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

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.

Download Full-text

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

10.1101/564740 ◽

2019 ◽

Cited By ~ 1

Author(s):

Philip J Robinson ◽

Shingo Kanemura ◽

Xiaofei Cao ◽

Neil J Bulleid

Keyword(s):

Secondary Structure ◽

Stochastic Model ◽

Cell Biology ◽

Disulfide Bonds ◽

Protein Secondary Structure ◽

Structural Features ◽

Protein Domain ◽

Translation System ◽

Structural Elements ◽

Disulfide Formation

AbstractHow and when disulfides form in proteins during their folding is a fundamental question in cell biology. Two models describe the relationship between disulfide formation and folding, the folded precursor model, in which formation of nascent structure occurs prior to the disulfides and the quasi-stochastic model where disulfides form prior to complete domain folding. Here we investigate oxidative folding within a cellular milieu of three structurally diverse substrates in order to understand the folding mechanisms required to achieve correct cysteine coupling. We use a eukaryotic translation system in which we can manipulate the redox conditions and produce stalled translation intermediates representative of different stages of translocation. We identify different disulfide bonded isomers by non-reducing SDS-PAGE. Using this approach, we determined whether each substrate followed a folding driven or disulfide driven mechanism. Our results demonstrate that the folding model is substrate-dependent with disulfides forming prior to complete domain folding in a domain lacking secondary structure, whereas disulfide formation was absent at this stage in proteins with defined structural elements. In addition, we demonstrate the presence and rearrangement of non-native disulfides specifically in substrates following the quasi-stochastic model. These findings demonstrate why non-native disulfides are prevented from forming in proteins with well-defined secondary structure.Significance statementA third of human proteins contain structural elements called disulfide bonds that are often crucial for stability and function. Disulfides form between cysteines in the specialised environment of the endoplasmic reticulum (ER), during the complex process of protein folding. Many proteins contain multiple cysteines that can potentially form correct or incorrect cysteine pairings. To investigate how correct disulfide pairs are formed in a biological context, we developed an experimental approach to assess disulfide formation and rearrangement as proteins enter the ER. We found that a protein domain with atypical secondary structure undergoes disulfide orchestrated folding as it enters the ER and is prone to incorrect disulfide formation. In contrast, proteins with defined secondary structure form folding dependent, native disulfides. These findings show how different mechanisms of disulfide formation can be rationalised from structural features of the folding domains.

Download Full-text