Distinct roles and actions of protein disulfide isomerase family enzymes in catalysis of nascent-chain disulfide bond formation

Abstract

The mammalian endoplasmic reticulum (ER) harbors more than 20 members of the protein disulfide isomerase (PDI) family that act to maintain proteostasis. Herein, we developed an in vitro system for directly monitoring PDI- or ERp46-catalyzed disulfide bond formation in ribosome-associated nascent chains of human serum albumin. The results indicated that ERp46 more efficiently introduced disulfide bonds into nascent chains with a short segment exposed outside the ribosome exit site than PDI. Single-molecule analysis by high-speed atomic force microscopy further revealed that PDI binds nascent chains persistently, forming a stable face-to-face homodimer, whereas ERp46 binds for a shorter time in monomeric form, indicating their different mechanisms for substrate recognition and disulfide bond introduction. Thus, ERp46 serves as a more potent disulfide introducer especially during the early stages of translation, whereas PDI can catalyze disulfide formation when longer nascent chains emerge out from ribosome.

Keywords: Cell Biology; Molecular Biology; Structural Biology.

Graphical abstract

Figure 1

Disulfide bond introduction into RNC 69-aa and 82-aa by PDI and ERp46 (A) Schematic structure of plasmids constructed in this study. “uORF2” is an arrest sequence that serves to stall translation of the upstream protein and thereby prepare stable ribosome-nascent chain complexes (RNCs). The bottom cartoon represents the location of cysteines and disulfide bonds in HSA domain I. HSA domain I consists of 195 amino acids and contains five disulfide bonds and one free cysteine at residue 34. A green box indicates the pro-sequence. Orange circles and red lines indicate cysteines and native disulfide bonds, respectively. The region predicted to be buried in the ribosome exit tunnel is shown by a cyan box. (B) Domain organization of PDI and ERp46. Redox-active Trx-like domains with a CGHC motif are indicated by cyan boxes, whereas redox-inactive ones in PDI are by light green boxes. Note that the PDI b′ domain contains a substrate-binding hydrophobic pocket. (C and E) Time course of PDI-, ERp46-, and glutathione (no enzyme)-catalyzed disulfide bond introduction into RNC 69-aa (C) and 82-aa (E). “noSS” and “1SS” denote reduced and single-disulfide-bonded species of HSA nascent chains, respectively. Note that faint bands observed between “no SS” and “1SS” likely represent a species in which one of cysteines is not subjected to mal-PEG modification due to glutathionylation. In support of this, these minor bands are even fainter under the conditions of no GSH/GSSG. (D and F) Quantification of disulfide-bonded species for RNC 69-aa (D) and 82-aa (F) based on the results shown in (C) and (E), respectively (n = 3, mean ± SD).

Figure 2

Disulfide bond introduction into RNC 82-aa Cys mutants by PDI and ERp46 (A) Cartoon of RNC constructs used in this study. In each construct, a cysteine (represented by a black circle) was mutated to alanine. Note that RNC 82-aa C34A retains a native cysteine pairing (i.e., Cys53 and Cys62), whereas RNC 82-aa C53A and C62A retain a non-native pairing. (B and C) Time course of PDI- and ERp46-catalyzed disulfide bond introduction into RNC 82-aa C34A (top), C53A (middle), and C62A (bottom) mutants. Note that faint bands observed between “no SS” and “1SS” likely represent a species in which one of the cysteines is not subjected to mal-PEG modification due to glutathionylation. Quantification of disulfide-bonded species of RNC 82-aa Cys mutants is based on the results shown for the upper raw data (n = 3, mean ± SD). (D) Formation of a mixed disulfide bond between RNC 82-aa mono-Cys mutants and PDI (upper)/ERp46 (lower). “Mixed” and “No SS” denote a mixed disulfide complex between PDI/ERp46 and RNC mono-Cys mutants and isolated RNC 82-aa, respectively. Note that faint bands observed between “Mixed” and “no SS” are likely non-specific bands, as they were seen at the same position regardless of which 82-aa mono-Cys mutant was tested or whether an RNC was reacted with PDI or ERp46. (E) Quantification of mixed disulfide species based on the results shown in (D). ∗p < 0.05, ∗∗p < 0.01. n = 3. Error bars indicate SD. (F) The cartoon on the left shows possible steric collisions between ribosomes and PDI when Cys62 attacks the mixed disulfide between Cys53 on RNC 82-aa and PDI (left). The cartoon on the right shows that ERp46 can avoid this steric collision due to its higher flexibility and domain arrangement.

Figure 3

Correlation of the distance between Cys residues and the ribosome exit site with the efficiency of disulfide bond introduction by PDI/ERp46 (A) Cartoons of RNC constructs with [SG]-repeat insertions. An [SG]₅ or [SG]₁₀ repeat sequence was inserted into RNC-82 aa C34A immediately after Cys62. (B and D) PDI- (B) and ERp46 (D)-mediated disulfide bond introduction into RNC 82-aa C34A with insertion of [SG]₅ (upper) or [SG]₁₀ (lower) repeats after Cys62. (C and E) Quantification of disulfide-bonded species (1SS) based on the results shown in (B) and (D). n = 3 for PDI and 2 for ERp46. Error bars indicate SD. (F) Formation of a mixed disulfide bond between the 82-aa mono-Cys mutant with a [SG]₁₀ repeat and PDI (upper)/ERp46 (lower). Note that bands observed between “Mixed” and “no SS” are likely non-specific bands, as they were seen at the same position regardless of which 82-aa mono-Cys [SG]₁₀ mutant was tested or whether the RNCs were reacted with PDI or ERp46. (G) Quantification of mixed disulfide species based on the results shown in (F). n = 3. Error bars indicate SD.

