Bacterial Signal Peptides- Navigating the Journey of Proteins

Abstract

In 1971, Blobel proposed the first statement of the Signal Hypothesis which suggested that proteins have amino-terminal sequences that dictate their export and localization in the cell. A cytosolic binding factor was predicted, and later the protein conducting channel was discovered that was proposed in 1975 to align with the large ribosomal tunnel. The 1975 Signal Hypothesis also predicted that proteins targeted to different intracellular membranes would possess distinct signals and integral membrane proteins contained uncleaved signal sequences which initiate translocation of the polypeptide chain. This review summarizes the central role that the signal peptides play as address codes for proteins, their decisive role as targeting factors for delivery to the membrane and their function to activate the translocation machinery for export and membrane protein insertion. After shedding light on the navigation of proteins, the importance of removal of signal peptide and their degradation are addressed. Furthermore, the emerging work on signal peptidases as novel targets for antibiotic development is described.

Keywords: SecA; SecYEG translocase; Tat pathway; YidC; antibiotic targets; protein transport; signal peptidase; signal peptide.

FIGURE 1

Bacterial Signal peptides. Schematic representations of the Sec-type signal peptide, the twin-arginine (Tat) signal peptide, the lipoprotein signal peptide, and the prepilin signal peptide. The various regions of the signal peptides (n, h, c and basic regions) are indicated. The SP cleavage site is represented with a red arrow. N and C indicates amino and carboxyl-terminus, respectively.

FIGURE 2

Membrane targeting pathways. Overview of targeting of exported proteins and membrane proteins. After exported proteins are released from the ribosome, Sec-dependent proteins can be stabilized by the molecular chaperone SecB in an unfolded state and then targeted to SecA at the membrane, followed by translocation by the SecYEG complex. Alternatively, SecA can interact with the ribosome bound nascent chain and target the exported protein to the SecYEG complex. In case of Tat complex, the proteins fold in the cytoplasm before being exported by the Tat complex. In the event of co-translational targeting, the nascent membrane proteins form a complex with SRP, which target proteins to FtsY (SRP receptor) for membrane insertion either by the SecYEG complex or the YidC insertase. Created with BioRender.com.

FIGURE 3

The structures/models of the bacterial export and insertion machineries. Export of proteins across the membrane are catalyzed by (A) the Tat complex (resting complex shown) in a folded state (left side) (Habersetzer et al., 2017) or (C) by SecYEG/SecDF/YidC [adapted from Botte et al. (2016) PDB: 5MG3] energized by the SecA motor ATPase (not shown) in an unfolded state. TatA, TatB and TatC is shown in cyan, magenta and green, respectively. SecY, SecE, and SecG is shown in green, red, and magenta; SecD, SecF and YidC are shown in orange, blue and cyan. Membrane protein integration is catalyzed by the SecYEG/SecDF/YidC (C) complex or by the YidC insertase (B) [adapted from Kumazaki et al. (2014b) PDB: 3WVF]. The view is in the plane of the membrane with the periplasmic face at the top and the cytoplasmic face at the bottom.

FIGURE 4

Crystal structure of the SecYEβ complex in the resting state from Methanocaldococcus jannaschii [adapted from van den Berg et al. (2004) PDB: 1RHZ] (A). TM1-5 (red) and TM6-10 (cyan) are the halves of SecY. SecE and Sec61β are in yellow and purple, respectively. (B) The pore ring comprised of six residues (pink) and lateral gate (TM2b in red and TM7 in cyan) are highlighted. (C) The plug helix located above the pore ring is indicated in dark blue. (D) The SecYEβ complex from Thermus thermophiles (PDB: 5AWW). The lateral gate region comprised of TM2b (red) and TM7 (blue) is the site where the signal peptide or TM segments of membrane proteins exit the channel upon opening of the gate. The SecYEβ structures in (A–C) are shown perpendicular to the membrane.

FIGURE 5

The NMR structure of SecA from E. coli [adapted from Gelis et al. (2007) PDB: 2VDA]. (A) The various domains of SecA are highlighted (without the signal peptide). The nucleotide binding domains I (orange) and II (blue), the central helix subdomain of helical scaffold domain (HSD in purple), the preprotein crosslinking domain (PPXD green), the helical wing domain (HWD cyan), and the observed carboxyl-terminal linker domain (CTL). Also highlighted is the 2-helix finger (2HF tan) within the HSD domain. (B) The signal peptide (red) binds roughly perpendicular to 2HF based on NMR studies (Gelis et al., 2007). (C) The signal peptide is modeled parallel to the 2HF of the E. coli SecA NMR structure based on FRET, mutagenesis and genetic studies (Grady et al., 2012).

