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A Catalytically Essential Motif in External Loop 5 of the Bacterial Oligosaccharyltransferase PglB*

Open AccessPublished:November 25, 2013DOI:https://doi.org/10.1074/jbc.M113.524751
      Asparagine-linked glycosylation is a post-translational protein modification that is conserved in all domains of life. The initial transfer of a lipid-linked oligosaccharide (LLO) onto acceptor asparagines is catalyzed by the integral membrane protein oligosaccharyltransferase (OST). The previously reported structure of a single-subunit OST enzyme, the Campylobacter lari protein PglB, revealed a partially disordered external loop (EL5), whose role in catalysis was unclear. We identified a new and functionally important sequence motif in EL5 containing a conserved tyrosine residue (Tyr293) whose aromatic side chain is essential for catalysis. A synthetic peptide containing the conserved motif can partially but specifically rescue in vitro activity of mutated PglB lacking Tyr293. Using site-directed disulfide cross-linking, we show that disengagement of the structurally ordered part of EL5 is an essential step of the glycosylation reaction, probably by allowing sequon binding or glyco-product release. Our findings define two distinct mechanistic roles of EL5 in OST-catalyzed glycosylation. These functions, exerted by the two halves of EL5, are independent, because the loop can be cleaved by specific proteolysis with only slight reduction in activity.

      Introduction

      Glycosyltransferases utilize activated donor sugar substrates for the transfer to various targets, thereby forming glycosidic bonds. The transfer of glycans to the side chain of asparagines, a process termed N-linked protein glycosylation, is of major importance for protein folding, cell viability, and organism development (
      • Helenius A.
      • Aebi M.
      Roles of N-linked glycans in the endoplasmic reticulum.
      ,
      • Kornfeld R.
      • Kornfeld S.
      Assembly of asparagine-linked oligosaccharides.
      ,
      • Varki A.
      Biological roles of oligosaccharides. All of the theories are correct.
      ). Protein N-glycosylation occurs in all three domains of life (
      • Eichler J.
      Extreme sweetness. Protein glycosylation in archaea.
      ,
      • Nothaft H.
      • Szymanski C.M.
      Protein glycosylation in bacteria. Sweeter than ever.
      ,
      • Schwarz F.
      • Aebi M.
      Mechanisms and principles of N-linked protein glycosylation.
      ), and although there exist a number of variations, the general mechanism is conserved; a lipid-linked oligosaccharide (LLO)
      The abbreviations used are:
      LLO
      lipid-linked oligosaccharide
      EL1 and EL5
      external loop 1 and 5, respectively
      Ni-NTA
      nickel-nitrilotriacetic acid
      OST
      oligosaccharyltransferase
      TM
      transmembrane
      HSQC
      heteronuclear single-quantum correlation
      HMQC
      heteronuclear multiple-quantum correlation.
      is assembled in a multistep process before being translocated from the cytosolic to the luminal side of the endoplasmic reticulum membrane of eukaryotes or to the periplasmic/external side of the plasma membrane of prokaryotes. The integral membrane enzyme oligosaccharyltransferase (OST) then catalyzes the en bloc transfer of the oligosaccharide to asparagine residues located in the consensus sequon Asn-Xaa-Ser/Thr (with Xaa ≠ Pro) of acceptor polypeptides. The OST enzyme in higher eukaryotes is a multiprotein complex with Stt3 as the catalytic subunit (
      • Nilsson I.
      • Kelleher D.J.
      • Miao Y.
      • Shao Y.
      • Kreibich G.
      • Gilmore R.
      • von Heijne G.
      • Johnson A.E.
      Photocross-linking of nascent chains to the STT3 subunit of the oligosaccharyltransferase complex.
      ,
      • Yan Q.
      • Lennarz W.J.
      Studies on the function of oligosaccharyl transferase subunits. Stt3p is directly involved in the glycosylation process.
      ), but in kinetoplastids and prokaryotes, OST is a single-subunit enzyme that is homologous to Stt3 (
      • Hese K.
      • Otto C.
      • Routier F.H.
      • Lehle L.
      The yeast oligosaccharyltransferase complex can be replaced by STT3 from Leishmania major.
      ,
      • Nasab F.P.
