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. 2016 Jan 1;3(Pt 1):10-9.
doi: 10.1107/S2052252515019971.

Peptide Binding to a Bacterial Signal Peptidase Visualized by Peptide Tethering and Carrier-Driven Crystallization

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Free PMC article

Peptide Binding to a Bacterial Signal Peptidase Visualized by Peptide Tethering and Carrier-Driven Crystallization

Yi Tian Ting et al. IUCrJ. .
Free PMC article

Abstract

Bacterial type I signal peptidases (SPases) are membrane-anchored serine proteases that process the signal peptides of proteins exported via the Sec and Tat secretion systems. Despite their crucial importance for bacterial virulence and their attractiveness as drug targets, only one such enzyme, LepB from Escherichia coli, has been structurally characterized, and the transient nature of peptide binding has stymied attempts to directly visualize SPase-substrate complexes. Here, the crystal structure of SpsB, the type I signal peptidase from the Gram-positive pathogen Staphylococcus aureus, is reported, and a peptide-tethering strategy that exploits the use of carrier-driven crystallization is described. This enabled the determination of the crystal structures of three SpsB-peptide complexes, both with cleavable substrates and with an inhibitory peptide. SpsB-peptide interactions in these complexes are almost exclusively limited to the canonical signal-peptide motif Ala-X-Ala, for which clear specificity pockets are found. Minimal contacts are made outside this core, with the variable side chains of the peptides accommodated in shallow grooves or exposed faces. These results illustrate how high fidelity is retained despite broad sequence diversity, in a process that is vital for cell survival.

Keywords: Gram-positive pathogen; crystal structures; enzyme mechanisms; peptide complexes; peptide tethering; protein structure; signal peptidase; structural biology.

Figures

Figure 1
Figure 1
Structures of SpsB and LepB. The topology diagrams on the left are colour-coded as for the adjacent ribbon diagrams. The conserved catalytic domains are in green, whereas the noncatalytic domain has a core three-stranded β-sheet (blue) flanked by a highly divergent region (grey). Dashed lines represent regions that are not visible in the electron density. The positions of key catalytic residues are shown in circles or in stick form in the ribbon diagrams. N, N-terminus; C, C-terminus; MBP, maltose-binding protein. The LepB structure is from PDB entry 1b12 (Paetzel et al., 1998 ▸).
Figure 2
Figure 2
Structural comparison between SpsB and LepB. (a) Cartoon showing the conserved extracellular catalytic domains of SpsB (green; Pep3 complex) and LepB (magenta; PDB entry 1b12). The catalytic Ser and Lys residues (in stick form) are shown at the head of the peptide-binding cleft. The peptide Pep3 bound to SpsB is shown in yellow stick form. The divergent noncatalytic domains are shown in grey. Dashed lines represent parts of the protein that are not visible in the electron density. (b) Close-up view of the residues that form the S1 and S3 pockets of the peptide-binding cleft of SpsB and LepB. SpsB residues are labelled in black, while the corresponding LepB residues are shown in red italics.
Figure 3
Figure 3
Structure of an MBP-SpsB–inhibitor peptide complex. Cartoon diagram showing the overall structure of a representative MBP-SpsB–inhibitor peptide complex. MBP is coloured cyan, SpsB green and the peptide blue. The three-amino-acid linker (Ala-Gly-Ala) between MBP and SpsB is shown in red and the engineered thiol group (MBP Q78C) in yellow (marked with an asterisk). Shown in stick form (green), adjacent to the peptide, are the SpsB catalytic residues Ser36 and Lys77. The N- and C-­termini of the fusion protein are designated N and C, respectively. Disordered loops are shown as dashed lines. The stylized black line shows where the cell membrane would be relative to SpsB and the signal peptide in vivo. The inset shows a view of the thioether bond linking the N-­terminus of the Pep3 peptide (blue) to the engineered cysteine residue (Cys78, yellow) on MBP. Residues are encompassed by 2F oF c electron density contoured at 1.0σ, which is orientated to clearly show the continuous electron density between the peptide and Cys78. The peptide position is not constrained by crystal contacts. There is no interaction between the peptide and any adjacent monomers in the crystal lattice, with the nearest adjacent monomer ∼14 Å from the peptide.
Figure 4
Figure 4
Conserved binding mode of the signal-peptide C regions. (a), (b) and (c) show the peptide-binding cleft in surface representation with the peptides Pep1, Pep2 and Pep3 shown in stick mode in their 2F oF c electron density contoured at 1.0σ. Peptide C-region residues are labelled P1–P6. (a) The structure of Pep1 shows an absence of electron density for the mature peptide region (P1′–P3′), which has been cleaved by SpsB. The catalytic Ser is rotated away from the carboxylate group of the cleaved peptide. There is no interpretable electron density for the peptide between P7 and the MBP linkage. (b) As for Pep1, the Pep2 peptide structure shows no electron density for the mature peptide region and rotation of the catalytic Ser away from the carboxylate group of the cleaved peptide. The entire cleaved peptide from P1 to the MBP linkage is well ordered. (c) The Pep3 inhibitor (yellow) bound in the peptide-binding cleft. A proline at position P1′ prevents cleavage. (d) Overlay of Pep1 (cyan), Pep2 (blue) and Pep3 (yellow) signal peptides shows that all peptide and SpsB residues (β-strands 1 and 4) share virtually identical main-chain atom positions, with only the Pep3 P1 residue being significantly displaced. The peptides (P1–P5) are shown in stick form and the SpsB residues as lines. All side chains have been removed, except for the Ala residues at P1 and P3 and Pro at P5.
Figure 5
Figure 5
Specificity of SpsB for the signal-peptide C region. (a) Surface representation contoured to show the S1 and S3 pockets, which contain Ala side chains at P1 and P3 of the peptide. The peptide inhibitor Pep3 (yellow) is shown in its 2F oF c electron density contoured at 1.0σ, with its C-region residues labelled P1–P6 and P1′–P3′ (mature protein portion). The catalytic serine and lysine residues are in cyan adjacent to the cleavage site (marked with an asterisk).
Figure 6
Figure 6
Hydrogen-bond contacts between the signal peptide and SpsB. A stereo figure showing an inhibitory signal peptide (yellow) bound to SpsB. The peptide makes main-chain parallel β-­sheet hydrogen-bond interactions (dashes) with strands that line the peptide-binding cleft, but makes no contact with SpsB before residue P5 or after residue P3′. The side chain of Ser36 is directed at the plane of the P1 scissile bond, with its Oγ atom 2.9 Å from the carbonyl C atom. The rigid P1′ proline pyrrolidine ring Cα atom prevents the P1 carbonyl C atom from moving closer to Ser36. Water is shown as a red sphere.
Figure 7
Figure 7
General model of signal-peptide binding to SpsB. A surface view showing that the enzyme accommodates the diverse side chains of signal peptides in shallow grooves or on exposed faces (subsites S5, S6 and S1′–S3′), with only the alanine side chains of the canonical Ala-X-Ala buried in the S1 and S3 pockets. The core Ala-X-Ala motif both defines specificity and accounts for the majority of interactions between the peptide and the enzyme, while the ‘subsites’ are such that wide peptide diversity can be accommodated.

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