Crystal structure of human cytosolic aspartyl-tRNA synthetase

Human cytosolic aspartyl-tRNA synthetase (DRS) catalyzes the attachment of the amino acid aspartic acid to its cognate tRNA and it is a component of the multi-tRNA synthetase complex (MSC) which has been known to be involved in unexpected signaling pathways. Here, we report the crystal structure of DRS at 2.25 Å resolution. DRS is a homodimer with a dimer interface 3,750.5 Å2which comprises of 16.6% of the monomeric surface area. Our structure reveals the C-terminal end of the N-helix which is considered as a unique addition in DRS, and its conformation further supports the switching model of the N-helix for the transfer of tRNAAsp to elongation factor 1α. From our analyses of the crystal structure and post-translational modification of DRS, we suggest that the phosphorylation of Ser146 provokes the separation of DRS from the MSC and provides the binding site for an interaction partner with unforeseen functions.

Download Full-text

Crystal Structure of Circadian Clock Protein KaiA fromSynechococcus elongatus

Journal of Biological Chemistry ◽

10.1074/jbc.m400077200 ◽

2004 ◽

Vol 279 (19) ◽

pp. 20511-20518 ◽

Cited By ~ 75

Author(s):

Sheng Ye ◽

Ioannis Vakonakis ◽

Thomas R. Ioerger ◽

Andy C. LiWang ◽

James C. Sacchettini

Keyword(s):

Crystal Structure ◽

Circadian Clock ◽

Binding Site ◽

Regulation Mechanism ◽

Response Regulators ◽

Dimer Interface ◽

Helix Bundle ◽

Bacterial Response ◽

Four Helix Bundle ◽

Clock Protein

The circadian clock found inSynechococcus elongatus, the most ancient circadian clock, is regulated by the interaction of three proteins, KaiA, KaiB, and KaiC. While the precise function of these proteins remains unclear, KaiA has been shown to be a positive regulator of the expression of KaiB and KaiC. The 2.0-Å structure of KaiA ofS. elongatusreported here shows that the protein is composed of two independently folded domains connected by a linker. The NH2-terminalpseudo-receiver domain has a similar fold with that of bacterial response regulators, whereas the COOH-terminal four-helix bundle domain is novel and forms the interface of the 2-fold-related homodimer. The COOH-terminal four-helix bundle domain has been shown to contain the KaiC binding site. The structure suggests that the KaiB binding site is covered in the dimer interface of the KaiA “closed” conformation, observed in the crystal structure, which suggests an allosteric regulation mechanism.

Download Full-text

Structure of nondiscriminating glutamyl-tRNA synthetase fromThermotoga maritima

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444910019086 ◽

2010 ◽

Vol 66 (7) ◽

pp. 813-820 ◽

Cited By ~ 5

Author(s):

Takuhiro Ito ◽

Noriko Kiyasu ◽

Risa Matsunaga ◽

Seizo Takahashi ◽

Shigeyuki Yokoyama

Keyword(s):

Crystal Structure ◽

Amino Acid ◽

Thermotoga Maritima ◽

Trna Synthetase ◽

Characteristic Structure ◽

Trna Synthetases ◽

Aminoacyl Trna Synthetases ◽

Rossmann Fold

Aminoacyl-tRNA synthetases produce aminoacyl-tRNAs from the substrate tRNA and its cognate amino acid with the aid of ATP. Two types of glutamyl-tRNA synthetase (GluRS) have been discovered: discriminating GluRS (D-GluRS) and nondiscriminating GluRS (ND-GluRS). D-GluRS glutamylates tRNAGluonly, while ND-GluRS glutamylates both tRNAGluand tRNAGln. ND-GluRS produces the intermediate Glu-tRNAGln, which is converted to Gln-tRNAGlnby Glu-tRNAGlnamidotransferase. Two GluRS homologues fromThermotoga maritima, TM1875 and TM1351, have been biochemically characterized and it has been clarified that only TM1875 functions as an ND-GluRS. Furthermore, the crystal structure of theT. maritimaND-GluRS, TM1875, was determined in complex with a Glu-AMP analogue at 2.0 Å resolution. TheT. maritimaND-GluRS contains a characteristic structure in the connective-peptide domain, which is inserted into the catalytic Rossmann-fold domain. The glutamylation ability of tRNAGlnby ND-GluRS was measured in the presence of the bacterial Glu-tRNAGlnamidotransferase GatCAB. Interestingly, the glutamylation efficiency was not affected even in the presence of excess GatCAB. Therefore, GluRS avoids competition with GatCAB and glutamylates tRNAGln.

