N-terminal signal peptides have evolved as a means of sorting secretory preproteins from cytoplasmic ones, predominantly in post-translational Sec-dependent protein export.2, 3, 4 During cytoplasmic transit, signal peptides delay preprotein folding1, 5, 6 and facilitate interactions with chaperones.7, 8, 9 At the export site, they allosterically activate the translocase and ‘in synergy’ with mature domain targeting signals allow preprotein binding/export.10, 11 On the trans side of the membrane specialized peptidases cleave the signal peptide,12 while the mature domain continues trafficking or folds to its native state.13 In co-translational Sec-dependent export signal peptides are commonly more hydrophobic and recognized by the ribosome-bound Signal Recognition Particle (SRP), while preproteins remain sequestered by the ribosome or might additionally require SecA for translocation (e.g. DsbA).14, 15, 16, 17, 18

Despite the sequence variability of signal peptides, universal biophysical features are preserved.19 All are composed of 3 parts: N-terminal (N-), hydrophobic helical core (H-) and flexible C-terminal (C-) regions (Figure 1(A);.20 N-regions are ∼ 5 residues long20 and commonly have positively charged residue(s) that can interact with the SRP,21 SecB,19 or the negatively charged periphery of the signal peptide binding groove of SecA.22 H-regions are 7–15 residues long, consist of large hydrophobic residues (e.g. Leu, Met, Phe) with occasional helix breakers and small residues (Gly, Pro, Ser)19, 23 and affect SRP recognition24 and secretion efficiency.19 They promote interactions with chaperones like Trigger factor7 and SecB.8 When fitted in an elongated hydrophobic groove of SecYEG-bound SecA22 they become stabilized and drive closure of SecA’s preprotein clamp to initiate secretion.25 C-regions are 3–7 residues long and consist of neutral or polar residues that are disorder-promoting (e.g. Ser, Pro, Asn) and contain a signal peptidase cleavage site [-3-1, AxA for SPaseI for most secretory proteins; L(A/S)(G/A) for SPaseII for lipoproteins19, 26]. Signal peptides are fused to mature domains by flexible linkers that are mainly composed of disorder-promoting residues; also referred to as conformational “rheostats”, because they influence the folding behaviour of the whole mature domain, as seen in proPhoA.27

Translocation competence and slow folding behaviour seems to be intimately connected among secretory preproteins.28 Specific features, e.g. more polar residues and weaker hydrophobic patches, differentiate mature domains from cytoplasmic proteins and correlate with folding delays.4, 28, 29 Signal peptides were also reported to slow down folding of secretory proteins.30 By comparing the secretory PpiA to its cytoplasmic PpiB structural twin, we previously established that the folding delay of the secretory protein is due to a slightly altered order of initial foldon formation.1 This difference has been attributed to smaller, less branched, highly stabilized native contacts, more frustrated residues and a suboptimal β-turn in the first foldon in PpiA.1 Folding of PpiA was further delayed by its signal peptide.1 Quick folding of the first half of the signal peptide’s H-region while the second half remained highly dynamic, coincided with increased flexibility across the N-terminus of the mature domain, suppression of some initial foldons and slow-down of the remaining ones thus, introducing a loosely folded PpiA intermediate.1

As “signal peptide-mature domain combinations” co-evolved in preproteins, it was anticipated that the effect of signal peptide in delaying mature domain folding or/and the ability of the preprotein to be secreted (referred to as “secretability”) have become optimal. However, this is not the case with every combination tested. Fusing different signal peptides (SP) in front of PhoA led to a wide range of secretability (from inhibition by SPAmy1 to about threefold increase by SPDsbA), displaying the need for optimal matching.27 The SPPpiA was proven potent in delaying folding/allowing secretion of the cytoplasmic PpiB, albeit less so than with proPpiA.1 In the case of thioredoxin A, the SPPhoA was less efficient in promoting secretion than SPDsbA or SPMBP.6, 17, 31 Clearly, the question which signal peptide features synergize with the inherent non-folding behaviour of mature domains to allow optimal secretion remains elusive.

We addressed this question by analysing the folding behaviour of various “signal peptide-mature domain combinations’’ and mutant derivatives, using global and local HDX-MS and monitoring their secretability in vivo. We reveal that, increasingly long/hydrophobic H-regions in signal peptides block folding more efficiently. Hydrophobicity is necessary to delay folding and allow subsequent secretion. A more hydrophobic helical core leads to a more extensive structured signal peptide, corelating with folding delays. A shorter distance between the H-region and the mature domain further optimizes folding delay/secretability. Trans addition of signal peptide had no effect on mature domain folding, pointing towards a cis effect as a likely mechanism. The signal peptide’s effect was guided via its C-domain (decreasing disorder of the latter further blocked PpiA’s folding) and is transmitted towards the mature domain through its rheostat (a more rigid rheostat enhances the signal peptide effect). Our findings provide an in-depth structural understanding of how signal peptides can manipulate the folding of mature domains, affecting secretion. Sequence information outside a protein’s folding core may be a universal mechanism for manipulation of folding pathways.