Signal peptidase I: cleaving the way to mature proteins

Abstract

Signal peptidase I (SPase I) is critical for the release of translocated preproteins from the membrane as they are transported from a cytoplasmic site of synthesis to extracytoplasmic locations. These proteins are synthesized with an amino-terminal extension, the signal sequence, which directs the preprotein to the Sec- or Tat-translocation pathway. Recent evidence indicates that the SPase I cleaves preproteins as they emerge from either pathway, though the steps involved are unclear. Now that the structure of many translocation pathway components has been elucidated, it is critical to determine how these components work in concert to support protein translocation and cleavage. Molecular modeling and NMR studies have provided insight on how the preprotein docks on SPase I in preparation for cleavage. This is a key area for future work since SPase I enzymes in a variety of species have now been identified and the inhibition of these enzymes by antibiotics is being pursued. The eubacterial SPase I is essential for cell viability and belongs to a unique group of serine endoproteases which utilize a Ser-Lys catalytic dyad instead of the prototypical Ser-His-Asp triad used by eukaryotes. As such, SPase I is a desirable antimicrobial target. Advances in our understanding of how the preprotein interfaces with SPase I during the final stages of translocation will facilitate future development of inhibitors that display a high efficacy against SPase I function.

Figure 1

The features and alignment of bacterial signal peptides. The tripartite structure of Sec-dependent non-lipoprotein signal peptides is depicted in (A), where the N-terminus is characterized by the presence of positively-charged residues (blue), the core of the peptide is comprised of hydrophobic residues (orange), and the C-terminus is typically neutral, but polar and contains the cleavage site (green). The red arrow indicates the SPase cleavage site and the amino acid motif common to the cleavage site is given. The mature region (purple) of the preprotein follows the cleavage site. (B) Sec-dependent signal peptide sequence alignment. Signal peptide sequences compiled from the SPdb database. The -1,-3 residues (red) display sequence conservation of small aliphatic residues among a number of Sec- and Tat-dependent signal peptides. If present, the conserved proline is in bold. The site of cleavage is carboxy-terminal to the -1 residue. (C) The Tat-dependent non-lipoprotein signal peptide depicted in the same manner as panel (A). (D) Tat-dependent signal peptide sequence alignment. Depicted as in panel (B) and the residues highlighted in blue are the required arginine residues that give the Tat pathway its name.

Figure 2

Bacterial protein translocation pathways requiring SPase I. (A) The Sec-dependent general secretory pathway for secretory preproteins via post-translational translocation. Preproteins are bound in the cytosol by SecB or SecA. If bound by SecB, the SecB-preprotein complex then binds to SecA and transfers the preprotein. SecA carrying the preprotein binds the SecYEG translocon channel and using the energy from ATP hydrolysis may propel the preprotein through the channel. Once sufficient preprotein has been translocated to ensure no back-slippage, the SPase I cleaves off the signal peptide, allowing the mature protein to release from the membrane and undergo folding. (B) The Tat-pathway is a post-translational translocation pathway used for secretion of fully folded preproteins. The TatBC complex recognizes and binds the signal sequence of a Tat-dependent preprotein. This causes the recruitment of TatA and the formation of an appropriately sized TatA translocon. The fully-folded preprotein is then secreted into the periplasm, while the signal sequence remains in the membrane. The mature protein is released into the periplasm once the SPase I cleaves the signal peptide.

Figure 3

E. coli SPase I Δ2-75 apoenzyme crystal structure (PDB ID: 1KN9). (A) A solid surface representation of the SPase I Δ2-75 apoenzyme structure with the modeled signal peptide binding subsites labeled., Subsites are colored as follows: green S4′, lime green S4′ and S3′ overlap, blue S3′, magenta S1, red S1 and S2 overlap, orange S1, S2, and S3 overlap, purple S2 and S3 overlap, and yellow S3. (B) A ribbon representation of SPase I Δ2-75. The colored portion of the protein represents the conserved domain I, while the gray region is the nonconserved domain II as well as the Gram-negative β-ribbon insertion. Box domains B-D are color coordinated. Box B is shown in red, box C is shown in purple, box D is colored orange, and box E is in blue. The residues that are important for catalysis (S88, S90, K145, and S278) are labeled, colored by box domain, and displayed in ball and stick representation. The placement of G272 and K145 are shown to emphasize the importance of glycine at residue 272. Any other amino acid in that location would result in steric hindrance with K145. D280, and R282 form a salt bridge that is important for structural stability of the enzyme. The N- and C-termini are labeled and the structure is lacking residues 107-124, 136, 176-177, 200-202, and 305-312, which were not resolved in the 3D structure. (C) Superimposition of the SPase I Δ2-75 apoenzyme crystal structure with inhibitor-bound crystal structures to highlight differences in the position of the W300 and W310 side-chains in the various structures. The structures are colored as follows: cyan is the apoenzyme 1KN9 molecule A, green is the apoenzyme 1KN9 molecule D, magenta is the lipopetide-bound structure 1T7D molecule B, orange is the β-lactam-bound structure 1B12 molecule A, and blue is the arylomycin A₂ and β-sultam bound structure 3IIQ molecule A. (D) As in (C) except displaying positioning differences of the side chains for residues S88 and K145 within the active site.

Figure 4

Alignment of the conserved box domains for signal peptidase I from different species. The molecular weight (M_w) is given for each protein in g/mol. The % sequence identity is relative to the entire E. coli SPase I protein sequence. Box domains B–E are located in the catalytic domain. The serine nucleophile and lysine/histidine general base residues that are involved in signal peptide cleavage are indicated with an asterisk. Box A comprises the transmembrane segments and is not shown.

Figure 5

Proposed mechanism for SPase I cleavage of preproteins using a Ser-Lys catalytic dyad. The preprotein substrate (where P₁ is the amino acid at the -1 position of the signal peptide and P₁' is the amino acid in the +1 position of the mature protein) binds to the enzyme active site. The amino group of Lys 145 acts as the general base and deprotonates the hydroxyl group of Ser 90 (Michaelis complex). The Ser 90 Oγ atom now acts as the nucleophile, and attacks the substrate P₁ residue carbonyl group to form a tetrahedral intermediate I. This shift of electrons results in the formation of an oxyanion hole, involving the main chain amide group of Ser 90 and the side chain hydroxyl group of Ser 88,, which results in the stabilization of the substrate tetrahedral intermediate. Lys 145 donates a proton to the amino group of the N-terminus of the mature protein, allowing its release from the enzyme, and generates a signal peptide acyl-enzyme intermediate. The deacylating water molecule now comes into play, with the loss of one of its protons to the amino group of Lys 145, and the attack of its oxygen atom on the peptide carbonyl group, forming another tetrahedral intermediate. Again, Ser 88 and Ser 90 serve to stabilize the intermediate via hydrogen bonding. Finally, the amide group of Lys 145 donates a proton to the Oγ atom of Ser 90, leading to the breakdown of the tetrahedral intermediate and release of the signal peptide.