01-overview.md

SignalP 6.0 — Overview

What is a signal peptide?

A signal peptide (SP) is a short stretch of amino acids (typically ~15–30 residues) at the N-terminus of a protein that acts as a molecular "address label." It directs the nascent protein to the secretory pathway / translocation machinery in the membrane, and is then cleaved off by a signal peptidase, releasing the mature protein. Getting SP prediction right matters for:

Annotating secretomes and membrane/surface proteins
Designing recombinant proteins for secretion (biotech, antibody/enzyme production)
Finding vaccine and drug targets (secreted/surface antigens)
Metagenomics, where the source organism is unknown

Classic SP structure has three regions, all of which SignalP 6.0 predicts:

| Region | Property | Role | |--------|----------|------| | n-region | Positively charged (basic residues) | Interacts with the translocon / membrane surface | | h-region | Hydrophobic core | Inserts into the membrane | | c-region | Polar, ends in the cleavage site (often A-x-A motif) | Recognized by signal peptidase |

A key caveat printed on the DTU page: "the presence or absence of a signal peptide is not the whole story about localization." SignalP tells you whether a protein enters the secretory pathway, not its final destination — use DeepLoc / DeepLocPro for subcellular localization and NetGPI for GPI anchors.

The five signal-peptide types

The headline feature of v6.0 is that it discriminates all five SP types found across Archaea and Bacteria (Eukarya only have Sec/SPI). Each type is defined by its translocon (how the protein crosses the membrane) and its signal peptidase (which enzyme cuts it).

| Type | Output label | Translocon | Peptidase | Distinguishing feature | |------|--------------|-----------|-----------|------------------------| | Sec/SPI | SP | Sec | SPase I (Lep/LepB) | "Standard" secretory SP; full n/h/c structure | | Sec/SPII | LIPO | Sec | SPase II (Lsp/LspA) | Lipoprotein SP; conserved lipobox with an invariant cysteine at the +1 position | | Tat/SPI | TAT | Tat | SPase I (Lep) | Twin-arginine (S/T-R-R-x-F-L-K) motif in the n-region; transports folded proteins | | Tat/SPII | TATLIPO | Tat | SPase II (Lsp) | Twin-arginine motif and a lipobox (Tat lipoprotein) | | Sec/SPIII| PILIN | Sec | SPase III (PilD/PibD)| Pilin / pilin-like SPs (type IV pili); cleaved within the h-region, no classic c-region |

Sec vs Tat: the Sec translocon exports unfolded chains; the Tat (twin-arginine translocation) pathway exports already-folded proteins and is marked by the conserved twin-arginine motif.
SPase I / II / III: three different signal peptidases produce the three cleavage chemistries. SPase II (lipoproteins) leaves a lipid-modified cysteine; SPase III processes pilins.

SignalP 5.0 could only reliably detect three types (Sec/SPI, Sec/SPII, Tat/SPI). Version 6.0 adds Tat/SPII and Sec/SPIII, which had too few known examples to model before the protein-language-model approach.

Why 6.0 is a step change

All five types in one pass, including the two rare ones.
Organism-agnostic: works on sequences of unknown origin (metagenomic / metatranscriptomic data), because the language model generalizes rather than relying on organism-specific statistics.
Region decoding: returns not just yes/no + cleavage site but the internal n/h/c architecture of the SP.
Better generalization to distant homologs: at <60% identity it clearly outperforms SignalP 5.0.

See 02-architecture.md for how the model achieves this and 04-benchmarks-and-context.md for the numbers.