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* This work was supported by a grant from NCCR Structural Biology Zurich (to K. P. L.) and Swiss National Science Foundation Grants SNF 200020-125020 (to J. L. R.), SNF 200020-124663 (to R. Z.), and SNF 31003A-131075/1 and SNF 31003A-146191/1 (to K. P. L.). This article contains supplemental Tables S1 and S2 and Figs. S1–S6. 1 Both authors contributed equally to this work. 2 Present address: GlycoVaxyn AG, 8952 Schlieren, Switzerland.
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.
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 (
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 (
). 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 (
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 (
). 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 (
). 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.
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 (
). 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 (
). 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 (
), 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 (
). 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 (
). 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 (
). 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 (
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).
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) (
). 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 (
). 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 (
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 (
) 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 (
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.
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.