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Unique domain appended to vertebrate tRNA synthetase is essential for vascular development.

Xu X, Shi Y, Zhang HM, Swindell EC, Marshall AG, Guo M, Kishi S, Yang XL - Nat Commun (2012)

Bottom Line: A structure-based second-site mutation, designed to release the sequestered NLS, restored normal vasculature.Thus, the essential function of SerRS in vascular development depends on UNE-S.These results are the first to show an essential role for a tRNA synthetase-associated appended domain at the organism level, and suggest that acquisition of UNE-S has a role in the establishment of the closed circulatory systems of vertebrates.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

ABSTRACT
New domains were progressively added to cytoplasmic aminoacyl transfer RNA (tRNA) synthetases during evolution. One example is the UNE-S domain, appended to seryl-tRNA synthetase (SerRS) in species that developed closed circulatory systems. Here we show using solution and crystal structure analyses and in vitro and in vivo functional studies that UNE-S harbours a robust nuclear localization signal (NLS) directing SerRS to the nucleus where it attenuates vascular endothelial growth factor A expression. We also show that SerRS mutants previously linked to vasculature abnormalities either deleted the NLS or have the NLS sequestered in an alternative conformation. A structure-based second-site mutation, designed to release the sequestered NLS, restored normal vasculature. Thus, the essential function of SerRS in vascular development depends on UNE-S. These results are the first to show an essential role for a tRNA synthetase-associated appended domain at the organism level, and suggest that acquisition of UNE-S has a role in the establishment of the closed circulatory systems of vertebrates.

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Related in: MedlinePlus

Crystal structure of human SerRS and the hypothetic conformational change caused by F383V mutation.(a) Crystal structure of human SerRS showing one subunit of the dimer. Secondary structures are labelled on the ribbon diagram. Motifs 1, 2 and 3 within the aminoacylation domain are shown in orange, magenta and blue, respectively. Inset: SerRS dimer with the second subunit shown in grey. Dimerization is mediated through the aminoacylation domain. (b) Superposition of the three dimers of SerRS contained in each asymmetric unit. The aminoacylation domains are superimposed to show the flexibility of the N-terminal tRNA-binding domains. (c) Spatial location of residue F383. F383 is located in the loop region of the β10–β11 hairpin within the aminoacylation domain, and forms a hydrophobic pocket with residues H170 and F316. The hydrophobic pocket is close to the active site, which is indicated by a modelled Ser-AMP (aminoacylation reaction intermediate) molecule. The N-terminus of the UNE-S domain is located close to F383, and the rest of UNE-S including the NLS is disordered in the crystal structure. The F383V mutation might affect the conformation of UNE-S and obscure the NLS, as illustrated in a model of the F383V mutant shown on the right. (d) Stereo image of partial electron density around F383, F316, H170 and D378 (shown in sticks). The 2Fo-Fc electron density map at 1.5 σ is shown in grey, and the backbone traces of the structure are shown in light green.
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f4: Crystal structure of human SerRS and the hypothetic conformational change caused by F383V mutation.(a) Crystal structure of human SerRS showing one subunit of the dimer. Secondary structures are labelled on the ribbon diagram. Motifs 1, 2 and 3 within the aminoacylation domain are shown in orange, magenta and blue, respectively. Inset: SerRS dimer with the second subunit shown in grey. Dimerization is mediated through the aminoacylation domain. (b) Superposition of the three dimers of SerRS contained in each asymmetric unit. The aminoacylation domains are superimposed to show the flexibility of the N-terminal tRNA-binding domains. (c) Spatial location of residue F383. F383 is located in the loop region of the β10–β11 hairpin within the aminoacylation domain, and forms a hydrophobic pocket with residues H170 and F316. The hydrophobic pocket is close to the active site, which is indicated by a modelled Ser-AMP (aminoacylation reaction intermediate) molecule. The N-terminus of the UNE-S domain is located close to F383, and the rest of UNE-S including the NLS is disordered in the crystal structure. The F383V mutation might affect the conformation of UNE-S and obscure the NLS, as illustrated in a model of the F383V mutant shown on the right. (d) Stereo image of partial electron density around F383, F316, H170 and D378 (shown in sticks). The 2Fo-Fc electron density map at 1.5 σ is shown in grey, and the backbone traces of the structure are shown in light green.

Mentions: To understand how F383V affects nuclear localization, we determined the crystal structure of human SerRS at 2.9 Å resolution (Fig. 4a and Supplementary Table S1). The human protein shares overall 81% sequence identity with the fish ortholog, and F383 is a strictly conserved residue from fish to humans (Fig. 1 and Supplementary Fig. S1). Three independent homodimers of SerRS were found in the asymmetric unit of the crystal. While the conformation of the aminoacylation domain is almost the same for all three dimers in the asymmetric unit, the N-terminal tRNA-binding domains have more flexible structures (Fig. 4b). Interestingly, the C-terminal UNE-S domain (including the NLS) was mostly disordered in all six subunits, suggesting a dynamic conformation of the NLS that would enhance its accessibility to the nuclear transport machinery. F383 is located near the end of a β-strand (β10) that is part of the core seven-stranded antiparallel β-sheet (β1–β9–β10–β11–β13–β8–β7) of the aminoacylation domain, and spatially close to the active site and the flexible NLS (Fig. 4a,c). The side chain of F383 forms hydrophobic interactions with H170 and F316 to stabilize the β10–β11 hairpin as part of the central core (Fig. 4c). A stereo image of the electron density map surrounding F383 is shown in Figure 4d. We speculated that the F383V substitution would destabilize the hydrophobic core and, in some way, create an internal binding site for the NLS. As a result, the NLS would become less accessible, as illustrated in Figure 4c, and less able to facilitate nuclear localization.