      • Schulz B.L.
      • Gamarro F.
      • Parodi A.J.
      • Aebi M.
      All in one. Leishmania major STT3 proteins substitute for the whole oligosaccharyltransferase complex in Saccharomyces cerevisiae.
      ,
      • Wacker M.
      • Linton D.
      • Hitchen P.G.
      • Nita-Lazar M.
      • Haslam S.M.
      • North S.J.
      • Panico M.
      • Morris H.R.
      • Dell A.
      • Wren B.W.
      • Aebi M.
      N-Linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli.
      ). The crystal structure of a full-length, bacterial Stt3 homolog, the PglB protein of Campylobacter lari, revealed the architecture of this class of enzymes (
      • Lizak C.
      • Gerber S.
      • Numao S.
      • Aebi M.
      • Locher K.P.
      X-ray structure of a bacterial oligosaccharyltransferase.
      ). PglB contains an N-terminal transmembrane (TM) domain featuring 13 TM segments and a soluble domain facing the periplasm. The TM domain contains two large external loops (EL1 and EL5) that provide non-covalent interactions between the TM and the periplasmic domains. Whereas the role of EL1 appears to be mainly a structural one, EL5 was proposed to be involved in catalytic steps of the process. Recent functional studies have provided a quantitative basis for sequon recognition and binding by PglB; the +2 sequon Ser/Thr of acceptor substrates is recognized by a binding pocket provided by the WWD motif (a diagnostic motif among Stt3 homologs) and a neighboring Ile residue (Ile572), suggesting that the +2 Ser/Thr defines the specificity of N-linked glycosylation sites (
      • Gerber S.
      • Lizak C.
      • Michaud G.
      • Bucher M.
      • Darbre T.
      • Aebi M.
      • Reymond J.L.
      • Locher K.P.
      Mechanism of bacterial oligosaccharyltransferase. In vitro quantification of sequon binding and catalysis.
      ). The C. lari PglB enzyme was also useful for studying the mechanism of activation of the acceptor Asn side chain of sequon (
      • Lizak C.
      • Gerber S.
      • Michaud G.
      • Schubert M.
      • Fan Y.Y.
      • Bucher M.
      • Darbre T.
      • Aebi M.
      • Reymond J.L.
      • Locher K.P.
      Unexpected reactivity and mechanism of carboxamide activation in bacterial N-linked protein glycosylation.
      ).
      Arguably the least understood feature of the PglB structure (and OST mechanism) is the role of the external loop EL5. Both for substrate binding and Asn activation, the C-terminal half of this loop appears to play an important role by pinning the bound sequon against the periplasmic domain, providing the essential residue Glu319 to the catalytic site and positioning the acceptor Asn correctly for glycosylation (
      • Lizak C.
      • Gerber S.
      • Numao S.
      • Aebi M.
      • Locher K.P.
      X-ray structure of a bacterial oligosaccharyltransferase.
      ,
      • Gerber S.
      • Lizak C.
      • Michaud G.
      • Bucher M.
      • Darbre T.
      • Aebi M.
      • Reymond J.L.
      • Locher K.P.
      Mechanism of bacterial oligosaccharyltransferase. In vitro quantification of sequon binding and catalysis.
      ). The function of the N-terminal half of EL5, which was disordered in the structure of peptide-bound PglB (supplemental Fig. S1, top state), remained unclear.
      To explore the function of the N-terminal half of EL5 and to investigate conformational rearrangements in the C-terminal half during catalysis, we performed a detailed structure-function analysis of C. lari PglB. We used a synthetic, fluorescently labeled peptide that contained a bacterial glycosylation sequon and a previously established in vitro assay that not only provided high precision in determining glycosylation turnover but also allowed us to observe very low reaction rates of disfavored PglB mutants and determine changes in sequon binding affinities (
      • Gerber S.
      • Lizak C.
      • Michaud G.
      • Bucher M.
      • Darbre T.
      • Aebi M.
      • Reymond J.L.
      • Locher K.P.
      Mechanism of bacterial oligosaccharyltransferase. In vitro quantification of sequon binding and catalysis.
      ). We thus identified a previously unrecognized, catalytically essential motif, termed Tyr-plug, in the N-terminal half of EL5 that contains a conserved tyrosine residue (Tyr293). The motif is essential for catalysis but not for peptide binding.