Download Full-text

Crystal structure of the SARS-CoV-2 non-structural protein 9, Nsp9

10.1101/2020.03.28.013920 ◽

2020 ◽

Cited By ~ 3

Author(s):

D. R. Littler ◽

B. S. Gully ◽

R. N. Colson ◽

J Rossjohn

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Peptide Binding ◽

Structural Protein ◽

Dimer Interface ◽

X Ray ◽

3C Protease ◽

Peptide Binding Site ◽

High Level ◽

Viral Genomic Rna

AbstractMany of the proteins produced by SARS-CoV-2 have related counterparts across the Severe Acute Respiratory Syndrome (SARS-CoV) family. One such protein is non-structural protein 9 (Nsp9), which is thought to mediate both viral replication and virulence. Current understanding suggests that Nsp9 is involved in viral genomic RNA reproduction. Nsp9 is thought to bind RNA via a fold that is unique to this class of betacoronoaviruses although the molecular basis for this remains ill-defined. We sought to better characterise the SARS-CoV-2 Nsp9 protein and subsequently solved its X-ray crystal structure, in an apo-form and, unexpectedly, in a peptide-bound form with a sequence originating from a rhinoviral 3C protease sequence (LEVL). The structure of the SARS-CoV-2 Nsp9 revealed the high level of structural conservation within the Nsp9 family. The exogenous peptide binding site is close to the dimer interface and impacted on the relative juxtaposition of the monomers within the homodimer. Together we have established a protocol for the production of SARS-CoV-2 Nsp9, determined its structure and identified a peptide-binding site that may warrant further study from the perspective of understanding Nsp9 function.

Download Full-text

Amino Acid Residues in Elongation Factor Tu from Escherichia Coli near the Binding Site for the 3′ Terminus of Aminoacyl-tRNA

Gene Manipulation and Expression ◽

10.1007/978-94-011-6565-5_35 ◽

1985 ◽

pp. 497-508

Author(s):

J. Jonák ◽

T. E. Petersen ◽

B. Meloun ◽

I. Rychlík

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Binding Site ◽

Elongation Factor ◽

Elongation Factor Tu ◽

Amino Acid Residues

Download Full-text

Barley elongation factor 1α: genomic organization, DNA sequence, and phylogenetic implications

Genome ◽

10.1139/g97-073 ◽

1997 ◽

Vol 40 (4) ◽

pp. 559-565 ◽

Cited By ~ 3

Author(s):

Peter S. Nielsen ◽

Andris Kleinhofs ◽

Odd-Arne Olsen

Keyword(s):

Amino Acid ◽

Gene Family ◽

Genomic Organization ◽

Elongation Factor ◽

Total Proteins ◽

Elongation Factor 1Α ◽

Barley Endosperm ◽

Barley Cultivars ◽

Amino Acid Sequence Comparison ◽

Phylogenetic Implications

A full length cDNA clone encoding the 447 amino acid long barley (Hordeum vulgare cv. Bomi) endosperm elongation factor 1α (eF-1α) was isolated by a differential screening procedure. RFLP mapping of eF-1α showed that the barley genome contains a small eF-1α gene family of 4 copies, with 1 copy of the gene being located on each of chromosomes 2, 4, 6, and 7. Analysis of barley endosperm total proteins by Western blot with antibodies directed towards wheat eF-1α and the sea urchin 51 kDa proteins gave a single band of the expected molecular weight. Amino acid sequence comparison with other plant eF-1α sequences showed that the isolated barley endosperm eF-1α is more similar to the published wheat eF-1α sequence than to eF-1α sequences previously published for the barley cultivars Igri and Dicktoo. The phylogenetic analysis suggests that the barley eF-1α gene family can be divided into two subfamilies and that two ancestral genes existed before the divergence of monocotyledonous and dicotyledonous plants.Key words: endosperm, gene family, RFLP.

Download Full-text

Structure of the Pseudomonas aeruginosa transamidosome reveals unique aspects of bacterial tRNA-dependent asparagine biosynthesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1423314112 ◽

2014 ◽

Vol 112 (2) ◽

pp. 382-387 ◽

Cited By ~ 20

Author(s):

Tateki Suzuki ◽

Akiyoshi Nakamura ◽

Koji Kato ◽

Dieter Söll ◽

Isao Tanaka ◽

...

