November 29
PROGRAM

14

Glycoscience : diversity and integration

Structural biology ofthe N-glycosylation reaction
Daisuke Kohda, Ph.D.
Professor
Medical Institute of Bioregulation
Kyushu University, JAPANDaisuke Kohda
ProfileDaisuke Kohda received his Bachelor ofScience in Biochemistry from the University of Tokyo in 1981. He obtained Ph.D.
in Biochemistry from the same Universityin 1986. In the graduate course, he studied
under the supervision of Prof. Tatsuo Miyazawa and Dr. Shigeyuki Yokoyama. After
graduation, he joined the NMR laboratory
led by Prof. Fuyuhiko Inagaki in the TokyoMetropolitan Institute of Medical Science
as a research scientist. During his 10 years
in office, he spent one year at Oxford University, England, as a visiting scientist inProf. Iain D. Campbell’s laboratory. FromJanuary 1996, he joined Biomolecular Engineering Research Institute, Osaka, as asenior, then principal research scientist,and led an NMR group under the mentorship of Prof. Kosuke Morikawa. In 2002,Dr. Kohda accepted a position as Professor at Kyushu University, Fukuoka. He
has been working in the field of structural
biology using the NMR and X-ray crystallography. His targets include EGF, TGF-α,EGF receptor, SH3, PX, and oligosaccharyltransferases. The figure of the complex
structure of Tom20 and a mitochondrialpresequence is shown in four standard biochemistry textbooks, including Molecular
Biology of THE CELL (5th edition).

Protein Asn-glycosylation is widespread
not only in eukaryotes but also in archaea
and some eubacteria. Oligosaccharyltransferase (OST) catalyzes the transferof an oligosaccharide chain from a lipid
donor to an asparagine residue in theN-glycosylation sequon, Asn-X-Ser/Thr(X ≠ Pro) (Figure 1). OST is a multi-subunit membrane protein complex in higher
eukaryotes, and a single-subunit membrane protein in lower eukaryotes, archaea
and eubacteria. The catalytic subunit of theOST enzyme has a common evolutionaryorigin, but is referred to as STT3 (Staurosporine and Temperature sensitivity 3) ineukaryotes, AglB (Archaeal GlycosylationB) in archaea, and PglB (Protein Glycosylation B) in eubacteria. The STT3/AglB/PglB proteins consist of an N-terminal
multi-span transmembrane region (400to 600 residues) and a soluble C-terminal,
globular domain (150 to 500 residues).
The STT3 proteins share more than 40%
sequence identity, but exhibit limited
sequence identity with the AglB and PglB
proteins, typically less than 20%. Thus, ameaningful multiple sequence alignment
across the three domains of life was almost
impossible, with just one exception. The
C-terminal globular domain contains a
5-residue motif, WWDYG, and its strong
conservation enables multiple sequencealignment up to 100 residues, using theWWDYG motif as the pivot. As for thesequence alignment of the remaining
regions, the three-dimensional structures
are required as references. A comparative
approach is very effective under this circumstance by finding common structuralfeatures among STT3/AglB/PglB proteins.
We determined crystal structures of theC-terminal globular domains of AglBs from
Pyrococcus and Archaeoglobus1)2) and a
PglB from Campylobacter (Figure 2) 1)2).
These structures facilitated the multiple
sequence alignment in the C-terminal
globular domain region. Even though they
are catalytically inactive, the comparisonof the closely and distantly related AglB/46

the 20th Anniversary of the Mizutani Foundation for Glycoscience

PglB structures revealed the minimal
structural unit as a common architecture,beyond sequence comparison. We identified new, short motifs within a characteristic, kinked helix. A comprehensive phylogenetic analysis revealed conserved aminoacid motifs within the kinked helix at theequivalent spatial positions: MXXIXXX(I/V/W), DXXKXXX(M/I), and its variant,
E<>KXXX(M/I/P), where <> denotes
inserted 4 to 14 residue sequence. Thesemotifs were referred to as MI, DK, andDKi, respectively 3).
Recently, a crystal structure of the fulllength C. lari PglB in a complex with anacceptor peptide was reported (Lizak et al,Nature (2011) 474, 350-355). Our C. jejuni
PglB structure was used as a search modelfor the molecular replacement in the structure determination. This epochal structure
revealed a special binding pocket that recognizes +2 Ser/Thr residue in the N-glycosylation consensus. The Ser/Thr pocket
consists of the invariant Trp-Trp-Asp partof the conserved WWDYG motif and theFigure 2. Overall structures of the C-terminal globular domain of Archaeoglobus fulgidus AglB, Campylobacter jejuni PglB,and Pyrococcus furiosus AglB. TM, transmembrane; CC, central core (blue); IS, insertion (green); P1, peripheral 1 (orange); P2,peripheral 2 (red). The TM region not included in the structure determination is outlined in gray. The characteristic kinked helix
bearing the DK/DKi/MI motif is highlighted in light brown.