      We had speculated earlier that once the N-glycosidic linkage is formed, the glyco-polypeptide will need to dissociate from the enzyme, which will probably require a disengagement of EL5 during the catalytic cycle (supplemental Fig. S1). To investigate this hypothesis, we engineered disulfide cross-links in PglB by introducing cysteines in EL5 and the enzyme core at three distinct locations. The resulting PglB mutants were expressed and purified in large quantities, and sequon binding and glycosylation turnover rates were determined under oxidizing and reducing conditions. The results allowed us to define the dynamics of EL5 during catalysis.

      DISCUSSION

      The long periplasmic loop EL5 was a remarkable yet insufficiently understood feature of the PglB structure. The C-terminal half of EL5 is involved in generating a tight binding site for acceptor peptide, suggesting that in a peptide-free state, PglB might adopt a distinct conformation where EL5 was disengaged from the core enzyme, as depicted in a three-step reaction cycle (supplemental Fig. S1). We tested this hypothesis using cysteine cross-linking experiments to restrict the flexibility of EL5. We indeed found that a cross-link at the peptide binding site (Pos1) abolished sequon binding and catalysis. In this PglB variant, EL5 may be unable to achieve an open conformation that is required for peptide binding, or the sequon binding site might have been distorted by the cross-link. The other two cross-links (positions 2 and 3), which are not overlapping with the peptide binding site, showed a reduced but clearly measurable activity. Our data allow the following conclusions. 1) Sequon binding can occur even when EL5 is only partially disengaged from the enzyme. 2) Sequon binding is not the rate-limiting step during catalysis in vitro because the strong reduction of peptide binding affinity (22-fold for Pos3) only leads to a slight reduction (1.3-fold for Pos3) in turnover. This interpretation is in agreement with previous observations that a replacement of the +2 Thr in glycosylation sequons by Ser, which reduced peptide binding 4-fold, only lowered turnover 1.2-fold (
      • Gerber S.
      • Lizak C.
      • Michaud G.
      • Bucher M.
      • Darbre T.
      • Aebi M.
      • Reymond J.L.
      • Locher K.P.
      Mechanism of bacterial oligosaccharyltransferase. In vitro quantification of sequon binding and catalysis.
      ). 3) The larger the peptide substrate, the stronger the effect of EL5 disengagement (for a comparison of peptide versus GFP glycosylation, see Table 1). 4) Partial disengagement of EL5 is sufficient for the release of glyco-products from the enzyme. Notably, product dissociation can only occur if either the polypeptide or the sugar moiety passes through the opening provided by EL5. Our observation that glycosylated GFP can be released even in the presence of a disulfide cross-link at Pos3 suggests that the N-glycan moiety of glyco-products is pulled through the provided opening, because GFP would be unable to fit.
      X-ray structures of the archaeal Stt3 homolog AglB from Archeoglobus fulgidus in two crystal forms have recently been reported (
      • Matsumoto S.
      • Shimada A.
      • Nyirenda J.
      • Igura M.
      • Kawano Y.
      • Kohda D.
      Crystal structures of an archaeal oligosaccharyltransferase provide insights into the catalytic cycle of N-linked protein glycosylation.
      ). Neither AglB structure contained bound acceptor peptide or LLO substrate. The structures revealed a transmembrane fold that is similar to that of our C. lari PglB structure. The entire loop EL5 is disordered in the AglB structure with bound zinc and sulfate ions, in line with our finding that EL5 is disengaged from the enzyme core in the apo-state of PglB. In the other AglB structure, the entire loop EL5 is ordered, including the N-terminal half, which is located at a distance of ∼15–20 Å from the active site of the enzyme, suggesting that a considerable conformational change would be required for interaction with the reducing end sugar. In addition to large scale conformational rearrangements of EL5, conformational fluctuations within the extracellular domain of the archaeal Stt3 homolog from Pyrococcus furiosus (AglB) have been reported to be important for function (
      • Nyirenda J.
      • Matsumoto S.
      • Saitoh T.
      • Maita N.
      • Noda N.N.
      • Inagaki F.
      • Kohda D.
