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Publication Abstract from PubMed

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/aIF2gamma, 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 aIF2gamma, 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 muM, 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).

A SelB/EF-Tu/aIF2gamma-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNA(Cys.).,Yanagisawa T, Ishii R, Hikida Y, Fukunaga R, Sengoku T, Sekine S, Yokoyama S J Struct Funct Genomics. 2015 Mar;16(1):25-41. doi: 10.1007/s10969-015-9193-6., Epub 2015 Jan 25. PMID:25618148^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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Categories: Large Structures | Methanosarcina mazei | Fukunaga R | Ishii R | Sengoku T | Yanagisawa T | Yokoyama S