Unique domain appended to vertebrate tRNA synthetase is essential for vascular development.

Xu X, Shi Y, Zhang HM, Swindell EC, Marshall AG, Guo M, Kishi S, Yang XL - Nat Commun (2012)

Crystal structure of human SerRS and the hypothetic conformational change caused by F383V mutation.(a) Crystal structure of human SerRS showing one subunit of the dimer. Secondary structures are labelled on the ribbon diagram. Motifs 1, 2 and 3 within the aminoacylation domain are shown in orange, magenta and blue, respectively. Inset: SerRS dimer with the second subunit shown in grey. Dimerization is mediated through the aminoacylation domain. (b) Superposition of the three dimers of SerRS contained in each asymmetric unit. The aminoacylation domains are superimposed to show the flexibility of the N-terminal tRNA-binding domains. (c) Spatial location of residue F383. F383 is located in the loop region of the β10–β11 hairpin within the aminoacylation domain, and forms a hydrophobic pocket with residues H170 and F316. The hydrophobic pocket is close to the active site, which is indicated by a modelled Ser-AMP (aminoacylation reaction intermediate) molecule. The N-terminus of the UNE-S domain is located close to F383, and the rest of UNE-S including the NLS is disordered in the crystal structure. The F383V mutation might affect the conformation of UNE-S and obscure the NLS, as illustrated in a model of the F383V mutant shown on the right. (d) Stereo image of partial electron density around F383, F316, H170 and D378 (shown in sticks). The 2Fo-Fc electron density map at 1.5 σ is shown in grey, and the backbone traces of the structure are shown in light green.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3293412&req=5

f4: Crystal structure of human SerRS and the hypothetic conformational change caused by F383V mutation.(a) Crystal structure of human SerRS showing one subunit of the dimer. Secondary structures are labelled on the ribbon diagram. Motifs 1, 2 and 3 within the aminoacylation domain are shown in orange, magenta and blue, respectively. Inset: SerRS dimer with the second subunit shown in grey. Dimerization is mediated through the aminoacylation domain. (b) Superposition of the three dimers of SerRS contained in each asymmetric unit. The aminoacylation domains are superimposed to show the flexibility of the N-terminal tRNA-binding domains. (c) Spatial location of residue F383. F383 is located in the loop region of the β10–β11 hairpin within the aminoacylation domain, and forms a hydrophobic pocket with residues H170 and F316. The hydrophobic pocket is close to the active site, which is indicated by a modelled Ser-AMP (aminoacylation reaction intermediate) molecule. The N-terminus of the UNE-S domain is located close to F383, and the rest of UNE-S including the NLS is disordered in the crystal structure. The F383V mutation might affect the conformation of UNE-S and obscure the NLS, as illustrated in a model of the F383V mutant shown on the right. (d) Stereo image of partial electron density around F383, F316, H170 and D378 (shown in sticks). The 2Fo-Fc electron density map at 1.5 σ is shown in grey, and the backbone traces of the structure are shown in light green.
Mentions: To understand how F383V affects nuclear localization, we determined the crystal structure of human SerRS at 2.9 Å resolution (Fig. 4a and Supplementary Table S1). The human protein shares overall 81% sequence identity with the fish ortholog, and F383 is a strictly conserved residue from fish to humans (Fig. 1 and Supplementary Fig. S1). Three independent homodimers of SerRS were found in the asymmetric unit of the crystal. While the conformation of the aminoacylation domain is almost the same for all three dimers in the asymmetric unit, the N-terminal tRNA-binding domains have more flexible structures (Fig. 4b). Interestingly, the C-terminal UNE-S domain (including the NLS) was mostly disordered in all six subunits, suggesting a dynamic conformation of the NLS that would enhance its accessibility to the nuclear transport machinery. F383 is located near the end of a β-strand (β10) that is part of the core seven-stranded antiparallel β-sheet (β1–β9–β10–β11–β13–β8–β7) of the aminoacylation domain, and spatially close to the active site and the flexible NLS (Fig. 4a,c). The side chain of F383 forms hydrophobic interactions with H170 and F316 to stabilize the β10–β11 hairpin as part of the central core (Fig. 4c). A stereo image of the electron density map surrounding F383 is shown in Figure 4d. We speculated that the F383V substitution would destabilize the hydrophobic core and, in some way, create an internal binding site for the NLS. As a result, the NLS would become less accessible, as illustrated in Figure 4c, and less able to facilitate nuclear localization.

Bottom Line: A structure-based second-site mutation, designed to release the sequestered NLS, restored normal vasculature.Thus, the essential function of SerRS in vascular development depends on UNE-S.These results are the first to show an essential role for a tRNA synthetase-associated appended domain at the organism level, and suggest that acquisition of UNE-S has a role in the establishment of the closed circulatory systems of vertebrates.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

ABSTRACT
New domains were progressively added to cytoplasmic aminoacyl transfer RNA (tRNA) synthetases during evolution. One example is the UNE-S domain, appended to seryl-tRNA synthetase (SerRS) in species that developed closed circulatory systems. Here we show using solution and crystal structure analyses and in vitro and in vivo functional studies that UNE-S harbours a robust nuclear localization signal (NLS) directing SerRS to the nucleus where it attenuates vascular endothelial growth factor A expression. We also show that SerRS mutants previously linked to vasculature abnormalities either deleted the NLS or have the NLS sequestered in an alternative conformation. A structure-based second-site mutation, designed to release the sequestered NLS, restored normal vasculature. Thus, the essential function of SerRS in vascular development depends on UNE-S. These results are the first to show an essential role for a tRNA synthetase-associated appended domain at the organism level, and suggest that acquisition of UNE-S has a role in the establishment of the closed circulatory systems of vertebrates.

Show MeSH
Related in: MedlinePlus