ABSTRACT
Alanyl-tRNA synthetase (AlaRS) catalyzes synthesis of Ala-tRNAAla and hydrolysis of mis-acylated Ser- and Gly-tRNAAla at 2 different catalytic sites. Here, we describe the monomer structures of C-terminal truncated archaeal AlaRS, with both activation and editing domains in the apo form, in complex with an Ala-AMP analog, and in a high-resolution lysine-methylated form. The structures show docking of the editing domain to the activation domain opposite from the predicted tRNA-binding surface. Thus, the editing site is positioned >35 A from the activation site, prompting us to model 2 different tRNA complexes: one binding tRNA at the activation site, and the other binding tRNA at the editing site. Interestingly, a gel-shift assay also implies the presence of 2 types of tRNA complex with different mobility. These results suggest that tRNA translocation via a canonical CCA flipping is unlikely to occur in AlaRS. The structure also demonstrated the binding of zinc in the editing site, in which the specific coordination of zinc would be facilitated by a conserved GGQ motif, implying that the editing mechanism may not be the same as in ThrRS. As Asn-194 in eubacterial AlaRS important for Ser misactivation is replaced by Thr-213 in archaeal AlaRS, a different Ser accommodation mechanism is proposed.
FIGURE CAPTION
The tRNA complexes.
(A) tRNAAsp (orange) was modeled by superposition of AspRS-tRNA complex onto the AD-HN region.
Surface representation of N752 without the α8-α10 insertion is shown and was colored by the sequence conservation among archaeal AlaRS using the program ConSurf 3.0 (23):
from low to high, cyan, white, and purple. The α8-α10 insertion is shown as a ribbon diagram (green).
The proposed direction of conformational change of the α8-α9 region is indicated by a black dashed arrow.
(B) tRNAThr (blue) is modeled by superposition of ThrRS-tRNA complex onto ED.
Surface representation of the whole N752 (including the α8-α10 insertion) is shown as in A.
Possible orientation of the CCA tail is shown as blue dashed lines.
Proposed orientation of OD is shown as gray dashed circle connecting to the C terminus of N752 by dashed lines.
REFERENCES
The structure of alanyl-tRNA synthetase with editing domain.
Masaaki Sokabe, Toyoyuki Ose, Akiyoshi Nakamura, Keita Tokunaga, Osamu Nureki, Min Yao, and Isao Tanaka.
Proc. Natl. Acad. Sci. 106, 11028-11033 (2009)