A new class of hybrid secretion system is employed in Pseudomonas amyloid biogenesis

Gram-negative bacteria possess specialised biogenesis machineries that facilitate the export of amyloid subunits for construction of a biofilm matrix. The secretion of bacterial functional amyloid requires a bespoke outer-membrane protein channel through which unfolded amyloid substrates are translocated. Here, we combine X-ray crystallography, native mass spectrometry, single-channel electrical recording, molecular simulations and circular dichroism measurements to provide high-resolution structural insight into the functional amyloid transporter from Pseudomonas, FapF. FapF forms a trimer of gated β-barrel channels in which opening is regulated by a helical plug connected to an extended coil-coiled platform spanning the bacterial periplasm. Although FapF represents a unique type of secretion system, it shares mechanistic features with a diverse range of peptide translocation systems. Our findings highlight alternative strategies for handling and export of amyloid protein sequences.


Supplementary
Surface antibody detection immunoblot. Intact bacteria were resuspended in PBS buffer to an OD600 of 1.5 and 2 µL dotted onto a nitrocellulose membrane. The same sample was diluted to an OD600 of 1.5 with SDS and boiled for 5 minutes to fully lyse the cells. The intact cells do not display surface binding of antihis HRP indicating that no histag is exposed on the extracellular surface. This is consistent with a periplasmic N-terminal domain for FapF.

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Supplementary Figure 8. Stability of parallel trimeric coiled coil built using CCBuilder V1.0. The peptide D3 to Q40 was used. Starting residue D3 was consensus 'e' register as found by multiple coiled coil prediction software. The core remained stable during short 40 ns of atomistic molecular dynamics simulation (right). The RMSD of all mainchain atoms of each peptide chain is shown (left). Each chain is shown separately coloured according to the figure on the right. The C-terminal region was least stable, which would be attached to the linker region in the full length protein.
Supplementary Figure 9. Method for generating a model of full length FapF. The crystal structure presented in this work was combined with the idealised parallel trimeric coiled coil shown in Supplementary Figure 7. Modeller (www.salilab.org) was used to generate a linker with random coil secondary structure for residues 40 to 87. This was then converted into standard MARTINI v2.2 coarse grained representation for coarse grained molecular dynamics simulations in a POPE/POPG lipid membrane.
Supplementary Figure 10. Conservation of detergent binding sites amongst monomeric subunits.
Here the detergent molcules are placed mapped onto each chain A (blue), B (yellow) and C (green). The only consistent binding site between all three chains is the detergent binding site above the helix plug (lower panel). All other observed binding sites were only resolved for 1 or 2 subunits.  Supplementary Figure 14. Steered molecular dynamics (SMD) simulations in which the helix plug is pulled from the pore. An imaginary harmonic spring is applied to the centre of mass of each of the three helices for the wildtype (WT, blue) and the F102A mutant (red). A harmonic force is applied to the spring, inceasing proportional to the distance between the plug and the starting point. The plot indicates the force vs time for WT (solid line and blue data points) and F102A mutant (dashed line, red data points) applied to each helix. The snapshots 2, 3, and 4 correspond to the time each plug is removed for the F126A mutant and highlight the position of the F102 remaining within the barrel in the WT. F126 remains as an anchoring point in the WT and more force is required to remove the helix fully from the pore than for F102A. The final snapshot (5) demonstrates that F102 is still within the pore whilst the rest of the helix is unfolded for the WT. In the case of the F102A mutant the whole plug is removed from the pore. Taken together this data shows the F102 causes a barrier to plug removal and forms the main anchoring point of the helix plug within the barrel. The lines shown are a moving average of the data points over 0.1 ns intervals.