Tethering an N-Glycosylation Sequon-Containing Peptide Creates a

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Tethering an N-Glycosylation-Sequon Containing Peptide Creates a Catalytically Competent Oligosaccharyltransferase Complex Shunsuke Matsumoto, Yuya Taguchi, Atsushi Shimada, Mayumi Igura, and Daisuke Kohda Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b01089 • Publication Date (Web): 20 Dec 2016 Downloaded from http://pubs.acs.org on December 22, 2016

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Tethering an N-Glycosylation-Sequon Containing Peptide Creates a Catalytically Competent Oligosaccharyltransferase Complex This work was supported by JSPS KAKENHI Grant Numbers JP24370047 and JP26119002 (to D.K.).

Shunsuke Matsumoto, †,¶,# Yuya Taguchi, †, # Atsushi Shimada, † Mayumi Igura, † and Daisuke Kohda *,†,‡,§

†

Division of Structural Biology, Medical Institute of Bioregulation, ‡ Research Center

for Advanced Immunology, and §Research Center for Live-Protein Dynamics, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan

* Medical Institute of Bioregulation, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: [email protected]. Phone: 81-92-642-6968. Fax: 81-92-642-6833. 1 ACS Paragon Plus Environment

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ABSTRACT: Oligosaccharyltransferase (OST) transfers an oligosaccharide chain to the Asn residue in the Asn-X-Ser/Thr sequon in proteins, where X is not proline. A sequon was tethered to an archaeal OST enzyme via a disulfide bond. The positions of the cysteine residues in the OST protein and the sequon-containing acceptor peptide were selected by reference to the eubacterial OST structure in a non-covalent complex with an acceptor peptide. We determined the crystal structure of the cross-linked OST-sequon complex. The Ser/Thr-binding pocket recognizes the Thr residue in the sequon, and the catalytic structure referred to as ‘carboxylate dyad’ interacted with the Asn residue. Thus, the recognition and the catalytic mechanism of the sequon are conserved between the archaeal and eubacterial OSTs. We found that the tethered peptides in the complex were efficiently glycosylated in the presence of the oligosaccharide donor. The stringent requirements are greatly relaxed in the cross-linked state: The two conserved acidic residues in the catalytic structure were each dispensable, although the double mutation abolished the activity. A Gln residue at the Asn position in the sequon functioned as an acceptor, and the hydroxy group at the +2 position was not required. In the standard assay using short free peptides, strong amino-acid preferences were observed at the X position, but the preferences, except for Pro, completely disappeared in the cross-linked state. By skipping the

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initial binding process and stabilizing the complex state, the catalytically competent cross-linked complex offers a unique system for studying the oligosaccharyl transfer reaction.

Asparagine-linked glycosylation is one of the most ubiquitous post-translational modifications of proteins, and is conserved in all domains of life. 1 All eukaryotic and archaeal organisms have the N-glycosylation system, and N-glycosylation also occurs in some eubacterial organisms. 2, 3 The N-glycans on proteins play pivotal roles in many important biological phenomena, including endoplasmic reticulum-associated protein quality control in eukaryotic cells, 4, 5 and the pathogenesis of eubacterial infection. 6 The oligosaccharide transfer is catalyzed by an integral membrane enzyme, oligosaccharyltransferase (OST). The oligosaccharide acceptor is the asparagine residue in the N-glycosylation sequon, Asn-X-Ser/Thr, where X ≠ Pro, in polypeptide chains. 7 Eubacteria use an extended 5-residue sequon, Asp/Glu-X1-Asn-X2-Ser/Thr, where X1/X2 ≠ Pro. 8, 9 The OST enzyme is a hetero-oligomeric membrane protein complex in most eukaryotes, but the lower eukaryotic protozoan OSTs and the archaeal and eubacterial OSTs are single-subunit membrane enzymes. OST is located in the endoplasmic reticulum membrane of eukaryotic cells, and in the plasma membranes of archaeal 3 ACS Paragon Plus Environment

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and eubacterial cells. The catalytic subunit is a polypeptide chain referred to as STT3 (staurosporine and temperature sensitivity 3) in Eukaryota, AglB (archaeal glycosylation B) in Archaea, and PglB (protein glycosylation B) in Eubacteria. Despite their different names, the presence of short, well-conserved motifs revealed that they have the same evolutionary origin. 10 An oligosaccharide chain is preassembled on a lipid-phospho carrier, to form an oligosaccharide donor called a lipid-linked oligosaccharide (LLO). 11 The LLO structure is a dolichol-diphosphate-oligosaccharide in Eukaryota, and a polyprenol-diphosphate-oligosaccharide in Eubacteria. In contrast, Archaea use two different types of LLOs, dolichol-diphosphate-oligosaccharide and dolichol-monophosphate-oligosaccharide. 12, 13 The difference in the number of phosphate groups closely matches the phylogenetic tree of Archaea: The phylum Crenarchaeota uses the diphosphate-type LLO and the phylum Euryarchaeota uses the monophosphate-type LLO. This finding is consistent with the hypothesis that the ancestor of Eukaryota is rooted within the TACK (Thaum-, Aig-, Cren-, and Korarchaeota) superphylum, which includes Crenarchaea. 14 The crystal structure of the PglB protein from a Gram-negative eubacterium, Campylobacter lari, was determined in the complex with an acceptor peptide. 15 This structure provided invaluable insights into the catalytic mechanism of the oligosaccharyl transfer reaction. Two conserved acidic residues were identified on the extracellular loops in the N-terminal 4 ACS Paragon Plus Environment

