Dimer-dimer interaction of the bacterial selenocysteine synthase SelA promotes functional active-site formation and catalytic specificity.
The 21st amino acid, selenocysteine (Sec), is incorporated translationally into proteins and is synthesized on its specific tRNA (tRNA(Sec)). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNA(Sec), formed by seryl-tRNA synthetase, to Sec-tRNA(Sec). SelA, a member of the fold-type-I pyridoxal 5'-phosphate-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a molecular mass of about 500kDa. Our previously determined crystal structures of Aquifex aeolicus SelA complexed with tRNA(Sec) revealed that the ring-shaped decamer is composed of pentamerized SelA dimers, with two SelA dimers arranged to collaboratively interact with one Ser-tRNA(Sec). The SelA catalytic site is close to the dimer-dimer interface, but the significance of the dimer pentamerization in the catalytic site formation remained elusive. In the present study, we examined the quaternary interactions and demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined the crystal structures of "depentamerized" SelA variants with mutations at the dimer-dimer interface that prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site and confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I pyridoxal 5'-phosphate-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary structures.
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; Laboratory of Membrane and Cytoskeleton Dynamics, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
Yuzuru
Y
Itoh
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
Markus J
MJ
Bröcker
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
Shun-ichi
S
Sekine
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA; Department of Chemistry, Yale University, New Haven, CT 06520-8114, USA.
Dieter
D
Söll
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; RIKEN Structural Biology Laboratory, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan. Electronic address: yokoyama@riken.jp.
Shigeyuki
S
Yokoyama
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000378:metabolism; D014166:Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012346:RNA, Transfer, Amino Acyl; C112705:selenocysteinyl-tRNA; D014166:Transferases; C060600:selenium transferase
21st amino acid; fold-type-I PLP-dependent enzyme; pentamer of dimers; selenium metabolism; tRNA(Sec)