      Crystallographic and NMR evidence for flexibility in oligosaccharyltransferases and its catalytic significance.
      ), suggesting that the extracellular/periplasmic domain probably adjusts its conformation upon substrate binding and EL5 engagement.
      In contrast to the C-terminal half, any role of the N-terminal half of EL5 in catalysis was completely unknown, and the corresponding segment was disordered in the x-ray structure of sequon-bound C. lari PglB (
      • Lizak C.
      • Gerber S.
      • Numao S.
      • Aebi M.
      • Locher K.P.
      X-ray structure of a bacterial oligosaccharyltransferase.
      ). Through a combination of systematic mutagenesis and in vivo activity analysis, we now identified a previously unrecognized motif that proved essential for catalytic activity. Unexpectedly, mutations in the newly found Tyr-plug had stronger effects on PglB activity than many of the previously described mutations at the active site, even of catalytically essential residues (
      • Gerber S.
      • Lizak C.
      • Michaud G.
      • Bucher M.
      • Darbre T.
      • Aebi M.
      • Reymond J.L.
      • Locher K.P.
      Mechanism of bacterial oligosaccharyltransferase. In vitro quantification of sequon binding and catalysis.
      ,
      • Lizak C.
      • Gerber S.
      • Michaud G.
      • Schubert M.
      • Fan Y.Y.
      • Bucher M.
      • Darbre T.
      • Aebi M.
      • Reymond J.L.
      • Locher K.P.
      Unexpected reactivity and mechanism of carboxamide activation in bacterial N-linked protein glycosylation.
      ). However, replacing the Tyr-plug sequence did not affect substrate peptide binding. This suggested a role in the interaction with the LLO molecule, the glycan donor of the reaction. Within the Tyr-plug, Tyr293 has a critical effect, but it was dependent on the immediate sequence context. The observation that Tyr293 could be replaced by other aromatic residues supported the notion that residue 293 might be involved in an interaction with a sugar moiety of the LLO substrate, because aromatic residues are frequently involved in π-stacking with carbohydrates (
      • Morales J.C.
      • Reina J.J.
      • Díaz I.
      • Aviñó A.
      • Nieto P.M.
      • Eritja R.
      Experimental measurement of carbohydrate-aromatic stacking in water by using a dangling-ended DNA model system.
      ,
      • Oldham M.L.
      • Khare D.
      • Quiocho F.A.
      • Davidson A.L.
      • Chen J.
      Crystal structure of a catalytic intermediate of the maltose transporter.
      ,
      • Weis W.I.
      • Drickamer K.
      Structural basis of lectin-carbohydrate recognition.
      ). However, our experiments excluded a direct interaction of Tyr293 with one of the distal sugar residues of the C. jejuni LLO, which agrees both with the observation that C. jejuni PglB can transfer diverse carbohydrate structures in vivo (
      • Feldman M.F.
      • Wacker M.
      • Hernandez M.
      • Hitchen P.G.
      • Marolda C.L.
      • Kowarik M.
      • Morris H.R.
      • Dell A.
      • Valvano M.A.
      • Aebi M.
      Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli.
      ,
      • Wacker M.
      • Feldman M.F.
      • Callewaert N.
      • Kowarik M.
      • Clarke B.R.
      • Pohl N.L.
      • Hernandez M.
      • Vines E.D.
      • Valvano M.A.
      • Whitfield C.
      • Aebi M.
      Substrate specificity of bacterial oligosaccharyltransferase suggests a common transfer mechanism for the bacterial and eukaryotic systems.
      ) and with in vitro studies of eukaryotic OST that identified the dolichol-linked monosaccharide Dol-PP-GlcNAc as the minimal glycosylation unit (
      • Tai V.W.
      • Imperiali B.
      Substrate specificity of the glycosyl donor for oligosaccharyl transferase.
      ).