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transmembrane (TM) region. Their side-chain carboxylate groups contacted the carboxyamide group of the acceptor Asn side chain, and simultaneously coordinated a divalent metal ion. The bipartite interactions with the two acidic residues in the ‘carboxylate dyad structure’ twist the planar carboxyamide group of the Asn side chain, and the resulting tetrahedral geometry of the twisted amide allows the lone-pair electrons of the amide nitrogen atom to nucleophilically attack the C1 carbon of the reducing-end sugar of the LLO. 16 The C-terminal half of the C. lari PglB (ClPglB) forms a soluble, globular domain, which contains a binding site for the Ser/Thr residues at the +2 position in the sequon. The N-glycosylation sequon (Asn-Gly-Thr) in the bound form adopted an extended conformation, which is inconsistent with the direct involvement of the hydroxy group of the +2 Ser/Thr residues in the catalysis. We also determined the crystal structure of the AglB protein from a hyperthermophilic archaeon, Archaeoglobus fulgidus. 17, 18 In contrast to the C. lari genome, the A. fulgidus genome encodes three AglB paralogs, and our structure corresponds to the longest one (AF_0380). We designated it as AglB-L, to distinguish it from the other two shorter AglB paralogs, AglB-S1 and AglB-S2, but will use AglB for clarity hereafter. The overall structure of A. fulgidus AglB (AfAglB) shared high structural similarity to that of ClPglB, despite the low amino acid sequence identity (< 20 %). Unfortunately, we did not obtain co-crystals with an acceptor peptide, probably due to its low affinity.


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The covalent crosslinking via a disulfide bond effectively increases the local concentration of the ligands/substrates at the targeted binding site, and allows discovery of druggable targets,19, 20 and trapping unstable complex states.21, 22 The disulfide bonds can be broken under mild conditions to recover captured molecules for further analyses. We and other groups have successfully used the disulfide bond tethering to study binding properties of ligands/substrates, such as, specificity and geometry in the binding sites.23-29 In the present study, we tethered an acceptor peptide to the AfAglB protein via an engineered disulfide bond to overcome the low-affinity problem. The crystal structure of the cross-linked AfAglB-peptide complex was determined to 3.5 Å resolution. The N-glycosylation sequon bound to the AfAglB in the same manner as in the ClPglB-peptide complex. We found that the tethered peptide served as an efficient substrate that receives the oligosaccharide chain from the LLO. The oligosaccharyl transfer is a single turnover reaction, since it proceeds within the covalently cross-linked complex. The catalytically competent cross-linked complex provides a unique assay system for analyses of the N-oligosaccharyl transfer reaction.

EXPERIMENTAL PROCEDURES Protein Expression and Purification. The amino acid sequence of A. fulgidus AglB(-L) is available through UniProtKB under UniProt O29867. The A. fulgidus AglB mutants were 6 ACS Paragon Plus Environment

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generated with a KOD plus mutagenesis kit (TOYOBO). The procedure for the expression and purification of the AfAglB protein and its mutants was described previously. 18 Briefly, the transformed E. coli C43 (DE3) cells (Lucigen) were grown at 310 K in Terrific Broth expression medium, supplemented with 100 mg L-1 ampicillin. After overnight induction with 0.5 mM IPTG at 298 K, the cells were harvested by centrifugation, and disrupted by sonication in TS buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl). The lysate was centrifuged at 8,500 × g for 15 min to remove debris. The supernatant was ultracentrifuged at 100,000 × g for 2 h, and the pellets were solubilized in TS buffer containing 1 % (w/v) n-dodecyl-β-D-maltopyranoside (DDM). After ultracentrifugation at 100,000 × g for 1 h, the DDM-solubilized recombinant protein in the supernatant was purified by affinity chromatography on nickel Sepharose High Performance resin (GE Healthcare) in TS buffer containing 0.1 % (w/v) DDM. Cross-Linked AfAglB-Peptide Complex for Assay. The peptide sequences used are listed in Table 1. For disulfide-bond tethering, the purified AfAglB G617C in DDM was incubated with an acceptor peptide at pH 8.0, at a molar ratio of 1 : 5. After an overnight incubation at room temperature, the AfAglB-peptide complex was separated from the unreacted peptide monomers and the by-product peptide dimers by membrane filtration, in 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.05 % (w/v) DDM. The disulfide-bond formation was verified by SDS-PAGE and in-gel fluorescent detection of the carboxytetramethylrhodamine (TAMRA) dye attached to the 7 ACS Paragon Plus Environment

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N-terminus of the peptides. The incomplete disulfide bond formation was monitored by the mobility shift on SDS-PAGE, using the Mal-PEG alkylation method. 30 Lipid-Linked Oligosaccharide from A. fulgidus Cells. Archaeoglobus fulgidus strain NBRC 100126 was obtained from the NITE Biological Resource Center (Chiba, Japan). The A. fulgidus medium was prepared according to the recipe in the first column of Table 2 in the previous report. 31 A. fulgidus cells were grown in 1-liter culture bottles anaerobically without shaking for 5 days, in an oven at 80 °C. Typically, 0.3 gram of pelleted cells was obtained from a 1-liter culture. A. fulgidus LLO was prepared according to the procedure used for the Haloferax volcanii LLO extraction. 32 Oligosaccharyl Transfer Assay. The oligosaccharyl transfer assay was performed by the PAGE method. 33 The reaction mixture (total 10 µl) contained 50 mM Tris-HCl buffer, pH 7.5, 10 mM MnCl2, LLO prepared from A. fulgidus cells, and 0.5 µM cross-linked AfAglB-peptide complex solubilized in 0.05 % (w/v) DDM. In a special case (Figure 1D and Figure S4), the tethered peptide was released from AfAglB by the pre-incubation with 100 mM dithiothreitol (DTT) for 30 min on ice, before the addition of LLO. The requisite amount of crude LLO in chloroform:methanol:water solvent was dried, and redissolved in the reaction solution, which contained DDM to solubilize the LLO. The reaction was performed in an oven at 65 °C, and stopped by the addition of 1 µl of 200 mM EDTA. Then, if necessary, 1 µl of 1 M DTT was 8 ACS Paragon Plus Environment

