The suppressor of copper sensitivity protein C from Caulobacter crescentus is a trimeric disulfide isomerase that binds copper(I) with subpicomolar affinity

Abstract

The introduction of disulfide bonds into periplasmic proteins is a critical process in many Gram-negative bacteria. The formation and regulation of protein disulfide bonds have been linked to the production of virulence factors. Understanding the different pathways involved in this process is important in the development of strategies to disarm pathogenic bacteria. The well characterized disulfide bond-forming (DSB) proteins play a key role by introducing or isomerizing disulfide bonds between cysteines in substrate proteins. Curiously, the suppressor of copper sensitivity C proteins (ScsCs), which are part of the bacterial copper-resistance response, share structural and functional similarities with DSB oxidase and isomerase proteins, including the presence of a catalytic thioredoxin domain. However, the oxidoreductase activity of ScsC varies with its oligomerization state, which depends on a poorly conserved N-terminal domain. Here, the structure and function of Caulobacter crescentus ScsC (CcScsC) have been characterized. It is shown that CcScsC binds copper in the copper(I) form with subpicomolar affinity and that its isomerase activity is comparable to that of Escherichia coli DsbC, the prototypical dimeric bacterial isomerase. It is also reported that CcScsC functionally complements trimeric Proteus mirabilis ScsC (PmScsC) in vivo, enabling the swarming of P. mirabilis in the presence of copper. Using mass photometry and small-angle X-ray scattering (SAXS) the protein is demonstrated to be trimeric in solution, like PmScsC, and not dimeric like EcDsbC. The crystal structure of CcScsC was also determined at a resolution of 2.6 Å, confirming the trimeric state and indicating that the trimerization results from interactions between the N-terminal α-helical domains of three CcScsC protomers. The SAXS data analysis suggested that the protomers are dynamic, like those of PmScsC, and are able to sample different conformations in solution.

Bacterial suppressor-of-copper-sensitivity (Scs) proteins exhibit diverse thiol-disulfide oxidoreductase cellular functions

“…The gene cluster encodes four proteins, ScsA, ScsB, ScsC, and ScsD. A putative copper-binding site and a thioredoxin-like motif containing the catalytic Cys-X-X-Cys site characteristic of Dsb proteins are present in most Scs proteins characterised to date (Petit et al, 2022, Gupta et al, 1997, Shepherd et al, 2013, Furlong et al, 2017, Cho et al, 2012. Limited information on the biological function of ScsA and ScsD suggested a role in S. Typhimurium resistance to H 2 O 2 and copper (López et al, 2018).…”

“…I n vitro evidence suggests ScsC scavenges toxic copper in the cells and delivers it to the copper-binding metallochaperone CueP, thereby reducing cellular toxicity in S . Typhimurium (Shepherd et al ., 2013, Subedi et al ., 2021, Petit et al ., 2022).…”

“…On the other hand, crystal structures of the ScsC homologues from P. mirabilis (PmScsC) and C. crescentus (CcScsC) revealed that their N-terminal regions oligomerize to form strikingly similar trimeric arrangements (Fig 1A-B) (Furlong et al ., 2017, Petit et al ., 2022). Nonetheless, the catalytic domains in each ScsC protomer resembles that of monomeric E. coli DsbA (Martin et al ., 1993) (Fig 1B-C).…”

“…Trimerization and catalytic domains are displayed in blue and slate, respectively. B, Protomer structures of PmScsc (blue/slate; PDB 5id4 (Furlong et al ., 2017)), CcScsC (green; PDB 7rgv (Petit et al ., 2022), this protein is a biological trimer), StScsC (pink; PDB 4gxz (Shepherd et al ., 2013), Monomeric) And EcDsba (Yellow; PDB 1Fvk (Guddat et al ., 1998), Monomeric). THE α- helix/loop differences in the globular domains are indicated with an arrow.…”

“…The three structurally characterised ScsC proteins, StScsC, CcScsC, and PmScsC, adopt distinct oligomeric states and exhibit distinct oxidoreductase activities in vitro , despite their catalytic domains sharing a striking structural resemblance with monomeric EcDsbA (Fig. 1) (Furlong et al ., 2017, Petit et al ., 2022, Shepherd et al ., 2013, Cho et al ., 2012, Subedi et al ., 2021). Furthermore, similar to other Dsb proteins, upon the loss of the N-terminal trimeric module, monomeric PmScsC protein (PmScsCΔN) was reported to lose disulfide reductase activity and gain dithiol oxidase activity in vitro (Furlong et al ., 2017).…”