1. Volume 22, Issue 4, Pages 590-601 (April 2014)
Structural Basis of Substrate Specificity of Human Oligosaccharyl Transferase Subunit N33/Tusc3 and Its Role in Regulating Protein N-Glycosylation 
Elisabeth Mohorko, Robin L. Owen, Goran Malojčić, Maurice S. Brozzo, Markus Aebi, Rudi Glockshuber 
Structure 
Volume 22, Issue 4, Pages (April 2014)
DOI: /j.str
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2. Structure 2014 22, 590-601DOI: (10.1016/j.str.2014.02.013)
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3. Figure 1
Subunit Composition of the Human OST Complex, Sequence Alignment of N33/Tusc3 with IAP/MagT1 and the Yeast Homologs Ost3 and Ost6, and Redox Properties of N33/Tusc3
(A) Human OST is composed of seven different subunits and exists in four alternative isoforms. The glycan transfer from a lipid-linked oligosaccharide onto an asparagine residue in a glycosylation sequon is catalyzed by the mutually exclusive subunits Stt3A or Stt3B, and either the redox-active subunit N33/Tusc3 or its homolog IAP/MagT1 is incorporated into the OST complex. Structural information on the human OST complex is available for the small membrane protein Ost4 (Gayen and Kang, 2011) and N33/Tusc3 from this study.
(B) Structure-based sequence alignment (performed with T-coffee, Notredame et al., 2000; and MUSTANG, Konagurthu et al., 2006, 2010) of the thioredoxin-like ER domains of human N-N33/Tusc3 and IAP/MagT1 with their yeast homologs Ost3 and Ost6. The invariant active-site cysteines are highlighted in yellow. The secondary structure elements of N-N33/Tusc3 (this study) and yeast Ost6p (Schulz et al., 2009) as identified with MUSTANG (Konagurthu et al., 2006, 2010) are indicated at the top and bottom, respectively. The amino acid numbering at the top and the bottom corresponds to the residue numbers in the solved structures of N-N33/Tusc3 and Ost6p, respectively. The residue numbers at the right correspond to the sequences of the full-length proteins (including their predicted, N-terminal signal sequences). See also Figure S1.
(C) Redox equilibrium between N-N33/Tusc3 and mixtures of reduced and oxidized dithiothreitol (DTTred and DTTox) at pH 7.0 and 25°C. Oxidized and reduced N-N33/Tusc3 were separated after acid-quenching by RP-HPLC and quantified. The fraction of reduced N-N33/Tusc3 (black circles) was plotted against the DTTred/DTTox ratio and fitted according to Equation 1 (solid black line), yielding an equilibrium constant (Keq) of 0.154 ± and a redox potential of −283 ± 1 mV for N-N33/Tusc3. The same redox potential within experimental error (−282 ± 1 mV) was obtained for the N-N33/Tusc3 variant Cys82Ser (blue squares, dotted blue line; Keq = 0.144 ± 0.025), showing that the unpaired Cys82 in wild-type N-N33/Tusc3 is not redox-active. The indicated errors for Keq are the errors from the fits to Equation 1.
(D) Oxidized N-N33/Tusc3 (blue circles) is more stable against thermal unfolding at pH 7.0 than reduced N-N33/Tusc3 (red circles). Thermal unfolding was followed by the change in the circular dichroism signal at 222 nm. Samples with reduced protein contained 5 mM DTTred. Because the transitions were not fully reversible and accompanied by partial aggregation, only apparent melting temperatures were extracted from fitting the data to a reversible transition (solid lines; indicated errors are fitting errors; see Supplemental Experimental Procedures for details).
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4. Figure 2 Peptide Binding Specificity of N-N33/Tusc3
The kinetics of mixed disulfide formation between N-N33/Tusc3C61S and peptide segments of potential N-N33/Tusc3 substrates with a single cysteine (peptides 1–4) are dictated by the peptide sequence (see also Figure S3).
(A) Reaction scheme of the competition experiment in which peptides 1–4 compete for mixed disulfide formation with N-N33/Tusc3C61S. N-N33/Tusc3C61S (gray sphere) was activated with Ellman’s reagent (reaction 1), and the purified, activated N33/Tusc3C61S-TNB complex was mixed with a mixture of peptides 1–4 (each at 2-fold excess over N33/Tusc3C61S-TNB; reaction 2). After removal of unbound peptides by gel filtration, the mixture of N33/Tusc3C61S-peptide complexes was reduced by DTTred (reaction 3), and the released peptides were separated by RP-HPLC and quantified.
(B) Comparison of the kinetics of mixed disulfide formation with the individual peptides 1–4, the tripeptide glutathione (GSH) or β-mercaptoethanol (β-ME). Reactions at pH 7.0 were initiated with stopped-flow mixing and performed under pseudo first-order conditions with initial concentrations of N-N33/Tusc3C61S-TNB (5 μM) and peptide (35 μM). Reactions were followed by the increase in tryptophan fluorescence upon release of TNB and evaluated according to pseudo first-order kinetics (Equation 3, solid lines).
(C) Thermal unfolding transitions at pH 7.0 of the purified mixed disulfide complexes between N-N33/Tusc3C61S and peptides 1–4 (blue, green, red and orange data points, respectively) compared to free N-N33/Tusc3C61S (black). Solid lines correspond to fits according to Equation 4 (see Supplemental Experimental Procedures).
(D) RP-HPLC analysis of the mixture of mixed disulfide complexes before (top) and after (bottom) treatment with DTTred (the mixture of mixed disulfide complexes [top] is eluted as a single peak).