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added to cleave the intermolecular disulfide bond, and the reaction solution was incubated on ice for 30 min. For SDS-PAGE analysis, 2.4 µl of 5 × SDS sample buffer (without DTT) was added. The in-gel fluorescence images of the SDS-PAGE gels were recorded with an LAS-3000 multicolor image analyzer (Fuji Photo Film), with green LED excitation. For time-course assay experiments, the reaction solutions were loaded on a ZORBAX Eclipse Plus C18 RRHD reverse-phase column (1.8 µm, 2.1 mm × 50 mm, Agilent) in 0.1 % trifluoroacetic acid and 20 % acetonitrile. A linear gradient of acetonitrile was applied at a flow rate of 0.5 ml/min, and the eluted materials were detected with an Agilent 1260 Infinity Fluorescence Detector (Ex 553 nm, Em 580 nm). The peak areas were used for the calculation of the reaction rate constants. The first-order reaction rate constant, k, was calculated by the curve fitting of a single exponential equation, ξ = A (1 - e-kt), where ξ was the extent of the reaction, and A was the ξ value at the incubation time t = ∞. The ξ value was calculated according to the equation, ξ = ‘fluorescent intensity of glycopeptide’ / (‘fluorescent intensity of glycopeptide’ + ‘fluorescent intensity of unreacted peptide’). Note that slightly different reaction rates were obtained for the same control cross-linked complex: 0.68 ± 0.11, 1.46 ± 0.13, and 1.05 ± 0.07 min-1 in Figures 3B, 4C and S4, respectively. This is because the amount of the LLO in different master mix solutions could not be fully controlled due to the highly volatile property of the crude LLO stock solution. In


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contrast, a single master mix of the reaction solution was divided into 10 µl aliquots, which guaranteed the self-consistent ξ values in one time-course experiment. Crystallization. The AfAglB G617C mutant protein was purified by gel filtration chromatography in the presence of 0.06 % (w/v) lauryldimethylamine N-oxide (LDAO), and mixed with an acceptor peptide, Ac-Arg-Tyr-Asn-Val-Thr-Ala-Cys-NH2, at pH 8.8, in the presence of 10 mM MgCl2. The final concentrations of the protein and the peptide were 5 µM and 50 µM, respectively. The cross-linked protein was concentrated to 15 mg/ml, and dialyzed against 10 mM Tris-HCl, pH 7.5, 0.1 M NaCl, and 0.06 % (w/v) LDAO. Initial crystallization screening was performed by the sitting drop vapor diffusion method, using MemGold I, MemGold II and MemStart + MemSys Kits (Molecular Dimensions). Crystals grew from a hanging drop with the reservoir solution (0.01 M MgCl2, 0.1 M Bis-Tris, pH 6.5, 22 % (w/v) polyethylene glycol 550MME, 5 % (v/v) Jeffamine M-600, pH 7.0) at 293 K. For cryoprotection, the crystals were transferred to a solution containing 0.01 M MgCl2, 0.1 M Bis-Tris, pH 6.5, 30 % (v/v) polyethylene glycol 550MME, and 0.06 % (w/v) LDAO, and then cryocooled in liquid nitrogen. Structure Determination. X-ray diffraction data were collected at beamline BL44XU of SPring-8 (Harima, Japan), and were processed to a resolution of 3.50 Å using the program HKL2000. 34 The program phenix.automr from the GUI of PHENIX 35 was used for the 10 ACS Paragon Plus Environment

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molecular replacement, with the structure of the apo form of A. fulgidus AglB (PDB 3WAK) as the search model. The asymmetric unit contained one protein molecule. The calculated solvent content was 63.2 % (Vm = 3.35 Å3 Da−1). After initial refinement using the program phenix.refine from the GUI of PHENIX, clear electron densities corresponding to the tethered peptide appeared in the expected region. Manual model building was performed with the program COOT, 36 and further crystallographic refinement was performed with the program phenix.refine from the GUI of PHENIX. Data collection and refinement statistics are summarized in Table S1. Other Programs. Figures were generated with the program PyMOL (Schrödinger). Nonlinear curve fitting was performed with the program xcrvfit, version 5.0.3 (http://www.bionmr.ualberta.ca/bds/software/xcrvfit/). MS Analysis. The reaction mixture (total 50 µl) contained 50 mM Tris-HCl buffer, pH 7.5, 10 mM MnCl2, AfLLO, and 500 pmol cross-linked AfAglB-peptide complex solubilized in 0.05 % (w/v) DDM. The reaction was performed in an oven at 65 °C until it reached maximal levels, and stopped by the addition of 5 µl of 200 mM EDTA. Then, 5 µl of 1 M DTT was added, and the reaction solution was incubated on ice for 1 h. The released glycopeptide product was separated on a COSMOSIL 5C18-AR-II reverse phase column (Nacalai Tesque, Kyoto, Japan), run in 0.1% trifluoroacetic acid and acetonitrile. The glycopeptide was eluted by a linear gradient 11 ACS Paragon Plus Environment

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of acetonitrile, collected, and dried in a SpeedVac concentrator. The dried glycopeptide sample was dissolved in 0.1% formic acid and 50% methanol. The direct infusion ESI-MS/MS analysis was performed with a QSTAR Elite mass spectrometer (ABSciex) in the positive ion mode. The triply-charged precursor ion was selected, and subjected to MS/MS analyses with a collision energy of 40 V. The data were acquired in the multichannel analyzer (MCA) mode.