second signature residue, Lys or Ile, ofthe DK/DKi/MI motif. Thus, the catalytic
subunit of OST can be classified into twogroups: one group consists of eukaryotes
and most archaea, and the other groupconsists of eubacteria and the remainingarchaea. The former STT3/AglB proteins
possess the DK-type Ser/Thr pocket, andthe latter AglB/PglB proteins possess theMI-type Ser/Thr pocket. This classification
provides a useful framework for future
OST studies.
Detailed structural comparison of thecrystal structures exhibited conformational differences of a segment including the WWDYG motif, due to crystal
contact effects. This finding suggested
an unusual plasticity in the C-terminal
globular domain. 15N NMR relaxation
study using A. fulgidus AglB showed that
this plastic segment was actually mobilein solution. Finally, we designed an engi-

neered disulfide bond connecting
two α-helices to restrict the flexibility in the C-terminal globulardomain (Figure 3). The formationof the disulfide bond completely
inhibited the enzymatic activity of P. furiosus AglB, while
the activity was fully recovered
upon the reduction of the disulfide bond. This indicates that thedynamic feature of the Ser/Thr
pocket in the C-terminal globular domain is essential for thecatalysis 4). Thus, we propose thatthe Ser/Thr pocket catches andreleases the Ser and Thr residues
in the N-glycosylation consensus,
and its dynamic nature enables
the efficient scanning of a nascent
polypeptide chain when coupledwith the ribosomal protein synthesis.

Figure 3. A disulfide bond was engineered in P. furiosus AglB to restrict the flexibility in the C-terminal
domain. The position of the disulfide bond is schematically shown by magenta sticks in the crystal structure ofC. lari PglB (PDB entry, 3RCE). The two shaded circles
indicate the position of the +2 Ser/Thr pocket in the Cterminal globular domain, and that of the catalytic center in the N-terminal transmembrane region.

References

Figuer 1. Oligosaccharyltransferase (OST)
catalyzes the transfer of an oligosaccharide
chain from lipid-linked oligosaccharide donors to the asparagine in a nascent polypeptide chain. This figure shows the N-glycantransfer by a eukaryotic OST.

1) 	Igura M, Maita N, Kamishikiryo J, Yamada M, Obita T, Maenaka K, Kohda
D. Structure-guided identification of a new catalytic motif of oligosaccharyltransferase. EMBO J (27): 234-243, 2008.
2) 	Maita N, Nyirenda J, Igura M, Kamishikiryo J, Kohda D. Comparative structural biology of eubacterial and archaeal oligosaccharyltransferases. J Biol
Chem (285): 4941-4950, 2010.
3) 	Matsumoto S, Igura M, Nyirenda J, Matsumoto M, Yuzawa S, Noda N, Inagaki F, Kohda D. Crystal structure of the C-terminal globular domain ofoligosaccharyltransferase from Archaeoglobus fulgidus at 1.75 Å resolution.
Biochemistry (in press), 2012.

4) 	Nyirenda J, Matsumoto S, Saitoh T, Maita N, Noda NN, Inagaki F, Kohda D.
Crystallographic and NMR evidence for flexibility in oligosaccharyltransferases and its catalytic significance. submitted. (51): 4157-4166, 2012.
5) 	Igura M, Kohda D. Quantitative assessment of the preferences for the aminoacid residues flanking archaeal N-linked glycosylation sites. Glycobiology(21): 575-583, 2011.
6) 	Igura M, Kohda D. Selective control of oligosaccharide transfer efficiency for
the N-glycosylation sequon by a point mutation in oligosaccharyltransferase.
J Biol Chem (286): 13255-13260, 2011.

Commemorative Symposium

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