A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNACys
Tatsuo Yanagisawa,Ryohei Ishii,Yasushi Hikida,Ryuya Fukunaga,Ryuya Fukunaga,Toru Sengoku,Shun-ichi Sekine,Shigeyuki Yokoyama +7 more
TLDR
It is proposed that MM1309 functions as either a guardian protein that protects the Cys moiety from oxidation or an alternative translation factor for Cys-tRNACys, and a hydrolysis protection assay revealed thatMM1309 binds cysteinyl (Cys)-t RNACys and protects the aminoacyl bond from non-enzymatic hydrolyism.Abstract:
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 A). 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.read more
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Cooperative energetic effects elicited by the yeast Shwachman-Diamond syndrome protein (Sdo1) and guanine nucleotides modulate the complex conformational landscape of the elongation factor-like 1 (Efl1) GTPase.
TL;DR: A structural-based analysis of the binding signatures indicates that E FL1 has a large structural flexibility, and the mutual effects of Sdo1 and nucleotides on Efl1 modulate in a very specific and robust way the complex conformational landscape of EFL1, resembling the behavior observed with other GTPases and their cofactors.
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