RESULTS Oligosaccharyl Transfer Reaction in the Cross-Linked State. An acceptor peptide was tethered to the AfAglB protein through an engineered disulfide bond (Figure 1A). The position of the Cys residue in the AfAglB protein was chosen by reference to the ClPglB-peptide complex structure (Figure S1). 15 Note that the native sequence of AfAglB lacks cysteine residues. First, we searched amino acid residues that were exposed on the molecular surface and located close to the Ser/Thr pocket. The four residues in the AfAglB protein were located in good positions: E613, G617, K618, and A621, but E613 and K618 were not tested further, because the two positions correspond to evolutionally conserved residues. The G617C and A621C mutant proteins were expressed in E. coli membrane fractions, and isolated in the presence of 0.1 % DDM. The purified mutant proteins were incubated with an excess amount of a peptide that contains the N-glycosylation sequon and a Cys residue. The position of the Cys residue (+4


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position) in the acceptor peptide sequence was chosen by inspection of the ClPglB-peptide complex structure as the template. An intermolecular disulfide bond was formed by air oxidation under slightly alkaline pH conditions. We also tested the different positions of the Cys residue (+3 and +5 positions) in a preliminary experiment, and found that these positions had similar efficiency of the disulfide cross-linking (data not shown). Thus, we selected the middle position (+4) for further experiments. The protein-peptide complexes were separated from the unreacted peptides by membrane filtration or dialysis, and subjected to crystallization screening. The crystals of the G617C-peptide and A621C-peptide complexes were obtained, but only the G617C-pepide crystals provided analyzable X-ray diffraction data set. In summary, we selected the G617C mutant and the Cys residue at +4 position in the acceptor peptide for detailed structural and enzymatic studies. We confirmed that the G617C mutation did not affect the oligosaccharyl transfer activity (Figure S2). The gel-filtration peak profiles of the G617C mutant with and without the acceptor peptide were almost the same as the wild type protein, indicating that the thermostability of the protein was not affected by the mutation or the peptide cross-linking (Figure S3). For fluorescent detection, a TAMRA dye group was attached to the N-terminus of the acceptor peptide. The band with an apparent molecular weight of 75 kDa showed the intermolecular disulfide-bond formation, and the absence of a band at the tracking dye front indicated clean 13 ACS Paragon Plus Environment

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separation from the unreacted peptides (Figure 1B, lane 2). The addition of a reducing agent, DTT, to the SDS sample buffer cleaved the disulfide bond and released the tethered peptide, as shown by the band at the tracking dye front position on the SDS-PAGE gel (lane 4). The oligosaccharyl transfer reaction was initiated by mixing the cross-linked complex with A. fulgidus LLO. The attachment of the N-glycan increased the molecular mass of the cross-linked complex by 1.2 kDa, corresponding to 7 monosaccharides, but the mobility shift was too small to detect by SDS-PAGE (lane 1). The addition of DTT to the SDS samples released the glycopeptide from the AglB protein (lane 3). The MS analysis confirmed the attachment of the heptasaccharide to the peptide (Figure 1C). Note that the intermolecular disulfide bond formation was not complete: The Mal-PEG alkylation analysis indicated that the final preparation contained about 10 % of the peptide-free AglB protein. The unreacted AglB could not be removed by chromatographic methods, but it did not interfere with the following analyses, since all of the peptide was attached to the AglB protein and the fluorescence detection of the peptide was employed. We considered that the oligosaccharyl transfer reaction would proceed in an intramolecular fashion within the cross-linked complex. For confirmation, we compared the reaction rate of the tethered peptide with that of the peptide in the free state at the same concentration. A pre-incubation with DTT was performed, to release the tethered peptide into the reaction 14 ACS Paragon Plus Environment

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solution. After the pre-incubation with DTT, the reaction rate was 30-fold slower than that of the reaction pre-incubated without DTT (Figure 1D). In this assay, the concentration of the cross-linked AfAglB-peptide complex was decreased 10-fold, to enhance the difference between the intra- and inter-molecular reaction rates. Under normal reaction conditions, the reaction rate was almost the same (Figure S4). Curiously, the extent of the reaction within the cross-linked complex reached a plateau at about 0.8, even after a prolonged incubation time (Figure 1D and Figure S4). In other words, about 20 % of the peptide in the complex remained unglycosylated. This is not due to the shortage of the donor substrate LLO, since the extent of the reaction reached 1.0 under the pre-incubation conditions with DTT (i.e., the acceptor peptide was in the free state). We checked the mass value of the unglycosylated peptide in the complex after the reaction, and confirmed that no chemical modifications, such as the deamination of the carboxyamide group, occurred (data not shown). Even though the exact nature of the inactive cross-linked complex is unknown, we consider our evaluation of the oligosaccharyl transfer activity of the cross-linked complex to be valid, by comparing the first-order reaction rate constants. Crystal Structure of the Cross-Linked AglB-Peptide Complex. We prepared the AfAglB protein cross-linked with a 7-residue acceptor peptide, Ac-RYNVTAC-NH2, in the presence of 0.06 % LDAO. Note that the peptide used for crystallization did not contain the TAMRA 15 ACS Paragon Plus Environment

