Photon Factory Activity Report 2014 #32 (2015) B

BL-1A, BL-5A, BL-17A, AR-NW12A, AR-NE3A/2013G023

Crystal structures of an archaeal oligosaccharyltransferase provide insights into thecatalytic cycle of N-linked protein glycosylation
Shunsuke Matsumoto1, Atsushi Shimada1, James Nyirenda1, Mayumi Igura1, Yoshiaki Kawano2, andDaisuke Kohda1, *1
Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka
812-8582, Japan2
RIKEN/SPring-8 Center, Hyogo 679-5148, Japan
1 Introduction
Asparagine-linked glycosylation (N-glycosylation) ofproteins is the most ubiquitous protein modification. The
oligosaccharyl transfer from the donor substrate, thelipid-linked oligosaccharide (LLO), to the asparagine
residues in the N-glycosylation sequon (Asn-X-Ser/Thr,X ≠ Pro) is catalyzed by a membrane-bound enzyme,
oligosaccharyltransferase (OST). In eukaryotes, thereaction occurs on the luminal side of ER, whereas it
occurs on the exterior surface of the plasma membranes
in archaea and eubacteria. The catalytic subunit of theOST enzyme is a polypeptide chain referred to as STT3in eukaryotes, AglB in archaea, and PglB in eubacteria,
although they originated from a common ancestor. The
eukaryotic OST is a multisubunit protein complex
containing STT3, but the archaeal and eubacterial OSTs,
are single subunit enzymes consisting only of the AglBand PglB proteins.
2 ExperimentProtein Expression and Purification. The DNA
encoding A. fulgidus AglB-L (O29867_ARCFU) wasamplified from the genomic DNA. The protein wasexpressed in Escherichia coli C43 (DE3) cells with a Cterminal His10-tag after a thrombin cleavage site. Themembrane fractions were prepared in Tris-Saline (TS)buffer (50 mM Tris·HCl, pH 8.0, 100 mM NaCl)containing 1% (wt/vol) DDM. The AglB-L protein waspurified by affinity chromatography on nickel Sepharose
resin in TS buffer containing 0.1% DDM. After removalof the C-terminal His tag, the protein was subjected to gel
filtration chromatography, using a Superdex200 column
in TS buffer containing 1% (wt/vol) OG or 0.06%(wt/vol) LDAO. The eluted protein was concentrated to15 mg·mL −1, in 20 mM Tris·HCl buffer, pH 8.0, forcrystallization.
Crystallization. In the presence of 1% (wt/vol) OG,crystals grew from a hanging drop with a 1:1 volume
ratio (total volume, 2 μL) of the protein stock solution
and the reservoir solution (0.2 M zinc sulfate, 0.1 M
sodium acetate buffer, pH 4.6, 15% (vol/vol)polyethylene glycol 4000) at 293 K. In the presence of
0.06% (wt/vol) LDAO, crystals grew from a hanging
drop with the reservoir solution (0.02 M Tris·HCl, pH 8.0,22% (wt/vol) polyethylene glycol 550MME) at 293 K.
Structure Determination. The molecular replacement
was performed with the structure of the C-terminal

globular domain of A. fulgidus AglB-L [Protein Data
Bank (PDB) 3WAI] as the search model [1].
3 Results and Discussion
We determined the crystal structures of the full-length
AglB from a hyperthermophilic archaeon, Archaeoglobus
fulgidus (Fig. 1) [2]. This is the second solved catalytic
subunit structure of OST, after the eubacterial PglB.

Fig. 1. (A) Domainorganizations of theAglB protein. CC,
centralcore;
IS,insertion; P1, peripheral
1. (B) The AglB-L
structure (OG form).
The TM helices arenumbered. Dashed lines
indicate the disorderedexternal and internalloops in the electron
density maps.
The AglB protein consists of an N-terminaltransmembrane region, which contains the catalytic center
consisting of conserved acidic residues and a divalent
metal ion, and a C-terminal globular domain, whichcontains a binding site for the Ser and Thr residues in theN-glycosylationsequon.
Inadditiontothecrystallographic data, NMR and biochemical studies
suggested the essential flexibility of one long loop in theTM region and the Ser/Thr pocket in the C-terminal
globular domain for the enzymatic activity [3]. It is likelythat the dynamic nature facilitates the efficient scanningof a nascent polypeptide chain for the N-glycosylationsequons when coupled with ribosomal protein synthesis.
The atomic coordinates and structure factors have been
deposited in the PDB (3WAJ and 3WAK).
References[1] S. Matsumoto et al, BMC Struct. Biol. 13, 11 (2013).
[2] S. Matsumoto et al, Proc. Natl. Acad. Sci., USA 110,17868 (2013).
[3] J. Nyirenda et al, Structure 21, 32 (2013)* kohda@bioreg.kyushu-u.ac.jp