      On the one hand, the exact position of the Tyr-plug within EL5 seemed to be irrelevant, because insertions, replacements, and even deletions in its vicinity were tolerated. On the other hand, the essential function of Tyr293 could not be taken over by any of the other aromatic residues within the Tyr-plug (Tyr288, Phe292, or Phe295). The inescapable conclusion was that the Tyr-plug adopts a defined three-dimensional conformation, at least during one of the rate-limiting steps in catalysis. We could indeed rescue (albeit to a limited extent) the effect of a Tyr-plug replacement in PglB by adding a synthetic peptide containing the Tyr-plug motif. However, this peptide did not adopt a defined structure on its own, as we could show by solution NMR analysis. This suggests that the Tyr-plug only adopts a defined conformation in the context of the enzyme and in the presence of bound LLO (Fig. 7A).
      Figure thumbnail gr7
      FIGURE 7Proposed function of the Tyr-plug in PglB. A, surface representation of PglB colored as in A. Binding of LLO donor substrate results in an interaction between the reducing end sugar and the Tyr-plug (thick red line). Red hexagon, diNAcBac; yellow square, N-acetylgalactosamine; blue circle, glucose; circled P, phosphate group; gray line, undecaprenyl (Und). B, mechanistic hypothesis of Tyr-plug function. Yellow dashed line indicates the sequon backbone. Hydrogen bonds between PglB residues Asp56 and Glu319 and the acceptor Asn contribute to the activation of the carboxamide group for the nucleophilic attack on the anomeric carbon (orange circle). The C-2 acetamido group of the reducing end sugar can be accommodated in a cavity of conserved residues in the active site of PglB (blue dashed line). The Tyr-plug is proposed to interact with the reducing end sugar and with the PglB surface. This is expected to restrict the rotation of the O-glycosidic bond and the first O–P bond (red arrows), increasing catalytic efficiency. R, oligosaccharyl; Und, undecaprenyl.
      What might the essential catalytic role of the Tyr-plug be? Fig. 7 illustrates our mechanistic hypothesis. Once LLO substrate is bound to PglB, its pyrophosphate moiety is bound by interactions with the conserved residue Arg375 and the divalent metal cation, both of which are essential for catalysis (Fig. 7B) (
      • Lizak C.
      • Gerber S.
      • Numao S.
      • Aebi M.
      • Locher K.P.
      X-ray structure of a bacterial oligosaccharyltransferase.
      ,
      • Gerber S.
      • Lizak C.
      • Michaud G.
      • Bucher M.
      • Darbre T.
      • Aebi M.
      • Reymond J.L.
      • Locher K.P.
      Mechanism of bacterial oligosaccharyltransferase. In vitro quantification of sequon binding and catalysis.
      ). Although the pyrophosphate is now fixed, the reducing end sugar is not, because a rotation around the O-glycosidic bond and around the O–P bond of the pyrophosphate moiety would be allowed (red arrows in Fig. 7B), both of which would prevent proper placement of the glycan for a nucleophilic attack by the activated carboxamide group of the Asn side chain of the sequon. We therefore propose that the function of the Tyr-plug is to engage in contacts both with the active site of PglB and with the reducing end sugar of LLO, forcing the reducing end sugar into a reactive conformation and thus increasing the rate of the transfer reaction. The aromatic side chain of Tyr293 is essential and might directly stack against the reducing end sugar diNAcBac (Fig. 7B). In addition, Tyr293 might interact with the C-4 acetamido group of diNAcBac, because methyl groups of acetamido moieties are often recognized by aromatic residues in different lectins (
      • Weis W.I.
      • Drickamer K.
      Structural basis of lectin-carbohydrate recognition.
      ,
      • Weis W.
      • Brown J.H.
      • Cusack S.
      • Paulson J.C.
      • Skehel J.J.
      • Wiley D.C.
      Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid.
      ,
      • Wright C.S.
      Structural comparison of the two distinct sugar binding sites in wheat germ agglutinin isolectin II.
      ,
      • Wright C.S.
      2.2 Å resolution structure analysis of two refined N-acetylneuraminyl-lactose–wheat germ agglutinin isolectin complexes.
      ,
      • Schubert M.
      • Bleuler-Martinez S.
      • Butschi A.
      • Wälti M.A.
      • Egloff P.
      • Stutz K.
      • Yan S.
      • Collot M.
      • Mallet J.M.
      • Wilson I.B.
      • Hengartner M.O.
      • Aebi M.
      • Allain F.H.
      • Künzler M.
      Plasticity of the β-trefoil protein fold in the recognition and control of invertebrate predators and parasites by a fungal defence system.