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fluorescent dye (Table 1). We used a more alkaline pH and a higher molar ratio of the peptide than those used in the cross linking for the oligosaccharyl transfer assay, to reduce the fraction of the non-cross-linked AglB protein. The Mal-PEG alkylation analysis suggested that the occupancy of the bound peptide was not complete, but substantially greater than 90 %. We collected an X-ray diffraction dataset to a resolution of 3.5 Å (Table S1). Initial phases were estimated by the molecular replacement method, using the structure of AfAglB (PDB 3WAK) as the search model. The electron density of the Asn-Val-Thr sequon was observed in the Fo-Fc difference electron density map, and the model was built. The cross-linked AglB-peptide complex structure was refined to an Rwork/Rfree = 20.7 %/27.8 % (Table S1 and Figure 2A). The N-terminal TM region of AfAglB comprises 13 TM helices (cyan). The C-terminal globular domain contains three structural units, CC (C-terminal core, salmon), IS (insertion, green), and P1 (peripheral 1, yellow). The acceptor peptide resides at the boundary between the N-terminal TM region and the C-terminal globular domain (Figure 2C). The bound peptide adopted an extended conformation in the Asn-Val-Thr sequon. For comparison, the structure of ClPglB in the sequon peptide-bound form is also shown (Figures 2B and 2D). 15 There are two notable interactions between the peptides and the AglB/PglB proteins. First, the side-chain carboxyamide group of the Asn residue in the sequon interacts with two conserved acidic residues in the TM regions (D47 and E360 in AfAglB, and D56 and E319 in ClPglB), and forms a catalytic structure 16 ACS Paragon Plus Environment

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together with a bound metal ion. These Asp and Glu residues are both conserved and belong to the DXD motif (GND in AfAglB and TND in ClPglB) in the first external loop (EL1), and the TIXE motif (TIAE in AfAglB and TIME in ClPglB) in the fifth external loop (EL5), respectively. 18, 37

The metal ion is not directly involved in the catalysis, but appears to be structurally

important to form the ‘carboxylate dyad’ structure. In fact, the removal of the metal ion by a chelating reagent, EDTA, abolished the enzymatic activity. 18 Secondly, the short conserved motif, W550-W551-D552, and another conserved residue, K618 in AfAglB and I572 in ClPglB, contribute to the formation of the Ser/Thr-binding pocket, which recognizes the Ser/Thr residue at the +2 position in the N-glycosylation sequon. The interaction between the +2 Ser/Thr residue in the sequon and the Ser/Thr-binding pocket contributes to the proper positioning of the sequon in the binding site, and eventually to the formation of the catalytic structure for the Asn side-chain activation. The conformational state of the EL5 loop (blue) is particularly interesting. The N-terminal half (Ser335-Gln350) of the EL5 loop was disordered in the cross-linked AfAglB-sequon structure, whereas the C-terminal half (Pro351-Thr373) was ordered (Figure 2A). The same partially ordered conformation of the EL5 loop was observed in the ClPglB-peptide complex (Figure 2B). Thus, the partially ordered state is apparently an intrinsic property of the EL5 loops of AglB/PglB in their complexes with acceptor peptides.


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Effects of Mutations of the Conserved Acidic Residues on the Enzymatic Activity in the Cross-Linked State. The two acidic residues, D47 and E360, of the AfAglB G617C protein were mutated to alanine individually, and their effects on the oligosaccharyl transfer activity were examined (Figure 3). The single mutations, D47A and E360A, both retained the oligosaccharyl transfer activity, but with reduced rates (Figure 3B). D47A had a more severe effect than E360A. By contrast, the double alanine mutation, D47A/E360A, completely abolished the catalytic activity (Figure 3A). N-Glycosylation Consensus Requirement in the Cross-Linked State. We examined the requirement of the consensus residues in the N-glycosylation sequon for the oligosaccharyl transfer activity in the cross-linked state (Figure 4A). First, the replacement of the Asn residue by a Gln residue in the acceptor peptide did not abolish the activity. The MS analysis indicated the presence of the heptasaccharide structure (Figure 4B), even though the transfer rate was very slow (Figure 4C). A control experiment using a cross-linked complex tethered to the AVT sequence did not generate the corresponding glycopeptide product, confirming the indispensable role of the Asn or Gln side-chain carboxyamide group in the reaction. Second, the replacement of the Thr residue at the +2 position by an Ala residue did not impair the activity. The transfer rate in the NVA complex was much slower than that in the NVT complex (Figure 4C). This result ruled out the possibility of the direct involvement of the hydroxy group of the Ser/Thr 18 ACS Paragon Plus Environment

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residue in the N-glycosylation reaction. The two experiments using the non-canonical sequons, QVT and NVA, clearly indicated that the requirement of the N-glycosylation consensus sequence was greatly relaxed in the oligosaccharyl transfer reaction in the cross-linked state. Non-Selectivity of the Side Chain at the X Position in the Sequon. In the previous in vitro analyses, the amino-acid variations at the X position in the sequon strongly affected the oligosaccharyl transfer efficiency catalyzed by Pyrococcus furiosus AglB-L (PfAglB) and Campylobacter jejuni PglB (CjPglB). 8, 38 We repeated the same peptide library experiment using AfAglB (Table 1, Figure 5A). A strong preference for particular amino-acid residues at the X position was observed: Glu and Gln were the most favored amino acid residues, whereas Arg, Lys, and Trp were the least favored ones. Then, we assessed the effects of amino acid variations at the X position in the cross-linked state. We prepared cross-linked complexes containing tethered peptides with 19 amino acid residues other than Cys at the X position (Table 1), and measured the extents of the reactions at 5 min and 15 min (Figure 5B). All of the cross-linked complexes except for the NPT-bearing complex showed comparable, efficient glycosylation. Thus, the X position effects disappeared in the cross-linked complex, except for the rejection of Pro.

