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A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNA(Cys)
The putative translation elongation factor Mbar_A0971 from the methanogenic archaeon Methanosarcina barkeri was proposed to be the pyrrolysine-specific paralogue of EF-Tu (“EF-Pyl”). In the present study, the crystal structures of its homologue from Methanosarcina mazei (MM1309) were determined in t...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Springer Netherlands
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4329189/ https://www.ncbi.nlm.nih.gov/pubmed/25618148 http://dx.doi.org/10.1007/s10969-015-9193-6 |
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author | Yanagisawa, Tatsuo Ishii, Ryohei Hikida, Yasushi Fukunaga, Ryuya Sengoku, Toru Sekine, Shun-ichi Yokoyama, Shigeyuki |
author_facet | Yanagisawa, Tatsuo Ishii, Ryohei Hikida, Yasushi Fukunaga, Ryuya Sengoku, Toru Sekine, Shun-ichi Yokoyama, Shigeyuki |
author_sort | Yanagisawa, Tatsuo |
collection | PubMed |
description | The putative translation elongation factor Mbar_A0971 from the methanogenic archaeon Methanosarcina barkeri was proposed to be the pyrrolysine-specific paralogue of EF-Tu (“EF-Pyl”). In the present study, the crystal structures of its homologue from Methanosarcina mazei (MM1309) were determined in the GMPPNP-bound, GDP-bound, and apo forms, by the single-wavelength anomalous dispersion phasing method. The three MM1309 structures are quite similar (r.m.s.d. < 0.1 Å). The three domains, corresponding to domains 1, 2, and 3 of EF-Tu/SelB/aIF2γ, are packed against one another to form a closed architecture. The MM1309 structures resemble those of bacterial/archaeal SelB, bacterial EF-Tu in the GTP-bound form, and archaeal initiation factor aIF2γ, in this order. The GMPPNP and GDP molecules are visible in their co-crystal structures. Isothermal titration calorimetry measurements of MM1309·GTP·Mg(2+), MM1309·GDP·Mg(2+), and MM1309·GMPPNP·Mg(2+) provided dissociation constants of 0.43, 26.2, and 222.2 μM, respectively. Therefore, the affinities of MM1309 for GTP and GDP are similar to those of SelB rather than those of EF-Tu. Furthermore, the switch I and II regions of MM1309 are involved in domain–domain interactions, rather than nucleotide binding. The putative binding pocket for the aminoacyl moiety on MM1309 is too small to accommodate the pyrrolysyl moiety, based on a comparison of the present MM1309 structures with that of the EF-Tu·GMPPNP·aminoacyl-tRNA ternary complex. A hydrolysis protection assay revealed that MM1309 binds cysteinyl (Cys)-tRNA(Cys) and protects the aminoacyl bond from non-enzymatic hydrolysis. Therefore, we propose that MM1309 functions as either a guardian protein that protects the Cys moiety from oxidation or an alternative translation factor for Cys-tRNA(Cys). |
format | Online Article Text |
id | pubmed-4329189 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Springer Netherlands |
record_format | MEDLINE/PubMed |
spelling | pubmed-43291892015-02-20 A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNA(Cys) Yanagisawa, Tatsuo Ishii, Ryohei Hikida, Yasushi Fukunaga, Ryuya Sengoku, Toru Sekine, Shun-ichi Yokoyama, Shigeyuki J Struct Funct Genomics Article The putative translation elongation factor Mbar_A0971 from the methanogenic archaeon Methanosarcina barkeri was proposed to be the pyrrolysine-specific paralogue of EF-Tu (“EF-Pyl”). In the present study, the crystal structures of its homologue from Methanosarcina mazei (MM1309) were determined in the GMPPNP-bound, GDP-bound, and apo forms, by the single-wavelength anomalous dispersion phasing method. The three MM1309 structures are quite similar (r.m.s.d. < 0.1 Å). The three domains, corresponding to domains 1, 2, and 3 of EF-Tu/SelB/aIF2γ, are packed against one another to form a closed architecture. The MM1309 structures resemble those of bacterial/archaeal SelB, bacterial EF-Tu in the GTP-bound form, and archaeal initiation factor aIF2γ, in this order. The GMPPNP and GDP molecules are visible in their co-crystal structures. Isothermal titration calorimetry measurements of MM1309·GTP·Mg(2+), MM1309·GDP·Mg(2+), and MM1309·GMPPNP·Mg(2+) provided dissociation constants of 0.43, 26.2, and 222.2 μM, respectively. Therefore, the affinities of MM1309 for GTP and GDP are similar to those of SelB rather than those of EF-Tu. Furthermore, the switch I and II regions of MM1309 are involved in domain–domain interactions, rather than nucleotide binding. The putative binding pocket for the aminoacyl moiety on MM1309 is too small to accommodate the pyrrolysyl moiety, based on a comparison of the present MM1309 structures with that of the EF-Tu·GMPPNP·aminoacyl-tRNA ternary complex. A hydrolysis protection assay revealed that MM1309 binds cysteinyl (Cys)-tRNA(Cys) and protects the aminoacyl bond from non-enzymatic hydrolysis. Therefore, we propose that MM1309 functions as either a guardian protein that protects the Cys moiety from oxidation or an alternative translation factor for Cys-tRNA(Cys). Springer Netherlands 2015-01-25 2015 /pmc/articles/PMC4329189/ /pubmed/25618148 http://dx.doi.org/10.1007/s10969-015-9193-6 Text en © The Author(s) 2015 https://creativecommons.org/licenses/by/4.0/ Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. |
spellingShingle | Article Yanagisawa, Tatsuo Ishii, Ryohei Hikida, Yasushi Fukunaga, Ryuya Sengoku, Toru Sekine, Shun-ichi Yokoyama, Shigeyuki A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNA(Cys) |
title | A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNA(Cys) |
title_full | A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNA(Cys) |
title_fullStr | A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNA(Cys) |
title_full_unstemmed | A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNA(Cys) |
title_short | A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNA(Cys) |
title_sort | selb/ef-tu/aif2γ-like protein from methanosarcina mazei in the gtp-bound form binds cysteinyl-trna(cys) |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4329189/ https://www.ncbi.nlm.nih.gov/pubmed/25618148 http://dx.doi.org/10.1007/s10969-015-9193-6 |
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