Geobacillus thermodenitrificans YjbH recognizes the C-terminal end of Bacillus subtilis Spx to accelerate Spx proteolysis by ClpXP

Abstract

Proteolytic control can govern the levels of specific regulatory factors, such as Spx, a transcriptional regulator of the oxidative stress response in Gram-positive bacteria. Under oxidative stress, Spx concentration is elevated and upregulates transcription of genes that function in the stress response. When stress is alleviated, proteolysis of Spx catalysed by ClpXP reduces Spx concentration. Proteolysis is enhanced by the substrate recognition factor YjbH, which possesses a His-Cys-rich region at its N terminus. However, mutations that generate H12A, C13A, H14A, H16A and C31/34A residue substitutions in the N terminus of Bacillus subtilis YjbH (BsYjbH) do not affect functionality in Spx proteolytic control in vivo and in vitro. Because of difficulties in obtaining soluble BsYjbH, the Geobacillus thermodenitrificans yjbH gene was cloned, which yielded soluble GtYjbH protein. Despite its lack of a His-Cys-rich region, GtYjbH complements a B. subtilis yjbH null mutant, and shows high activity in vitro when combined with ClpXP and Spx in an approximately 30 : 1 (ClpXP/Spx : GtYjbH) molar ratio. In vitro interaction experiments showed that Spx and the protease-resistant Spx DD (in which the last two residues of Spx are replaced with two Asp residues) bind to GtYjbH, but deletion of 12 residues from the Spx C terminus (SpxDC) significantly diminished interaction and proteolytic degradation, indicating that the C terminus of Spx is important for YjbH recognition. These experiments also showed that Spx, but not GtYjbH, interacts with ClpX. Kinetic measurements for Spx proteolysis by ClpXP in the presence and absence of GtYjbH suggest that YjbH overcomes non-productive Spx-ClpX interaction, resulting in rapid degradation.

Adaptor bypass mutations of Bacillus subtilis spx suggest a mechanism for YjbH‐enhanced proteolysis of the regulator Spx by ClpXP

“…Recently, the C-terminal tail of Spx was shown to be important for binding to the proteolytic adaptor, YjbH, and for ClpXP protease recognition. More specifically, a deletion of 12 residues from the Spx C-terminus (SpxΔC) significantly decreased the affinity of Spx for YjbH interaction (Chan et al , 2012), and Spx resisted ClpXP proteolysis when its last two amino acids were replaced with aspartic acids (Spx DD ) (Nakano et al , 2003a). To investigate which structural features of the Spx C-terminal tail might function in proteolytic control, an alignment of Spx sequences from 21 organisms was inspected, uncovering a conserved motif (IRRFLP) at their C-termini (Fig.…”

“…All of these controls ensure the Spx is upregulated when needed but is maintained in low levels during unperturbed growth. Deletion of the yjbH gene results in elevated Spx concentrations (Larsson et al , 2007), and YjbH protein accelerates Spx proteolysis in a reaction containing purified ClpXP (Chan et al , 2012, Garg et al , 2009). Unlike the adaptor, SspB, YjbH in affinity pull-down reactions, shows no detectable affinity for the protease ClpXP (Chan et al , 2012).…”

“…The last 13 amino acids of Spx (119–131) are missing from the crystal structure probably due to structural disorder. Previous studies showed that the C-terminus of Spx is important for adaptor YjbH binding and ClpXP proteolysis (Chan et al , 2012, Nakano et al , 2003a). Changing the last two amino acids to aspartic acid in Spx (Spx DD ) resulted in resistance to proteolysis by ClpXP (Nakano et al , 2003a).…”

“…Changing the last two amino acids to aspartic acid in Spx (Spx DD ) resulted in resistance to proteolysis by ClpXP (Nakano et al , 2003a). Also, deletion of the last 12 amino acids from Spx significantly decreased its interaction with YjbH (Chan et al , 2012) and resulted in Spx protein stability in vivo (Lin and Zuber, 2012). At present, the mechanism of YjbH-mediated, ClpXP-catalyzed Spx proteolysis is not known, however.…”

Role of Adaptor TrfA and ClpPC in Controlling Levels of SsrA-Tagged Proteins and Antitoxins in Staphylococcus aureus

“…These proteins lack the AAAϩ ATPase activity found in chaperones and instead facilitate motif identification either through direct interactions with a target (24), through selective motif occlusion (25), or by modification of a protein to alter its degradation rate (26). On the basis of homology to adaptors identified in Bacillus subtilis, there are at least three adaptors in S. aureus, namely, McsB (SA0482), YjbH (SA0860), and TrfA (SA0857).…”