DISCUSSION


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We developed a new assay system that is useful to study the oligosaccharyl transfer reaction catalyzed by oligosaccharyltransferase. The disulfide bond was used to generate the productive cross-linked complex of the AglB protein and an acceptor peptide (Figure 1A). The positions of the Cys residues on the AglB protein and in the peptide sequence were selected by reference to the crystal structure of the ClPgB-sequon complex. 15 The sequon-containing peptide attached to the AglB protein was efficiently glycosylated in the presence of LLO (Figure 1C). The glycopeptide product was cleaved from the complex by reduction, and the extent of the oligosaccharyl transfer reaction was monitored by the mobility shift in the fluorescent image of the SDS-PAGE gel (Figure 1B). The transfer reaction proceeded within the cross-linked complex, because the release of the tethered peptide greatly decreased the reaction rate (Figure 1D). We determined the crystal structure of the cross-linked AfAglB-sequon complex to a resolution of 3.5 Å (Figure 2). The peptide sequence contained a typical N-glycosylation sequence, Asn-Val-Thr, and Cys at the +4 position for tethering via disulfide bond. The tethering was necessary for the crystallization of the complex state. In contrast, the PglB structure was determined to a comparable resolution (3.4 Å) without tethering. 15 For the AfAglB protein, the structure of the apo form was determined previously. 18 The comparison revealed that the overall structures of AfAglB in the peptide-bound state and in the apo state were almost identical, with 20 ACS Paragon Plus Environment

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Biochemistry

an rmsd (root-mean-square deviation) of 0.43 Å over 851 aligned Cα atoms (Figure 6A). One interesting difference is the conformational state of the EL5 loop (Figure 6B). The EL5 loop was fully ordered, and E360 in the EL5 loop was not involved in the metal-ion coordination in the apo state (orange model). Upon the acceptor peptide binding, the N-terminal half of the EL5 loop became unstructured, and E360 moved to participate in the bipartite interactions with the acceptor Asn side chain and the metal ion to form the carboxylate dyad structure (green model). This dynamic behavior of the EL5 loop was previously speculated to occur, based on the heterologous comparison between the apo form of AfAglB and the peptide-bound form of ClPglB, 18 but was now observed for the first time within the same protein molecule. Interestingly, the different roles of the N-terminal and C-terminal halves of the EL5 loop were suggested by the intensive study on the EL5 loop of ClPglB. 39 The N-terminal half recognizes the oligosaccharide part of the LLO molecule, whereas the C-terminal half functions as a conformational switch involved in sequon-binding or product release. We previously analyzed the effects of the mutations of the two conserved acidic residues, D47 and E360, on the enzymatic activity of AfAglB without tethering. 18 The two single alanine mutations led to the complete loss of the oligosaccharyl transfer activity. In this study, we analyzed the effects of these mutations in the cross-linked complex. To our surprise, the D47A and E360A mutants retained reduced but significant activity (Figure 3). Thus, the tethering of the 21 ACS Paragon Plus Environment

Biochemistry

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acceptor peptide compensated for the lack of either of the two carboxylate groups. This result implies that the formation of the carboxylate dyad structure is essential for the binding step, but is dispensable for the catalytic step in the cross-linked complex. As expected, the double alanine mutation, D47A/E360A, completely deactivated AfAglB, suggesting that at least one carboxylate group is necessary for the catalysis. In the reaction in the cross-linked state, the stringent requirement for the N-glycosylation consensus was greatly relaxed: the extension of the Asn side chain by one methylene group (i.e., Gln) was tolerable as an acceptor, and the hydroxy group at the +2 position was dispensable (Figure 4). Lizak et al. reported that eubacterial ClPglB could glycosylate Gln (-CH2-CH2-CO-NH2), homoserine (-CH2-CH2-OH), and hydroxamate (-CH2-CO-NHOH) residues, but with low efficiency. 16 The relative in vitro activities showed 200,000-, 900-, and 20-fold reductions, respectively, as compared to Asn (-CH2-CO-NH2). They also revealed that ClPglB could use atypical sequons, DQNAC, DQNAA and DQNAV, at low efficiencies. 40 Their relative in vitro activities were reduced by 400-, 4,000-, and 7,000-fold, respectively, as compared to the best DQNAT sequon. The eukaryotic OST can also catalyze the glycosylation of similar atypical sequons. Glycoproteome analyses of several mouse tissues revealed the N-glycosylation of atypical sequences, NXC, NGX, and NXV (X≠Pro), at 1.3 %, 0.5 %, and 0.4 % of the glycosylated sites, respectively. 41 Gln-linked (QGT) and non-consensus Asn-linked 22 ACS Paragon Plus Environment

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(SXN and TXN) oligosaccharides were also reported in human recombinant antibodies at 0.02 % and 0.01~1.1 % of the atypical sequences, respectively. 42 In summary, although it has been widely believed that the Asn-X-Ser/Thr sequon (X≠Pro) is necessary for protein N-glycosylation, the previous and present studies revealed that the consensus sequence is not absolutely required for N-glycosylation in the three domains of life. Although the canonical N-glycosylation sequon is highly preferred, a variety of atypical sequons are acceptable in special cases. In the previous in vitro oligosaccharyl transfer assays using short peptides, strong amino-acid preferences were observed at the X position for the glycosylation catalyzed by PfAglB and CjPglB. 8, 38 In this study, we confirmed a similar strong preference of AfAglB at the X position (Figure 5A). These observations are apparently inconsistent with statistical studies, which showed that glycosylated sites had no significant preference at the X position other than the rejection of Pro. 43-45 Intriguingly, the preference completely disappeared in the reaction in the cross-linked state (Figure 5B), suggesting that the disulfide tethering of the acceptor peptide mimics the co-translational oligosaccharyl transfer reaction coupled with the membrane permeation of proteins. The structural basis of the non-preference is the extended conformation of the bound sequon. The sequon was stretched out by the two simultaneous interactions between the Asn side chain and the carboxylate dyad structure, and the Ser/Thr side chain with the 23 ACS Paragon Plus Environment

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Ser/Thr-binding pocket. As a result, there are few contacts around the side chain of the X residue in the AglB/PglB-sequon complexes (Figures 2C and 2D).