FIGURE 6

Structures of substrate engaged SecYE or Sec61 complexes. (A) Crystal structure of SecYE-SecA [adapted from Li et al. (2016) PDB: 5EUL] with a portion of the preprotein (comprised of the OmpA signal sequence and a few residues in the mature region) fused into the 2HF (navy blue) by insertion between 741 and 744 of SecA. SecA (in light blue) was from B. subtilis and SecYE was from Geobacillus thermodenitrificans. Nanobody (chartreuse) bound to the periplasmic side of SecY (tan). (B) CryoEM structure of SecYEG-SecA complexed with a proOmpA sfGFP [adapted from Ma et al. (2019) PDB: 6ITC] fusion protein. The structure was performed with SecYE in a lipid nanodisc. An anti-GFP nanobody was inserted at the C-terminus of SecA to recognize and stabilize the fused sfGFP of the substrate. In addition, a disulfide was created between a cysteine at position 8 in the early mature region of the proOmpA GFP fusion protein and a cysteine placed in the plug domain of SecY. Finally, a SecY nanobody that recognizes the periplasmic SecY region was added to stabilize the complex. SecA was from B. subtilis and SecYE was from Geobacillus thermodenitrificans. The nanobody is shown in green in (A,B). (C) CryoEM structure [adapted from Voorhees and Hegde, 2016 PDB: 3JC2] in detergent of the canine ribosome Sec61 channel engaged with the N-terminal 86-amino acid preprolactin region. (D) CryoEM structure [adapted from Gogala et al. (2014) PDB: 4CG6] of the canine Sec61 channel engaged with a hydrophobic TM segment (light green) of a leader peptidase (lep) arrested intermediate. The TM segment was modeled within the opened TM2/TM7 lateral gate. (E) CryoEM structure (adapted from Bischoff et al. (2014) PDB: 5ABB) of a stalled E. coli ribosome SecYE complex engaged with proteorhodopsin (TM indicated in light green). TM2 and TM7 of the lateral gate are shown in magneta and cyan, respectively in (A–E). The signal peptide (red) is indicated in (A–C). The plug helix is indicated in yellow in (D,E).

FIGURE 7

The Tat complex components and a model of TatC-signal peptide complex. (A) The single span TatA (PDB: 2MN7) and TatB (PDB: 2MI2) proteins were determined by NMR (Rodriguez et al., 2013; Zhang et al., 2014). The structure of 6 membrane spanning TatC [adapted from Rollauer et al. (2012) PDB: 4B4A] was solved by X-ray crystallography (Rollauer et al., 2012; Ramasamy et al., 2013). (B) The model of TatC binding with the Tat signal peptide in the groove adapted from Ramasamy et al. (2013). Only signal peptide and early mature region of the preprotein are indicated.

FIGURE 8

The YidC insertase [adapted from Kumazaki et al. (2014b) PDB: 3WVF]. (A) The E. coli YidC has a large periplasmic domain, a coiled cytoplasmic domain, and a conserved core region comprising of 5 TM helices (TMs 2–6) that form a hydrophilic groove open to the cytoplasm and lipid bilayer. The hydrophilic groove has a strictly conserved arginine that helps to keep the region hydrated. (B) A close-up view of the greasy slide (TM3 and TM5) that contacts the TM region of YidC substrates during insertion. The residues that contact the substrate TM segment (s) are indicated in dark blue. (C) During membrane insertion of the Pf3 coat, the TM segment moves up the greasy slide with the N-tail region captured transiently in the hydrophilic groove. The periplasmic domain of YidC is omitted in (B,C).

FIGURE 9

Peptidases involved in the removal of signal peptides and their degradation. (A) Signal peptidase 1 [adapted from Paetzel et al. (2004) PDB: 1T7D] is a novel Ser-Lys protease that cleaves the preprotein at the membrane surface on the periplasmic side. Signal peptidase 2 [adapted from Vogeley et al. (2016) PDB: 5DIR] is an aspartic acid protease that cleaves a diacyl glyceride modified preproteins within the plane of the membrane. (B) SppA [adapted from Kim et al. (2008) PDB: 3BF0] is a tetrameric protein that degrades signal peptides which are released from the membrane into the periplasmic space. SppA employs a Ser-Lys dyad and is anchored to the membrane by an amino terminal TM segment. (C) RseP signal peptide peptidase [from Methanocaldococcus jannaschii adapted from Feng et al. (2007) PDB: 3B4R] in open state. Both water molecules and peptide substrates reach the active site containing Zn²⁺ ion (blue) during its open state.

FIGURE 10

Structures of Signal peptidase inhibitors (Rao et al., 2014). (A) allyl (5S,6S)-6 [(R)-acetoxyethyl]-penem-3-carboxylate. (B) Arylomycin A. (C) Decanoyl PTANA aldehyde. (D) GO775, an optimized arylomycin. (E) Globomycin. (F) Myxovirescin.