiently secreted and was biologically active (2). Because subunit dissociation would lead to inactivation of the heterodimer, a single-chain form could have higher biological activity.Although the gonadotropin dimers have similar structural features, they are nevertheless unique. The ␣ subunit in the dimers has a different conformation which is manifested by distinct immunological and spectral characteristics (3-6). In addition, the carbohydrates on the ␣ subunits are not the same in all the gonadotropin dimers (7-9) and the receptor contact sites on the ␣ subunit differ among the hormones (10, 39, 40). Thus, a priori one cannot predict based on the CG model that other members of the glycoprotein hormone family can be converted to single chains.Tethering a variety of multisubunit complexes into single chains has been performed by several laboratories to increase protein stability or activity (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). In such studies, a linker sequence was designed to give optimal alignment of determinants. In the case of the glycoprotein hormones, we presumed that a linker would be required for successful expression of the correspondin...

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mentioning

confidence: 99%

“…Tethering a variety of multisubunit complexes into single chains has been performed by several laboratories to increase protein stability or activity (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). In such studies, a linker sequence was designed to give optimal alignment of determinants.…”

mentioning

confidence: 99%

Expression of Biologically Active Fusion Genes Encoding the Common α Subunit and the Follicle-stimulating Hormone β Subunit

Sato

Kudo³

et al. 1996

Journal of Biological Chemistry

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“…In Escherichia coli, the enzyme is a monomer of 939 amino acids and is composed of a N-terminal catalytic domain and a C-terminal domain. Although no crystal structure is available for Ile-tRNA synthetase, limited homology with Met-tRNA synthetase, the structure of which has been determined (13), has permitted mapping of the structural domains and elements of secondary structure in Ile-tRNA synthetase (14). The N-terminal region of Ile-tRNA synthetase is predicted to have the nucleotide binding domain, a common structural motif among 10 aminoacyl-tRNA synthetases (14).…”

mentioning

confidence: 99%

“…Although no crystal structure is available for Ile-tRNA synthetase, limited homology with Met-tRNA synthetase, the structure of which has been determined (13), has permitted mapping of the structural domains and elements of secondary structure in Ile-tRNA synthetase (14). The N-terminal region of Ile-tRNA synthetase is predicted to have the nucleotide binding domain, a common structural motif among 10 aminoacyl-tRNA synthetases (14). The C-terminal domain contains an anticodon binding region and a zinc binding site, both of which are necessary for aminoacylation activity (12,(15)(16)(17).…”

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confidence: 99%

Dominant negative inhibition by fragments of a monomeric enzyme

Michaels

Schimmel

Shiba

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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Dominant negative inhibition is most commonly seen when a mutant subunit of a multisubunit protein is coexpressed with the wild-type protein so that assembly of a functional oligomer is impaired. By analogy, it should be possible to interfere with the functional assembly of a monomeric enzyme by interfering with the folding pathway. Experiments in vitro by others suggested that fragments of a monomeric enzyme might be exploited for this purpose. We report here dominant negative inhibition of bacterial cell growth by expression of fragments of a tRNA synthetase. Inhibition is fragment-specific, as not all fragments cause inhibition. An inhibitory fragment characterized in more detail forms a specific complex with the intact enzyme in vivo, leading to enzyme inactivation. This fragment also associated stoichiometrically with the full-length enzyme in vitro after denaturation and refolding, and the resulting complex was catalytically inactive. Inhibition therefore appears to arise from an interruption in the folding pathway of the wild-type enzyme, thus suggesting a new strategy to design dominant negative inhibitors of monomeric enzymes.

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“…The variable size of the CP1 insertion in class I enzymes possibly reflects the different origins of the motifs recruited into the catalytic domain for acceptor helix interactions (22). We wondered whether this insertion, once acquired, was under the same relative selective pressure as the classdefining catalytic core with which it is combined and, in addition, whether a similar selective pressure was exerted on the C-terminal anticodon-binding domain.…”

mentioning

confidence: 99%

Human cytoplasmic isoleucyl-tRNA synthetase: selective divergence of the anticodon-binding domain and acquisition of a new structural unit.

Shiba

Suzuki

Shigesada

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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We show here that the class I human cytoplasmic isoleucyl-tRNA synthetase is an exception large polypeptide (1266 aa) which, unlike its homologues in lower eukaryotes and prokaryotes, has a third domain oftwo repeats ofan -9O-aa sequence appended to its C-terminal end. The relationship between trinucleotides and amino acids spelled out by the genetic code is determined by the specificity of aminoacyl-tRNA synthetases, which catalyze attachment of amino acids to the cognate tRNAs bearing anticodon trinucleotides (1-4). The enzymes are among the most ancient proteins and, in their earliest versions, probably consisted of polypeptides that catalyzed formation of aminoacyladenylates (5-8). The sequences and structures of the adenylate-formation domain of tRNA synthetases are the basis for dividing them into two distinct classes of 10 enzymes each (1, 9-11). Several (at least 8) of the enzymes aminoacylate small RNA oligonucleotides that recapitulate part or all of the 7-bp acceptor stem of their cognate tRNAs (12). The specific sequence/structures in small RNA helices that confer aminoacylation constitute an operational RNA code for amino acids which presumably has a historical relationship to the genetic code (12).The class-defining catalytic domain which also encodes determinants for tRNA acceptor helix interactions is the functional unit needed for aminoacylation ofRNA oligonucleotides. Joined to the class-defining domain is a second domain, idiosyncratic to the synthetase, which provides interactions with the parts of the tRNA which are distal to the amino acid attachment site. In some synthetases, this second domain interacts directly with the anticodon (13, 14), while in other enzymes no contact is made between the second domain and the anticodon (15,16). Thus, to a first approximation, the two domains in tRNA synthetases interact with the two distinct domains ofthe L-shaped tRNA structure so that the parts ofthe synthetase and of the tRNA which are needed for the operational RNA code are segregated into discrete protein and RNA domains. In recent experiments, a fragment containing the conserved active-site domain of a tRNA synthetase has been shown to have the same activity as the full-length enzyme for aminoacylation ofan RNA oligonucleotide corresponding to the cognate tRNA acceptor stem (17).As a prototypical example ofa member ofa subgroup ofthe most recently evolved class I enzymes, we are particularly interested in the isoleucine enzyme. The five members ofthis subgroup, like all class I enzymes, have an N-terminal nucleotide-binding fold consisting of alternating (3-strands and a-helices and a C-terminal domain that is rich in a-helices and that contains residues needed for interactions with the parts ofthe tRNA distal to the amino acid attachment site (18,19). These enzymes, the cysteinyl-, isoleucyl-, leucyl-, methionyl-, and valyl-tRNA synthetases, are grouped together because they are more closely related in sequence to each other than to the other five members of class I (19, 20). In Esche...

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Intron locations and functional deletions in relation to the design and evolution of a subgroup of class I tRNA synthetases

Cited by 19 publications

References 48 publications

Expression of Biologically Active Fusion Genes Encoding the Common α Subunit and the Follicle-stimulating Hormone β Subunit

Expression of Biologically Active Fusion Genes Encoding the Common α Subunit and the Follicle-stimulating Hormone β Subunit

Dominant negative inhibition by fragments of a monomeric enzyme

Human cytoplasmic isoleucyl-tRNA synthetase: selective divergence of the anticodon-binding domain and acquisition of a new structural unit.

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