Figure 4

High-speed AFM analysis of ERp46 (A) AFM images (scan area, 300 × 300 Å; scale bar, 30 Å) for ERp46 V-shape (left) and O-shape (right) conformations. (B) Left upper: Histograms of circularity calculated from AFM images of ERp46. Values represent the average circularity (mean ± SD) calculated from curve fitting with a single- (middle and right) or two- (left) Gaussian model. Left lower: Histograms of height calculated from AFM images of ERp46. Values represent the average height (mean ± SD) calculated from curve fitting with a single-Gaussian model. Right: Two-dimensional scatterplots of the height versus circularity for ERp46 molecules observed by HS-AFM. (C) Time course snapshots of oxidized ERp46 captured by HS-AFM. The images were traced for 10 s. See also Video S1. (D) Time trace of the circularity of an ERp46 molecule. (E) Histogram of the circularity of ERp46 calculated from the time course snapshots shown in (D).

Figure 5

Single-molecule observation of PDI/ERp46 acting on 82-aa CA RNC by high-speed atomic force microscopy (A) The AFM images (scan area, 500 Å × 500 Å; scale bar, 100 Å) displaying 82-aa CA RNC in the absence of PDI family enzymes on a Ni²⁺-coated mica surface. The surface model on the right side of each AFM image illustrates ribosome whose view angle is approximately adjusted to the observed RNC particle. 40S and 60S ribosomal subunits are shown in red and blue, respectively. (B) Upper AFM images (scan area, 500 Å × 500 Å; scale bar, 100 Å) displaying 82-aa CA RNC in the presence of oxidized PDI (1 nM). PDI molecules that appear to bind 82-aa CA RNC are marked by red squares. Lower images (scan area, 250 Å × 250 Å; scale bar, 50 Å) highlight the regions surrounded by red squares in the upper images. See also Videos S2 and S3. (C) Upper AFM images (scan area, 750 Å × 750 Å; scale bar, 150 Å) displaying 82-aa CA RNC in the presence of oxidized ERp46 (1 nM). ERp46 molecules that appear to bind 82-aa CA RNC are marked by blue squares. Lower images (scan area, 250 Å × 250 Å; scale bar, 50 Å) highlight the regions surrounded by blue squares in the upper images. See also Video S4. (D) Histograms of the RNC binding time of the PDI monomer (left), the PDI dimer (middle), and ERp46 (right), calculated from the observed AFM images. (E) Histograms of the distance between the edge of the ribosome and the centers of RNC-neighboring PDI (left) and ERp46 (right) molecules, calculated from the observed AFM images. Values represent the average distance (mean ± SD) calculated from curve fitting with a single-Gaussian model.

Figure 6

Role of the PDI hydrophobic pocket in PDI-mediated disulfide bond introduction into RNC 82-aa (A) Disulfide bond introduction into RNC 82-aa by PDI I289A (upper) and ERp57 (lower). Note that faint bands observed between “no SS” and “1SS” likely represent a species in which one of cysteines is not subjected to mal-PEG modification due to glutathionylation. In support of this, these minor bands are even fainter under the conditions of no GSH/GSSG. (B) Quantification of disulfide-bonded species based on the results shown in (A). Quantifications for ERp46 and PDI are based on the results shown in Figures 1E and 1F. Statistical analysis has been made for the difference between PDI WT and PDI I289A at reaction times of 60 s, 180 s, and 360 s. ∗p < 0.05, ∗∗∗p < 0.001. n = 3. Error bars indicate SD. (C) HS-AFM analyses for binding of PDI I289A to RNC CA 82-aa. Upper AFM images (scan area, 800 Å × 800 Å; scale bar, 100 Å) display the PDI I289A molecules that bind 82-aa CA RNC, as marked by red squares. Lower images (scan area, 250 Å × 250 Å; scale bar, 50 Å) highlight the regions surrounded by red squares in the upper images. (D) Histograms show the distribution of the RNC binding time of the PDI I289A monomers (left) and dimers (right). (E) Histogram shows the distribution of the distance between the edge of the ribosome and the centers of RNC-neighboring PDI I289A molecules, calculated from the observed AFM images. Values represents the average distance (mean ± SD) calculated from curve fitting with a single-Gaussian model.

Figure 7

Proposed model of co-translational disulfide bond introduction into nascent chains by ERp46 and PDI During the early stages of translation, ERp46 introduces disulfide bonds through transient binding to a nascent chain. For efficient disulfide introduction by ERp46, a pair of cysteines must be exposed by at least ~8 amino acids from the ribosome exit site. By contrast, PDI introduces disulfide bonds by holding a nascent chain inside the central cavity of the PDI homodimer during the later stages of translation, where a pair of cysteines must be exposed by at least ~18 amino acids from the ribosome exit site. However, when a longer polypeptide is exposed outside the ribosome, ERp46- or PDI-mediated disulfide bond formation can be slower, possibly due to formation of higher-order conformation in the nascent chain. Longer nascent chains may allow PDI family enzymes to compete with each other for binding and acting on RNC.