Exploring structure and interactions of the bacterial adaptor protein YjbH by crosslinking mass spectrometry

Abstract

Adaptor proteins assist proteases in degrading specific proteins under appropriate conditions. The adaptor protein YjbH promotes the degradation of an important global transcriptional regulator Spx, which controls the expression of hundreds of genes and operons in response to thiol-specific oxidative stress in Bacillus subtilis. Under normal growth conditions, the transcription factor is bound to the adaptor protein and therefore degraded by the AAA+ protease ClpXP. If this binding is alleviated during stress, the transcription factor accumulates and turns on genes encoding stress-alleviating proteins. The adaptor protein YjbH is thus a key player involved in these interactions but its structure is unknown. To gain insight into its structure and interactions we have used chemical crosslinking mass spectrometry. Distance constraints obtained from the crosslinked monomer were used to select and validate a structure model of YjbH and then to probe its interactions with other proteins. The core structure of YjbH is reminiscent of DsbA family proteins. One lysine residue in YjbH (K177), located in one of the α-helices outside the thioredoxin fold, crosslinked to both Spx K99 and Spx K117, thereby suggesting one side of the YjbH for the interaction with Spx. Another lysine residue that crosslinked to Spx was YjbH K5, located in the long and presumably very flexible N-terminal arm of YjbH. Our crosslinking data lend support to a model proposed based on site-directed mutagenesis where the YjbH interaction with Spx can stabilize and present the C-terminal region of Spx for protease recognition and proteolysis. Proteins 2016; 84:1234-1245. © 2016 Wiley Periodicals, Inc.

Identification of Novel Spx Regulatory Pathways in Bacillus subtilis Uncovers a Close Relationship between the CtsR and Spx Regulons

“…Inspection of the Spx structure reveals that the first two (R14 and R91) are near the disulfide switch, and the latter two (R100 and R112) are near the putative sites of binding of the YjbH adaptor protein for ClpXP proteolysis (Fig. 3G) (28,29). The Spx paralog MgsR was phosphorylated on R17 and R95 (24), which align with Spx R14 and R92 in the protein structures.…”

Structural Basis for YjbH Adaptor-Mediated Recognition of Transcription Factor Spx

“…Cloning, Expression and Purification Construction of the vector for expression of G. kaustophilus YjbH, pNIC28_YjbHGk, has been described previously (Al-Eryani et al, 2016). To produce G. kaustophilus YjbH, cells of E. coli BL21 StarÔ(DE3) carrying the pNIC28_yjbHGK plasmid were grown in 3 l of Difco Terrific Broth (3 3 1 l in 5 l baffled E-flasks) containing kanamycin at 50 mg ml À1 .…”

“…In a previous study, the structure of YjbH was predicted computationally and CXMS was used to evaluate the model (Al-Eryani et al, 2016). The model has significant overall structural similarity (Ca RMSD = 2.0 Å for 197 matching residues) to the experimentally determined structure of YjbH.…”

“…To gain insight into the structure and interactions of YjbH with Spx and compare it with the interaction between Spx and aCTD, we have previously applied homology modeling combined with chemical crosslinking and mass spectrometry (CXMS) (Al-Eryani et al, 2016). The analysis suggested that YjbH has a structure related to the DsbA family of thiol-disulfide oxidoreductases.…”

Harnessing the potential of bacterial oxidative folding to aid protein production

“…For example, Synechococcus spp . encode a DsbA‐like protein fused to its DsbB‐like oxidative partner, VKOR (Li et al., 2010), the Porphyromonas gingivalis protein Hbp35 has a DsbA‐like domain fused to a hemin‐binding domain (Shiroza et al., 2008), and the DsbA‐like domain of the Bacillus subtilis YjbH is N‐terminal to a transcription factor‐binding protein (Al‐Eryani et al., 2016; Awad et al., 2019). These dual enzymes can perform both DsbA‐mediated oxidative folding and the function of their additional domains.…”