Long-Lived Folding Intermediates Predominate the Targeting-Competent Secretome

Abstract

Secretory preproteins carry signal peptides fused amino-terminally to mature domains. They are post-translationally targeted to cross the plasma membrane in non-folded states with the help of translocases, and fold only at their final destinations. The mechanism of this process of postponed folding is unknown, but is generally attributed to signal peptides and chaperones. We herein demonstrate that, during targeting, most mature domains maintain loosely packed folding intermediates. These largely soluble states are signal peptide independent and essential for translocase recognition. These intermediates are promoted by mature domain features: residue composition, elevated disorder, and reduced hydrophobicity. Consequently, a mature domain folds slower than its cytoplasmic structural homolog. Some mature domains could not evolve stable, loose intermediates, and hence depend on signal peptides for slow folding to the detriment of solubility. These unique features of secretory proteins impact our understanding of protein trafficking, folding, and aggregation, and thus place them in a distinct class.

Intrinsically disordered domains: Sequence ➔ disorder ➔ function relationships

“…72 Furthermore, recent work in bacteria shows that their secreted proteins need to be at least partially unfolded inside the cell to interact with the secretion apparatus and become folded outside the cell, where the extra-cellular folding is brought about not just by disulfide bond formation but by a variety of mechanisms. 73 Thus, secreted proteins represent a new cohort that are transiently disordered and have been called "delayed folding proteins." 74 The observation that the delayed folding of many secreted proteins is regulated by disulfide bond formation confirms our previous conjecture 62 that disordered domains that become folded by disulfide bond formation are likely secreted proteins that remain unstructured inside the cell and then gain structure outside the cell following secretion.…”

“…74 The observation that the delayed folding of many secreted proteins is regulated by disulfide bond formation confirms our previous conjecture 62 that disordered domains that become folded by disulfide bond formation are likely secreted proteins that remain unstructured inside the cell and then gain structure outside the cell following secretion. 69,[72][73][74] Proteins with ion binding functions are significantly enriched in IDPs. 75 Various types of ions, such as Ca 2+ , Zn 2+ , Cd 2+ , Co 2+ , Mg 2+ , Cu 2+ , and Na + , have been identified in the current study.…”

Investigating the stability of the SecA–SecYEG complex during protein translocation across the bacterial membrane

“…When analyzing these results, we also considered the potential effect of substrate folding and secondary structure. A recent study by Tsirigotaki et al (24) revealed important differences between the in-solution behavior of proOmpA and proPhoA. They show that proOmpA adopts some elements of secondary structure in solution whereas proPhoA does not (24).…”

Refined measurement of SecA-driven protein transport reveals indirect coupling to ATP turnover

“…One additional factor we believe will prove particularly critical to understanding the slow in vitro transport rates is the folding state of the pre-protein, which is known to be important for enabling transport Tsirigotaki et al, 2018). Chaperones generally capture pre-proteins in vivo as they are translated and deliver them to the membrane in an optimally translocation competent state.…”