In conclusion, the disulfide tethering of the acceptor peptide offers a unique system for studying the oligosaccharyl transfer reaction, by skipping the initial binding process and stabilizing the complex state. A mimetic of the co-translational oligosaccharyl transfer reaction coupled with the membrane permeation of proteins is an interesting application. We used the catalytically competent cross-linked complex for the enzymatic characterization of low-activity mutants and inefficient atypical sequons (Figures 3 and 4) and the structure determination of the enzyme-peptide complex (Figures 2 and 6). It is necessary to consider adverse effects of the disulfide-bond tethering. The strict rejection of Pro at the X position within the cross-linked complex, however, suggested that the tethering just increased the probability of rare events, and did not cause artificial preferences to happen (Figure 5).

ASSOCIATED CONTENT

Supporting Information


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The Supporting Information is available free of charge on the ACS Publication website at DOI: XXXXXX.

Design of the cysteine mutants (Figure S1), test assay of the G617C mutant (Figure S2), gel-filtration peak profiles of the G617C mutant with and without the acceptor peptide (Figure S3), comparison of the oligosaccharyl transfer rate under normal reaction conditions between in the cross-linked state and in the free state of the acceptor peptide (Figure S4), and a summary of crystallographic and refinement statistics (Table S1) (PDF)

Accession Code

The atomic coordinates and structure factors have been deposited in the Protein Data Bank as an entry 5GMY.

AUTHOR INFORMATION

Corresponding Author * Phone: 81-92-642-6968. Fax: 81-92-642-6833. E-mail: [email protected].


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Present Address ¶

Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo-Motoyama, Kita-ku, Kyoto

603-8555, Japan

Author Contributions #

These authors contributed equally.

SM and DK conceived and coordinated the study and wrote the paper. YT performed the time course assays and the MS/MS analyses. SM and AS measured the X-ray diffraction and determined the crystal structure. MI performed the preliminary tethering experiments described in Figures 1A and 1B. All authors reviewed the results and approved the final version of the manuscript.

Funding This work was supported by JSPS KAKENHI Grant Numbers JP24370047 and JP26119002 (to D.K.).

Notes The authors declare no competing financial interest. 26 ACS Paragon Plus Environment

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ACKNOWLEDGMENTS

We thank Ms. Yuki Matsuzaki (Laboratory for Technical Support, Medical Institute of Bioregulation, Kyushu University) for DNA sequencing. The experiments at the Photon Factory were performed with the approval of the Photon Factory Program Advisory Committee, as Proposal 2015G085, and those at SPring-8 were performed under the Cooperative Research Program of the Institute for Protein Research, Osaka University, Osaka, Japan, as Proposals 21046922 and 20156519.

ABBREVIATIONS AglB, archaeal glycosylation B; AfAglB, Archaeoglobus fulgidus AglB-L; CjPglB, Campylobacter jejuni PglB; ClPglB, Campylobacter lari PglB; DDM, n-dodecyl-β-D-maltopyranoside; DTT, dithiothreitol; EL5, external loop 5; LDAO, lauryldimethylamine N-oxide; LLO, lipid-linked oligosaccharide; OST, oligosaccharyltransferase; PfAglB, Pyrococcus furiosus AglB-L; PglB, protein glycosylation B; TAMRA, carboxytetramethylrhodamine; TM, transmembrane.


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Table 1.

Peptides used in this study

Peptide sequence a

X+1

Tethering position

Purpose

b

TAMRA-APYNVTACKR

+4

V

TAMRA-APYQVTACKR

+4

V

TAMRA-APYNVAACKR

+4

V

TAMRA-APYAVTACKR

+4

V

TAMRA-APYNPTACKR

+4

P

TAMRA-APYAAAACKR

+4

A

(C-TAMRA)-RGNXTAR

-

19 aa except C

library experiment in Figure 5A

TAMRA-APYNXTACKR

+4

19 aa except C

library experiment in Figure 5B

Ac-RYNVTAC-NH2

+4

a

V

sequon requirement in Figure 4

crystallization in Figure 2A

The N-glycosylation sequon is underlined. TAMRA denotes the direct coupling of

carboxytetramethylrhodamine to the N-terminal α-amino group, and C-TAMRA denotes the TAMRA-maleimide modification of the thiol group of the N-terminal Cys residue after peptide synthesis. Ac- and -NH2 indicate the modifications of the acetyl and amide groups at the α-amino and α-carboxyl groups, respectively. No other modifications were present unless otherwise indicated. b

The tethering position is defined as X−2-X−1-Asn0-X+1-Thr+2-X+3-X+4-X+5.


