Protein secondary structure determines the temporal relationship between folding and disulfide formation

Abstract

How and when disulfide bonds form in proteins relative to the stage of their folding is a fundamental question in cell biology. Two models describe this relationship: the folded precursor model, in which a nascent structure forms before disulfides do, and the quasi-stochastic model, where disulfides form prior to folding. Here we investigated oxidative folding of three structurally diverse substrates, β2-microglobulin, prolactin, and the disintegrin domain of ADAM metallopeptidase domain 10 (ADAM10), to understand how these mechanisms apply in a cellular context. We used a eukaryotic cell-free translation system in which we could identify disulfide isomers in stalled translation intermediates to characterize the timing of disulfide formation relative to translocation into the endoplasmic reticulum and the presence of non-native disulfides. Our results indicate that in a domain lacking secondary structure, disulfides form before conformational folding through a process prone to nonnative disulfide formation, whereas in proteins with defined secondary structure, native disulfide formation occurs after partial folding. These findings reveal that the nascent protein structure promotes correct disulfide formation during cotranslational folding.

The Mammalian Cytosolic Thioredoxin Reductase Pathway Acts via a Membrane Protein to Reduce ER-localised Proteins

“…4C, lanes 3 and 5). We have previously shown that the disulfide-bonded products formed form this ADAM10 construct are present in nascent chains translocated into the ER lumen (Robinson, 2019). Taken together these results show that a source of non-membrane permeable reducing agent can resolve the non-native disulfides formed in nascent chains within the ER lumen.…”

“…We previously demonstrated that non-native disulfides formed during the synthesis of β1-integrin require a cytosolic reductive pathway to ensure their isomerisation to the correct disulfides (Poet et al, 2017). To evaluate the ability of our reconstituted pathway to isomerise non-native disulfides, we generated stalled translocation intermediates of a protein that forms non-native disulfides following in vitro translation in the presence of semi-permeabilised (SP) cells (Robinson, 2019). By isolating SP-cells containing these intermediates we were then able to follow the rearrangement of disulfides post-translationally in the presence or absence of a TrxR1 reduction system (Fig.…”

“…4C, lane 1). These products correspond to the translocated, signal sequence cleaved chain and untranslocated preprotein (Robinson, 2019). When the translations were carried out in the absence of added G6P and a post-translational incubation carried out in the absence of G6P, a diffuse pattern emerged with the translation products forming both intra and inter-chain disulfides (lane 2).…”

“…The human ADAM10 construct codes for residues 456-550 and contains the human-β2M signal sequence residues (1-20). The generation of this construct and its transcription and translation were described previously (Robinson, 2019). Essentially, plasmid DNA was used as a template for PCR using an appropriate forward primer that adds a T7 promoter and a reverse primer that lacks a stop codon.…”

The mammalian cytosolic thioredoxin reductase pathway acts via a membrane protein to reduce ER-localised proteins

“…We previously demonstrated that non-native disulfides formed during the synthesis of β1-integrin require a cytosolic reductive pathway to ensure their isomerisation to the correct disulfides (Poet et al, 2017). To evaluate the ability of our reconstituted pathway to isomerise non-native disulfides, we generated stalled translocation intermediates of a protein that forms non-native disulfides following in vitro translation in the presence of semi-permeabilised (SP) cells (Robinson, Kanemura et al, 2020).…”