TY - JOUR
T1 - Crystallographic and NMR evidence for flexibility in oligosaccharyltransferases and its catalytic significance
AU - Nyirenda, James
AU - Matsumoto, Shunsuke
AU - Saitoh, Takashi
AU - Maita, Nobuo
AU - Noda, Nobuo N.
AU - Inagaki, Fuyuhiko
AU - Kohda, Daisuke
N1 - Funding Information:
This work was performed under the approval of the Photon Factory Program Advisory Committee as proposals 2009G208 and 2011G020, and the experiments at SPring-8 were performed under the Cooperative Research Program of the Institute for Protein Research, Osaka University, as proposals 2011A1904, 2011A6619, and 2011B6619. J.N. was supported by the Japanese Government (Monbukagakusho) Scholarship. The research was supported by Grant-in-Aids for Scientific Research on Innovative Areas 21121003 and for Scientific Research (B) 24370047 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
PY - 2013/1/8
Y1 - 2013/1/8
N2 - Oligosaccharyltransferase (OST) is a membrane-bound enzyme that catalyzes the transfer of an oligosaccharide to an asparagine residue in glycoproteins. It possesses a binding pocket that recognizes Ser and Thr residues at the +2 position in the N-glycosylation consensus, Asn-X-Ser/Thr. We determined the crystal structures of the C-terminal globular domains of the catalytic subunits of two archaeal OSTs. A comparison with previously determined structures identified a segment with remarkable conformational plasticity, induced by crystal contact effects. We characterized its dynamic properties in solution by 15N NMR relaxation analyses. Intriguingly, the mobile region contains the +2 Ser/Thr-binding pocket. In agreement, the flexibility restriction forced by an engineered disulfide crosslink abolished the enzymatic activity, and its cleavage fully restored activity. These results suggest the necessity of multiple conformational states in the reaction. The dynamic nature of the Ser/Thr pocket could facilitate the efficient scanning of N-glycosylation sequons along nascent polypeptide chains.
AB - Oligosaccharyltransferase (OST) is a membrane-bound enzyme that catalyzes the transfer of an oligosaccharide to an asparagine residue in glycoproteins. It possesses a binding pocket that recognizes Ser and Thr residues at the +2 position in the N-glycosylation consensus, Asn-X-Ser/Thr. We determined the crystal structures of the C-terminal globular domains of the catalytic subunits of two archaeal OSTs. A comparison with previously determined structures identified a segment with remarkable conformational plasticity, induced by crystal contact effects. We characterized its dynamic properties in solution by 15N NMR relaxation analyses. Intriguingly, the mobile region contains the +2 Ser/Thr-binding pocket. In agreement, the flexibility restriction forced by an engineered disulfide crosslink abolished the enzymatic activity, and its cleavage fully restored activity. These results suggest the necessity of multiple conformational states in the reaction. The dynamic nature of the Ser/Thr pocket could facilitate the efficient scanning of N-glycosylation sequons along nascent polypeptide chains.
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U2 - 10.1016/j.str.2012.10.011
DO - 10.1016/j.str.2012.10.011
M3 - Article
C2 - 23177926
AN - SCOPUS:84872149340
SN - 0969-2126
VL - 21
SP - 32
EP - 41
JO - Structure with Folding & design
JF - Structure with Folding & design
IS - 1
ER -