Biochemistry

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Figure 1. Preparation of the catalytically competent cross-linked AfAglB complex. (A) Outline of the preparation procedure. The AfAglB protein is a single-subunit membrane polypeptide containing 13 transmembrane helices in the N-terminal region (cyan rectangle) embedded in the membrane (purple rectangles). A single cysteine was introduced at residue 617 in the C-terminal globular domain (orange circle) (step 1). The purified G617C mutant was mixed with an acceptor peptide containing the N-glycosylation sequon (NxT), a Cys residue at the +4 position for disulfide crosslinking, and a fluorescent TAMRA dye (φ) attached to the N-terminus for detection (step 2). An intermolecular disulfide bond was formed by air oxidation (step 3), and 38 ACS Paragon Plus Environment

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Biochemistry

then unreacted excess peptides were removed by membrane filtration or dialysis (step 4). The addition of the donor substrate, A. fulgidus LLO (step 5), initiated the oligosaccharyl transfer reaction to generate the glycopeptide on the AfAglB protein (step 6). The addition of a reducing reagent, DTT, cleaved the disulfide bond (step 7), to release the glycopeptide product for fluorescent quantification (step 8). (B) After the AfAglB-peptide complex was incubated with or without AfLLO (LLO + or -), SDS sample buffer was added, and the reaction was incubated at room temperature in the presence or absence of DTT (DTT + or -). The reactions were fractionated by SDS-PAGE, and in-gel fluorescent imaging was used to analyze the products. (C) MS/MS analysis of the product in the cross-linked complex. The precursor ion is marked by the vertical arrow. The expected m/z values were observed within 0.02 of the theoretical values. (D) Comparison of the oligosaccharyl transfer reaction rate in the cross-linked state (pre-incubation without DTT) with that in the free state of the acceptor peptide (pre-incubation with DTT). DTT was added to the reaction solution before the reaction, to dissociate the tethered peptide from the complex.


Biochemistry

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Figure 2. Crystal structure of the cross-linked AfAglB-sequon complex, and comparison with the ClPglB structure in the non-covalent complex with a sequon peptide. Overall structures of AfAglB (A) and ClPglB (PDB 3RCE) (B), both in the acceptor-peptide bound states. The N-terminal transmembrane region consists of 13 transmembrane (TM) helices (cyan), and the C-terminal globular domain comprises structural units referred to as CC (salmon), IS (green), and P1 (yellow). The P1 unit is specific to AfAglB. The N-terminal half (Ser335-Gln350 in AfAglB,


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Biochemistry

and Leu283-Ala306 in ClPglB) of the external loop 5 (EL5) was structurally disordered (blue dashed lines), whereas the C-terminal half (Pro351-Thr373 in AfAglB, and Ala307-Val327 in ClPglB) was ordered. Acceptor peptides (yellow stick models) are bound at the boundary between the N-terminal TM region and the C-terminal globular domain of AfAglB (C) and ClPglB (D). For AfAglB, a simulated annealing Fo – Fc omit electron density map contoured at +2.7σ is shown as a green mesh. The atoms of the acceptor peptide and the C617 residue in the AfAglB protein were omitted in the map calculation.


Biochemistry

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Figure 3. Mutagenesis of the conserved acidic residues in the catalytic structure. The two acidic residues that participate in the formation of the carboxylate dyad structure, D47 and E360, were replaced by Ala. (A) Effects of the mutations on the oligosaccharyl transfer activity in the cross-linked state. The reaction was incubated for 1 h. (B) Time course of the glycopeptide formation in the cross-linked state. Triplicate measurements were performed for each time-point. The error bars represent the standard deviations. Note that G617C, G617C/D47A, and G617C/E360A are referred to as WT, D47A, and E360A, for clarity.


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Biochemistry

Figure 4. Non-stringent requirement of the N-glycosylation sequon in the cross-linked state. (A) Effects of the amino-acid substitutions in the N-glycosylation consensus sequence on the oligosaccharyl transfer reaction in the cross-linked state. (B) MS/MS analysis of the product in the complex cross-linked with a peptide containing QVT. The precursor ion is marked with the vertical arrow. The expected m/z values were observed within 0.02 of the theoretical values. (C) 43 ACS Paragon Plus Environment

Biochemistry

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Time course of the glycopeptide formation of the sequon lacking the Ser/Thr residue at the +2 position (NVA), and the sequon with Gln at the 0 position (QVT). Triplicate measurements were performed for each time-point. The error bars represent the standard deviations.


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Biochemistry

Figure 5. Effects of the amino acid substitution at the X position of the N-glycosylation sequon on the oligosaccharyl transfer activity. Amino-acid preference at the X position (+1 position) of the peptide substrate in the oligosaccharyl transfer reaction in the free state (A), and in the oligosaccharyl transfer reaction in the cross-linked state (B). The peptide sequences used are described in Table 1. In (A), the ratios of the oligosaccharyl transfer reaction rates were calculated for the NXT-containing peptides (X is 19 amino acids other than cysteine) relative to the NET-containing peptide. In (B), the open and filled bars indicate the extents of the reactions at 5-min and 15-min, respectively.


Biochemistry

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Figure 6. Comparison of the conformations of the EL5 loop between the peptide-bound and apo states of the AfAglB protein. Overall structure (A) and close-up view of the catalytic site (B). The EL5 loop is colored green in the peptide-bound form and orange in the apo form. N* and C* denote the N-terminal and C-terminal positions of the EL5 loop, respectively. The bound acceptor peptide is depicted as a yellow tube with a stick model of the Asn side chain. The side chains of the two conserved acidic residues, D47 and E360, are also shown in the stick model. The binding of the acceptor peptide induced the disordered conformation of the N-terminal half of the EL5 loop, and simultaneously the movement of E360 (shown by the dashed arrow) to form the carboxylate dyad structure, together with D47 and a divalent metal ion (M2+).


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Biochemistry

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Tethering an N-Glycosylation-Sequon Containing Peptide Creates a Catalytically Competent Oligosaccharyltransferase Complex Shunsuke Matsumoto, Yuya Taguchi, Atsushi Shimada, Mayumi Igura, and Daisuke Kohda*


Tethering an N-Glycosylation Sequon-Containing Peptide Creates a

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