Keyword(s):

Crystal Structure ◽

Pseudomonas Aeruginosa ◽

Protein Synthesis ◽

Genetic Code ◽

Trna Synthetase ◽

Complex Assembly ◽

Evolutionary Pathway ◽

Bacterial Type ◽

Direct Attachment ◽

Cognate Trna

Many prokaryotes lack a tRNA synthetase to attach asparagine to its cognate tRNAAsn, and instead synthesize asparagine from tRNAAsn-bound aspartate. This conversion involves two enzymes: a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) that forms Asp-tRNAAsn, and a heterotrimeric amidotransferase GatCAB that amidates Asp-tRNAAsn to form Asn-tRNAAsn for use in protein synthesis. ND-AspRS, GatCAB, and tRNAAsn may assemble in an ∼400-kDa complex, known as the Asn-transamidosome, which couples the two steps of asparagine biosynthesis in space and time to yield Asn-tRNAAsn. We report the 3.7-Å resolution crystal structure of the Pseudomonas aeruginosa Asn-transamidosome, which represents the most common machinery for asparagine biosynthesis in bacteria. We show that, in contrast to a previously described archaeal-type transamidosome, a bacteria-specific GAD domain of ND-AspRS provokes a principally new architecture of the complex. Both tRNAAsn molecules in the transamidosome simultaneously serve as substrates and scaffolds for the complex assembly. This architecture rationalizes an elevated dynamic and a greater turnover of ND-AspRS within bacterial-type transamidosomes, and possibly may explain a different evolutionary pathway of GatCAB in organisms with bacterial-type vs. archaeal-type Asn-transamidosomes. Importantly, because the two-step pathway for Asn-tRNAAsn formation evolutionarily preceded the direct attachment of Asn to tRNAAsn, our structure also may reflect the mechanism by which asparagine was initially added to the genetic code.

Download Full-text

Crystal Structure of Transglutaminase 2 with GTP Complex and Amino Acid Sequence Evidence of Evolution of GTP Binding Site

PLoS ONE ◽

10.1371/journal.pone.0107005 ◽

2014 ◽

Vol 9 (9) ◽

pp. e107005 ◽

Cited By ~ 24

Author(s):

Tae-Ho Jang ◽

Dong-Sup Lee ◽

Kihang Choi ◽

Eui Man Jeong ◽

In-Gyu Kim ◽

...

Keyword(s):

Crystal Structure ◽

Amino Acid ◽

Amino Acid Sequence ◽

Binding Site ◽

Transglutaminase 2 ◽

Gtp Binding

Download Full-text

Early Evolution of the Metazoa and Phylogenetic Status of Diploblasts as Inferred from Amino Acid Sequence of Elongation Factor-1α

Molecular Phylogenetics and Evolution ◽

10.1006/mpev.1996.0036 ◽

1996 ◽

Vol 5 (2) ◽

pp. 414-422 ◽

Cited By ~ 22

Author(s):

Mari Kobayashi ◽

Hiroshi Wada ◽

Noriyuki Satoh

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Elongation Factor ◽

Early Evolution ◽

Elongation Factor 1Α ◽

Phylogenetic Status

Download Full-text

Fine-Tuning of Alanyl-tRNA Synthetase Quality Control Alleviates Global Dysregulation of the Proteome

Genes ◽

10.3390/genes11101222 ◽

2020 ◽

Vol 11 (10) ◽

pp. 1222

Author(s):

Paul Kelly ◽

Arundhati Kavoor ◽

Michael Ibba

Keyword(s):

Amino Acids ◽

Quality Control ◽

Amino Acid ◽

Antibiotic Sensitivity ◽

Trna Synthetase ◽

Fine Tuning ◽

Trna Synthetases ◽

E Coli ◽

Suppressor Mutations ◽

Cognate Trna

One integral step in the transition from a nucleic acid encoded-genome to functional proteins is the aminoacylation of tRNA molecules. To perform this activity, aminoacyl-tRNA synthetases (aaRSs) activate free amino acids in the cell forming an aminoacyl-adenylate before transferring the amino acid on to its cognate tRNA. These newly formed aminoacyl-tRNA (aa-tRNA) can then be used by the ribosome during mRNA decoding. In Escherichia coli, there are twenty aaRSs encoded in the genome, each of which corresponds to one of the twenty proteinogenic amino acids used in translation. Given the shared chemicophysical properties of many amino acids, aaRSs have evolved mechanisms to prevent erroneous aa-tRNA formation with non-cognate amino acid substrates. Of particular interest is the post-transfer proofreading activity of alanyl-tRNA synthetase (AlaRS) which prevents the accumulation of Ser-tRNAAla and Gly-tRNAAla in the cell. We have previously shown that defects in AlaRS proofreading of Ser-tRNAAla lead to global dysregulation of the E. coli proteome, subsequently causing defects in growth, motility, and antibiotic sensitivity. Here we report second-site AlaRS suppressor mutations that alleviate the aforementioned phenotypes, revealing previously uncharacterized residues within the AlaRS proofreading domain that function in quality control.

Download Full-text