Idiosyncratic helix-turn-helix motif in Methanosarcina barkeri seryl-tRNA synthetase has a critical architectural role.
All seryl-tRNA synthetases (SerRSs) are functional homodimers with a C-terminal active site domain typical for class II aminoacyl-tRNA synthetases and an N-terminal domain involved in tRNA binding. The recently solved three-dimensional structure of Methanosarcina barkeri SerRS revealed the idiosyncratic features of methanogenic-type SerRSs; that is, an active site zinc ion, a unique tRNA binding domain, and an insertion of approximately 30 residues in the catalytic domain, which adopt a helix-turn-helix (HTH) fold. Here, we present biochemical evidence for multiple roles of the HTH motif; it is important for dimerization of the enzyme, contributes to the overall stability, and is critical for the proper positioning of the tRNA binding domain relative to the catalytic domain. The changes in intrinsic fluorescence during denaturation of the wild-type M. barkeri SerRS and of the mutated variant lacking the HTH motif combined with cross-linking and gel analysis of protein subunits during various stages of the unfolding process revealed significantly reduced stability of the mutant dimers. In vitro kinetic analysis of enzymes, mutated in one of the N-terminal helices and the HTH motif, shows impaired tRNA binding and aminoacylation and emphasizes the importance of this domain for the overall architecture of the enzyme. The role of the idiosyncratic HTH motif in dimer stabilization and association between the catalytic and tRNA binding domain has been additionally confirmed by a yeast two-hybrid approach. Furthermore, we provide experimental evidence that tRNA binds across the dimer.
Bilokapic
Silvija
S
Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, and Divisions of Physical Chemistry and Organic Chemistry and Biochemistry, Rudjer Boskovic Institute, Bijenicka c. 54, HR-10000 Zagreb, Croatia, Switzerland.
Ivic
Nives
N
Godinic-Mikulcic
Vlatka
V
Piantanida
Ivo
I
Ban
Nenad
N
Weygand-Durasevic
Ivana
I
D019281:Dimerization; D004795:Enzyme Stability; D019077:Helix-Turn-Helix Motifs; D017021:Methanosarcina barkeri / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012714:Serine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D012346:RNA, Transfer, Amino Acyl; D012714:Serine-tRNA Ligase