(E) Separation and quantification of the released peptides 1–4 (expanded representation of D).
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5. Figure 3
Structures of Oxidized N-N33/Tusc3 and N-N33/Tusc3C61S in Complex with Peptide 1 or 2
(A) Ribbon diagram of the 1.6 Å X-ray structure of the ER luminal domain of oxidized N-N33/Tusc3, which adopts a thioredoxin-like fold consisting of a four-stranded β sheet surrounded by alpha helices. The N-terminal segment of α helix 3 contains the catalytic disulfide bond (indicated in yellow) formed by the Cys58-Cys61 cysteine pair.
(B) Superposition of oxidized N-N33/Tusc3 (red) with oxidized yeast Ost6 (blue; Schulz et al., 2009). The thioredoxin-like fold of the human and the yeast protein is identical except for the segment between β4 and α5, which harbors a fifth strand in Ost6, and the loop segment N-terminal to helix α3 (both segments accentuated in green for Ost6; see also Figure S5).
(C) The 1.10 Å X-ray structure of the N-N33/Tusc3C61S-peptide 1 mixed disulfide complex. The termini of peptide 1 (dark gray) in the binding groove are indicated to illustrate the orientation of the peptide.
(D) The 1.6 Å X-ray structure of the N-N33/Tusc3C61S-peptide 2 mixed disulfide complex. Peptide 2 (light gray) binds to the same groove as peptide 1, but in the opposite orientation.
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6. Figure 4 Peptide Recognition Mode of N-N33/Tusc3
(A) Binding mode of peptide 1 to N-N33/Tusc3 in C-N orientation. Phe4 at the −2 position relative to the cysteine (Cys6) forming a mixed disulfide with Cys58 of N-N33/Tusc3 is specifically recognized by a hydrophobic side chain pocket. Asn8 of the glycosylation sequon contained in peptide 1 (marked in pink) is solvent exposed and not recognized specifically (see also Figures S4 and S6).
(B) Binding mode of peptide 2 to N-N33/Tusc3 in N-C orientation. Leu12 at the +2 position relative to Cys10 is accommodated by the hydrophobic side chain pocket. Again, the glycosylation sequon in peptide 2 (Asn7 and Thr9, marked in pink) is not recognized specifically (see also Figures S4 and S6).
(C) Close-up of the peptide-binding sites, showing the backbone atoms of peptides 1 and 2 that are bound in opposite orientation and are stabilized by the same hydrogen bond interactions with Asn104 and Ser105 of N33/Tusc3 (the side chain atoms of peptides 1 and 2 are omitted for clarity; see also Figures S4 and S6).
(D) Thermal unfolding transitions of mixed disulfides between N-N33/Tusc3C61S and peptide 2 (black) or peptide 2 variants in which the Leu12 residue at the +2 position relative to the cysteine residue in peptide 2 was replaced by Val (red), Phe (green), Tyr (pink), and Ala (blue). The mixed disulfide between hN33C61S and wild-type peptide 2 (with Leu at position 12) proved to be most stable against thermal unfolding. The hydrophobic residues Phe and Val also proved to stabilize the mixed disulfide complex relative to Ala at postion 12. The small stabilizing effect relative to Ala of Tyr at position 12 can be rationalized by potential steric clashes in the +2 side chain binding pocket of N-N33/Tusc3, because the pocket is too small to accommodate the aromatic hydroxyl group of Tyr. The thermal unfolding transitions of the mixed disulfide complexes were followed by the change in the circular dichroism signal at 222 nm, and normalized after fitting the data according to Equation 4 (see Supplemental Experimental Procedures). The indicated errors in ΔTm correspond to the sum of the respective fitting errors.
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7. Figure 5
Model of the Possible Modulation of Co- and Posttranslational Glycosylation Efficiency by N-N33/Tusc3 in Brain Tissue
(A) Cotranslational glycosylation: the OST complex is associated with the translocon and glycosylation is preferentially carried out by Stt3A (Ruiz-Canada et al., 2009). A cysteine thiolate from a polypeptide chain newly translocated into the ER attacks the disulfide bond of N33/Tusc3 and forms a transient mixed disulfide with the Cys58 sulfur of N33/Tusc3. The mixed disulfide is resolved again by attack from the Cys61 thiolate of N33/Tusc3. Mixed disulfide formation is favored when a hydrophobic residue (black arrow) is located two residues C-terminal to the attacking cysteine residue.
(B) Posttranslational recognition, in which Stt3B is the main catalytic subunit. Glycosylation sites close to the C terminus of nascent polypeptides can only be glycosylated after their release into the ER and rebinding of the translocated polypeptide to the OST complex (Ruiz-Canada et al., 2009; Shrimal et al., 2013a). Here, the polypeptide chain can also bind to N-N33/Tusc3 in an orientation opposite to that shown in (A), and a hydrophobic residue two residues N-terminal to the attacking cysteine would favor mixed disulfide formation under these conditions. In both (A) and (B), mixed disulfide formation is predicted to increase the efficiency of glycosylation by preventing the folding of the polypeptide substrate and increasing the accessibility of Stt3A to glycosylation sequons (indicated by “NxT”). Glycosylation efficiency may also be decreased when a glycosylation sequon close to a cysteine is bound to the peptide-binding groove of N-N33/Tusc3 and no longer accessible to Stt3A. The subunit IAP/MagT1 can be incorporated instead of N33/Tusc3 into the OST complex and may have a function similar to that of N33/Tusc3. Whether Stt3A and Stt3B can associate with both N33/Tusc3 and IAP/MagT1 is presently unknown.
Structure  , DOI: ( /j.str )
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