The Activity of Escherichia coli Chaperone SurA Is Regulated by Conformational Changes Involving a Parvulin Domain

Abstract

The periplasmic chaperone SurA is critical for the biogenesis of outer membrane proteins (OMPs) and, thus, the maintenance of membrane integrity in Escherichia coli. The activity of this modular chaperone has been attributed to a core chaperone module, with only minor importance assigned to the two SurA peptidyl-prolyl isomerase (PPIase) domains. In this work, we used synthetic phenotypes and covalent tethering to demonstrate that the activity of SurA is regulated by its PPIase domains and, furthermore, that its activity is correlated with the conformational state of the chaperone. When combined with mutations in the β-barrel assembly machine (BAM), SurA mutations resulting in deletion of the second parvulin domain (P2) inhibit OMP assembly, suggesting that P2 is involved in the regulation of SurA. The first parvulin domain (P1) potentiates this autoinhibition, as mutations that covalently tether the P1 domain to the core chaperone module severely impair OMP assembly. Furthermore, these inhibitory mutations negate the suppression of and biochemically stabilize the protein specified by a well-characterized gain-of-function mutation in P1, demonstrating that SurA cycles between distinct conformational and functional states during the OMP assembly process.

Importance: This work reveals the reversible autoinhibition of the SurA chaperone imposed by a heretofore underappreciated parvulin domain. Many β-barrel-associated outer membrane (OM) virulence factors, including the P-pilus and type I fimbriae, rely on SurA for proper assembly; thus, a mechanistic understanding of SurA function and inhibition may facilitate antibiotic intervention against Gram-negative pathogens, such as uropathogenic Escherichia coli, E. coli O157:H7, Shigella, and Salmonella. In addition, SurA is important for the assembly of critical OM biogenesis factors, such as the lipopolysaccharide (LPS) transport machine, suggesting that specific targeting of SurA may provide a useful means to subvert the OM barrier.

FIG 1

SurA has a modular architecture. The structure of full-length SurA (PDB accession no. 1M5Y) is shown in surface (left) and cartoon (right) forms. Amino acid numbers reference the nonprocessed peptide sequence.

FIG 2

SurA activity is inhibited by the P1 domain. Relative OMP levels were determined in MC4100 and in strains containing each of the surA mutations listed in either a bamA616 (A) or bamB::kan (B) background by SDS-PAGE and immunoblotting for the proteins indicated. (C) Gentle lysis (G. lysis; + lanes) was performed by releasing cellular contents with BugBuster extraction reagent and incubating extracts at 25°C prior to SDS-PAGE and immunoblotting for the periplasmic proteins SurA and maltose binding protein (MBP).

FIG 3

Disulfide bonds properly form between the SurA P1 domain and core module. (A) SurA disulfide tethers were designed using the disulfide database (DSDBASE) (28) and were depicted on the SurA crystal structure (PDB accession no. 1M5Y). The surA10 residue (S220) is represented by red spheres. In each mutant, the residues used for cysteine substitution are represented by black spheres and are boxed. (B) Whole-cell lysates were suspended in loading buffer with (+, left) or without (−, right) the reducing agent β-ME prior to SDS-PAGE and immunoblotting for the proteins indicated. MBP is shown as a control.

FIG 4

Cysteine tethers between the SurA P1 domain and core module lock SurA in a stable conformation. (A) Gentle lysis (G. lysis; + lanes) was performed by releasing cellular contents with BugBuster extraction reagent and incubating extracts at 25°C prior to SDS-PAGE and immunoblotting for the periplasmic proteins SurA and MBP. (B) Extracts from gentle lysis were suspended in loading buffer with (top panel, SurA_Red) or without (bottom panel, SurA^Ox) the reducing agent β-ME prior to SDS-PAGE and immunoblotting for the proteins indicated.

FIG 5

Cysteine tethers between the SurA P1 domain and core module inhibit SurA function. (A) Relative OMP and periplasmic protein levels were determined in MC4100 and in strains containing each of the surA mutations listed in a ΔsurA background by SDS-PAGE and immunoblotting for the proteins indicated. (B) Antibiotic sensitivity was assessed by spotting serial 10-fold dilutions of stationary-phase cultures of ΔsurA strains containing the mutations indicated on LB with or without vancomycin and incubated at 37°C.

FIG 6

Disulfide bonds between the SurA P1 domain and core module negate surA10 suppression. Relative OMP levels were determined in MC4100 and in strains containing each of the surA mutations listed in either a ΔsurA; bamA616 (A) or ΔsurA; bamB::kan (B) background by SDS-PAGE and immunoblotting for the proteins indicated.

FIG 7

Model for the structural and functional equilibrium of SurA. SurA exists in equilibrium between two (less active and more active) states that are correlated with its structural stability (stable and unstable, respectively). The surA10 mutation biases SurA toward a more structurally disorganized, but more active, conformation through the loosening, or opening, of the P1-core domain interface. The surAΔP2 deletion (indicated by the dashed arrow and dashed P2 domain border) and P1-core disulfide tethers (labeled surA P1-core), however, bias SurA toward a more structured and stable but less active state, where the P1 and core domains are locked in a closed conformation. This equilibrium is regulated by the BAM complex, as the surA10 and surAΔP2 phenotypes are only clear when the BAM complex function is disrupted, such as in a bamA616 or bamB-null mutant.