TY - JOUR
T1 - A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNACys
AU - Yanagisawa, Tatsuo
AU - Ishii, Ryohei
AU - Hikida, Yasushi
AU - Fukunaga, Ryuya
AU - Sengoku, Toru
AU - Sekine, Shun ichi
AU - Yokoyama, Shigeyuki
N1 - Funding Information:
We would like to thank the staff of the beamline BL41XU at SPring-8 (Harima, Japan), as well as the staff of the BL-5A and AR-NW12 beamlines at the Photon Factory (Tsukuba, Japan). We also thank Dr. Takuhiro Ito (The University of Tokyo) for assisting with the data collection, as well as for helpful discussions. We thank Dr. Tomomi Sumida (RIKEN) for technical assistance. We are grateful to Dr. Hirofumi Nakagawa (The University of Tokyo), and Drs. Takuma Kasai and Takashi Umehara (RIKEN) for ITC analyses. We would like to thank Azusa Ishii, Takako Imada, Kojiro Ake, and Tomoko Nakayama for clerical assistance. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and the RIKEN Structural Genomics/Proteomics Initiative (RSGI) in the National Project on Protein Structural and Functional Analyses, MEXT, and the Targeted Proteins Research Program (TPRP), MEXT.
Publisher Copyright:
© 2015, The Author(s).
PY - 2015/3
Y1 - 2015/3
N2 - 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·Mg2+, MM1309·GDP·Mg2+, and MM1309·GMPPNP·Mg2+ 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)-tRNACys 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-tRNACys.
AB - 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·Mg2+, MM1309·GDP·Mg2+, and MM1309·GMPPNP·Mg2+ 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)-tRNACys 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-tRNACys.
KW - Crystal structure
KW - GTP
KW - Translation factor
KW - tRNA
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U2 - 10.1007/s10969-015-9193-6
DO - 10.1007/s10969-015-9193-6
M3 - Article
C2 - 25618148
AN - SCOPUS:84925496906
SN - 1345-711X
VL - 16
SP - 25
EP - 41
JO - Journal of Structural and Functional Genomics
JF - Journal of Structural and Functional Genomics
IS - 1
ER -