Flexibility of the PPI2 domain influences the activity of the periplasmic chaperone SurA in vivo

The periplasm of Gram-negative bacteria is comprised of a complex chaperone network, facilitating the transport of unfolded proteins to the outer membrane (OM). One major chaperone is SurA, which interacts with the β-barrel assembly machinery (BAM) complex and prevents aggregation of unfolded outer membrane proteins (OMPs) during their transport across the periplasm. SurA consists of three distinct domains: the NC-core and two peptidyl-prolyl-isomerase (PPI) domains PPI1 and PPI2. Previous studies revealed flexible "closed to open" conformational dynamics of these two PPI domains (1). While the binding of substrates involves mainly interactions within the PPI1/NC-core, the PPI2 domain can be linked to BAM-complex binding in vitro (2). Molecular dynamics simulations suggested, that the PPI2 domain forms salt bridges with the NC-core. Destabilization of these bonds via alanine substitutions results in increased SurA activity in vitro. Knowledge about PPI2 and the function of its interaction with the NC-core function remained scarce in vivo. Hence, we wanted to find out how interference with these interactions might impact the function of SurA in whole cells.

To investigate the impact of weakened or stabilized interactions between PPI2 and the NC-core, we either created alanine substitutions or introduced disulfide bonds within the interaction surface and expressed the resulting SurA variants from the chromosome in an E. coli BW25113 ΔsurA strain. The activity of the SurA variants in vivo was then tested by probing OM permeability and the induction of a σE stress response using a PmicA-GFP reporter system. Thereby we aimed to quantify the cellular stress levels due to the accumulation of unfolded OMPs in the periplasm (3).

Taken together, we found distinct phenotypes regarding OM integrity and σE stress in the periplasm of E. coli expressing SurA variants carrying changes in the PPI2/NC core interaction interface, that were correlated with the degree of flexibility of the PPI2 domain.

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