      ). A tentative modeling of the LLO with the diNAcBac properly aligned for a nucleophilic attack places the C2-acetamido group into a PglB cavity that contains residues conserved in all Stt3 homologs. This cavity might also be involved in restricting sugar rotation, in particular rotation around the anomeric bond (Fig. 7B). The C2-acetamido group of the reducing end sugar is indeed essential both for bacterial and eukaryotic LLO substrates (
      • Wacker M.
      • Feldman M.F.
      • Callewaert N.
      • Kowarik M.
      • Clarke B.R.
      • Pohl N.L.
      • Hernandez M.
      • Vines E.D.
      • Valvano M.A.
      • Whitfield C.
      • Aebi M.
      Substrate specificity of bacterial oligosaccharyltransferase suggests a common transfer mechanism for the bacterial and eukaryotic systems.
      ,
      • Tai V.W.
      • O'Reilly M.K.
      • Imperiali B.
      Substrate specificity of N-Acetylglucosaminyl(diphosphodolichol) N-acetylglucosaminyl transferase, a key enzyme in the dolichol pathway.
      ).
      Does the Tyr-plug also exist in archaeal and eukaryotic Stt3? Although the chemical reaction and the basic mechanism are conserved among OST homologs, it is currently unclear if eukaryotic and archaeal enzymes use a similar Tyr-plug to restrict the motion of the reducing end sugar during the catalytic step. Sequence alignments would support this hypothesis, because Stt3 homologs also contain aromatic residues (including Tyr and Phe) in EL5 at a similar distance from the catalytically essential Glu319 residue, which is also conserved. However, it will be very challenging to study these effects in vivo or in vitro. Most eukaryotic OSTs contain seven additional subunits (
      • Kelleher D.J.
      • Gilmore R.
      An evolving view of the eukaryotic oligosaccharyltransferase.
      ) that might contribute to binding of the LLO and the correct positioning of the reducing end sugar moiety. Additionally, the glycan structure transferred by eukaryotic OST (GlcNAc2Man9Glc3) is different and much bigger than its bacterial counterpart, which could imply that steric effects restricting sugar motions play an additional role. Furthermore, the reducing end sugar is a GlcNAc in eukaryotic LLOs and even a hexose in some archaeal LLOs (
      • Eichler J.
      Extreme sweetness. Protein glycosylation in archaea.
      ,
      • Matsumoto S.
      • Shimada A.
      • Nyirenda J.
      • Igura M.
      • Kawano Y.
      • Kohda D.
      Crystal structures of an archaeal oligosaccharyltransferase provide insights into the catalytic cycle of N-linked protein glycosylation.
      ,
      • Calo D.
      • Kaminski L.
      • Eichler J.
      Protein glycosylation in Archaea. Sweet and extreme.
      ,
      • Abu-Qarn M.
      • Yurist-Doutsch S.
      • Giordano A.
      • Trauner A.
      • Morris H.R.
      • Hitchen P.
      • Medalia O.
      • Dell A.
      • Eichler J.
      Haloferax volcanii AglB and AglD are involved in N-glycosylation of the S-layer glycoprotein and proper assembly of the surface layer.
      ), suggesting mechanistic diversity.
      In summary, our study shows that EL5 of PglB has two distinct functions. Whereas the N-terminal half of EL5 is supposed to form a critical interaction with the LLO during catalysis, the C-terminal part contributes to peptide binding and contributes the conserved active site residue Glu319 that binds both the divalent metal and the acceptor Asn of the sequon. Intriguingly, the two functions of EL5 require no covalent link between the N- and C-terminal halves of the loop, as shown by our ability to specifically cleave EL5 with only minor effects on activity. Although the importance of the Tyr-plug for catalysis is now established, insight into the mechanistic details will require structural evidence, in particular a LLO-bound co-crystal structure of OST.

      Acknowledgments

      We thank E. Weber-Ban and R. Glockshuber for access to the fluorometer and M. Aebi for helpful discussions. We are grateful to the D-BIOL NMR Platform for access to NMR spectrometers. We thank A. Geerlof for providing the protocol for 3C protease expression and purification and A. Zeltina for providing HmuT-Q147C cells.

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