Mechanisms of Disulfide Bond Formation in Nascent Polypeptides Entering the Secretory Pathway

Abstract

Disulphide bonds are an abundant feature of proteins across all domains of life that are important for structure, stability, and function. In eukaryotic cells, a major site of disulphide bond formation is the endoplasmic reticulum (ER). How cysteines correctly pair during polypeptide folding to form the native disulphide bond pattern is a complex problem that is not fully understood. In this paper, the evidence for different folding mechanisms involved in ER-localised disulphide bond formation is reviewed with emphasis on events that occur during ER entry. Disulphide formation in nascent polypeptides is discussed with focus on (i) its mechanistic relationship with conformational folding, (ii) evidence for its occurrence at the co-translational stage during ER entry, and (iii) the role of protein disulphide isomerase (PDI) family members. This review highlights the complex array of cellular processes that influence disulphide bond formation and identifies key questions that need to be addressed to further understand this fundamental process.

Keywords: ER; PDI; disulphide formation; protein folding; protein secretion; protein synthesis.

Figure 1

Disulfide bond formation and conformational folding. (A) Schematic diagram of a nascent polypeptide, with the molecular structure of cysteine sidechains highlighted before (i) and after (ii) disulfide bond formation. (B) Mechanistic scheme to describe folding coupled disulfide formation. At the pre-folding stage, the nascent polypeptide exists as a dynamic random coil, in which disulfide interchange can take place to form transient disulfide bonded species (TDBS). If folding follows the folded precursor mechanism (red arrows), then formation of the nascent tertiary structure occurs first and promotes native disulfide formation. If folding follows the quasi-stochastic mechanism (blue arrows), then disulfide formation occurs first and drives the formation of the native tertiary structure. At the post-folding stage, the native fold is complete, and disulfide bonds are buried and protected.

Figure 2

Folding of a ribosome-associated nascent chain during endoplasmic reticulum (ER) entry. Schematic showing a translating ribosome targeted to the ER membrane. The nascent polypeptide (green) enters the ER via the sec complex, undergoes signal peptide cleavage, and begins to fold at the ER-exposed N-terminus. Both membrane-bound and soluble ER factors can interact with the nascent polypeptide as it undergoes translocation.

Figure 3

Model proteins to study co-translational disulfide bond formation. (A) Schematic showing an ER-targeted, ribosome-associated nascent chain complex in which disulfide formation takes place before translation is complete. (B) Ribbon diagram representing the three-dimensional (3D) structure of the haemagglutinin ectodomain (HA) monomer (Protein Data Bank (PDB) 1HA0). (C) Extensions to proteins at the C-terminus enable full translocation of the N-terminal domain while the C-terminus remains ribosome-attached. (D) Ribbon diagrams representing the 3D structures of (i) β2M (PDB 1A1M), (ii) prolactin (PDB 1RW5), and (iii) the disintegrin domain of ADAM10 (PDB 6BE6). Molecular graphics for figures were performed with UCSF Chimera version 1.14, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco [48].

Figure 4

The mechanism of thiol–disulfide exchange in protein disulfide isomerase (PDI)-catalysed disulfide formation. (A) Ribbon diagram representing the 3D structure of PDI (PDB 2B5E), which contains four thioredoxin domains. The locations of the CXXC motifs are highlighted. (B) The reaction mechanism for thiol–disulfide exchange between a substrate and PDI, in which PDI oxidises the substrate and in turn becomes reduced. (C) The linear trisulphur transition state that forms during the S_N2 reactions at steps 1 and 3 as shown in (B).