Intermolecular interactions in the periplasmic chaperone SurA can be linked to its activity in vitro

The periplasmic chaperone SurA shuttles outer membrane proteins (OMPs) across the periplasm in many Gram-negative bacteria. Loss or proper SurA function is linked to a reduction of virulence and outer membrane integrity (1, 2). SurA has three distinct domains, the NC-core, and the peptidyl-prolyl-isomerase domains PPI1 and PPI2, which sample various conformations in solution. NC-core and PPI1 predominantly interact with substrates whereas PPI2 and the very N-terminus can be linked to an interaction with the beta-barrel assembly machinery in vitro (4-9).

We established a novel SurA activity assay using a partially unfolded luciferase as a model substrate. This unfolded luciferase is catalytically inactive, upon refolding becomes active and can emit light in presence of ATP and Luciferin. Addition of SurA enhances its refolding. Molecular dynamics simulations in which we provided higher kinetic energy to overcome local minima in the SurA domain dynamics, revealed a to date unknown interaction between PPI2 and NC-core. To validate our simulations, we incubated SurA at elevated temperatures prior to the assay setup.

We observed an altered behavior of SurA after heat treatment. Instead of assisting (chaperoning) the refolding of the Luciferase, SurA inhibited refolding (holdase). Mutational analysis of the PPI2 NC-core interaction surface revealed a connection between interactions between PPI2 and NC-core and holdase activity.

In summary, we developed a novel assay to investigate the holdase activity of SurA and could show that interactions between the SurA NC-core and PPI2 domain regulate this holdase activity.

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