Crystal structure of a subtilisin-like autotransporter passenger domain reveals insights into its cytotoxic function

Abstract

Autotransporters (ATs) are a large family of bacterial secreted and outer membrane proteins that encompass a wide range of enzymatic activities frequently associated with pathogenic phenotypes. We present the structural and functional characterisation of a subtilase autotransporter, Ssp, from the opportunistic pathogen Serratia marcescens. Although the structures of subtilases have been well documented, this subtilisin-like protein is associated with a 248 residue β-helix and itself includes three finger-like protrusions around its active site involved in substrate interactions. We further reveal that the activity of the subtilase AT is required for entry into epithelial cells as well as causing cellular toxicity. The Ssp structure not only provides details about the subtilase ATs, but also reveals a common framework and function to more distantly related ATs. As such these findings also represent a significant step forward toward understanding the molecular mechanisms underlying the functional divergence in the large AT superfamily.

Fig. 1. Structure of Ssp.

a Linear schematic of Ssp’s primary sequence encompassing an N-terminal signal peptide (SP) and a C-terminal translocator domain flanking the central passenger. Active site residues in the subtilase domain are shown by red line, self-cleavage sites shown by green arrows. b Crystal structure showing overall architecture of Ssp passenger in cartoon representation. Subtilase (protease) domain is depicted in blue with active site residues shown as red spheres. The β-helical stalk domain is represented in yellow, with protruding β-helix Loop 1 and Loop 2, coloured in rust and green, respectively. The passenger-associated-transport-repeat (PATR) is displayed in pink and the RGD motif shown as pink sticks, the α-helical loop in orange and the β-hairpin cap at the base of the passenger are coloured in dark blue. Calcium ions are shown as pink spheres with those bound to the protease domain labelled using Dohnalek et al. nomenclature. c Protease domain of Ssp in cartoon representation showing the unique active site protrusions (E1–E3). Namely, short β-hairpin extension (E1, pink) long β-hairpin extension (E3, green), and extended loop extension with connected α-helix (E2, orange). Active site is displayed as red sticks. d Ssp protease domain with central β-sheet and α-helices shown in hot pink and jade, respectively. Disulfide bond is shown as yellow sticks.

Fig. 2. Comparison of the protease domain of Ssp and subtilisin BPN’.

a Overlay of the crystal structure of the subtilase domain of Ssp (blue) and subtilisin BPN’ (grey, PDB: 1LW6. Active site residues are shown in red sticks. Subtilisin BPN’ residues 98–109 which are missing in Ssp and make the substrate binding cleft wider are shown in purple. b Close up of active site residues with Ssp in blue and subtilisin BPN’ in grey. Ssp catalytic triad is labelled. c 2Fo−Fc electron density map contoured at 1σ encompassing the Ssp subtilase active site. d Top-down view of the active site of subtilisin BPN’ in complex with inhibitor CI2 (P5-P2’ residues displayed for clarity). Subtilisin BPN’ residues 98–109 are shown in purple. e Electrostatic surface of subtilisin BPN’ complexed with CI2 with protease. Subsites are labelled in the inset. f Top-down view of the active site of Ssp. Ssp’s unique active site extensions are highlighted, namely short β-hairpin extension (E1, pink) long β-hairpin extension (E3, green), and extended loop extension with connected α-helix (E2, orange). Active site is shown in red. g Electrostatic surface of Ssp protease domain. Putative protease subsites indicated in the inset. The electrostatic surface potentials were calculated with the APBS plugin in Pymol with electrostatic potential coloured from negative (red) to positive (blue) with a range of ±5 kT/e.

Fig. 3. Expression and protease activity of Ssp variants.

a Crystal structure of Ssp in cartoon representation displaying location of mutations. b SDS-PAGE analysis of culture supernatant from expression of Ssp variants in E. coli Top10. Data is representative of three independent experiments. c Protease activity of Ssp variants using a fluorescent casein substrate. Error bars represent standard deviation. Ssp variants include wildtype (WT), deletions of Loop 2 (ΔL2), deletion of active site protrusions E2 and E3 (ΔE2, ΔE3, ΔE2/ΔE3), and site-directed mutants of the active site Ser (S314A) and RGD motif (RAE). pBAD denotes vector only control. Mean is plotted with error bars representing the standard deviation of technical replicates (n = 3). Data is representative of three independent experiments.

Fig. 4. Effect of Ssp on HEp-2 cells.

a HEp-2 cells were incubated with concentrated culture supernatants of Ssp variants (25 μg/mL) for 30 min and imaged by DIC microscopy. Scale bar represents 100 μm. Ssp variants include wildtype (WT), deletion of active site protrusions E2 and E3 (ΔE2, ΔE3, ΔE2/ΔE3), and site-directed mutant of the active site (S314A). pBAD denotes vector only control. Cell rounding was observed WT ∼75%, pBAD ∼5%, S341A ∼5%, ΔE2 ∼20%, ΔE3 ∼50%, ΔE2/ΔE3 ∼10%. b Cytotoxicity of Ssp was measured by incubation with HEp-2 cells and the release of LDH measured. Mean is plotted with error bars representing the standard deviation of technical replicates (n = 3). Data is representative of three independent experiments.

Fig. 5. Internalisation of Ssp variants by HEp-2 cells.

Confocal microscopy images of HEp-2 cells incubated with concentrated culture supernatants of Ssp variants (25 μg/mL) for 5 h. Ssp was visualised with anti-Ssp polyclonal antibody followed by Alexa Fluor Plus 647 conjugated secondary antibody (yellow), actin cytoskeleton was stained with phalloidin (magenta) and nucleus stained with DAPI (cyan). Ssp variants include wildtype (WT), deletions of Loop 2 (ΔL2), deletion of active site protrusions E2 and E3 (ΔE2, ΔE3, ΔE2/ΔE3), and site-directed mutants of the active site Ser (S314A) and RGD motif (RAE). pBAD denotes vector only control. Images are representative of cells observed from at least three independent experiments. Scale bar represents 50 μm.