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

Abstract

Summary The global regulator, Spx, is under proteolytic control exerted by the adaptor YjbH and ATP-dependent protease ClpXP in Bacillus subtilis. While YjbH is observed to bind the Spx C-terminus, YjbH shows little affinity for ClpXP, indicating adaptor activity that does not operate by tethering. Chimeric proteins derived from B. subtilis AbrB and the Spx C-terminus showed that a 28 residue C-terminal section of Spx (AbrB28), but not the last 12 or 16 residues (AbrB12, AbrB16), was required for YjbH interaction and for ClpXP proteolysis, although the rate of AbrB28 proteolysis was not affected by YjbH addition. The result suggested that the YjbH-targeted 28 residue segment of the Spx C-terminus bears a ClpXP-recognition element(s) that is hidden in the intact Spx protein. Residue substitutions in the conserved helix α6 of the C-terminal region generated Spx substrates that were degraded by ClpXP at accelerated rates compared to wild type Spx, and showed reduced dependency on the YjbH activity. The residue substitutions also weakened the interaction between Spx and YjbH. The results suggest a model in which YjbH, through interaction with residues of α6 helix, exposes the C-terminus of Spx for recognition and proteolysis by ClpXP.

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

“…(A)]. The crosslinking between Spx K117 and YjbH is consistent with the notion that the C‐terminal 12 amino acids in Spx are important for recognition by YjbH, and it is close to a conserved IRRFLP motif in the C‐terminal end, that has been proposed to mediate interactions with YjbH promoting proteolysis of Spx by ClpXP …”

“…Crosslinks were detected between YjbH K177 and Spx K117 which is close to the IRRFL‐motif in the α6 helix in close proximity to the Spx C‐terminus. Our crosslinking data thus lend support, by an independent method, to data from mutational studies and suggest a model in which YjbH, through interaction with residues of the α6 helix, stabilizes the C‐terminus of Spx for recognition and proteolysis by ClpXP.…”

“…This adaptor protein is therefore involved in a number of different protein‐protein interactions and structural changes. The binding to YjbH is proposed to be the cause of the rapid degradation of the transcription factor Spx, through a conformational change and exposure of its C‐terminal region such that is becomes accessible to the ClpXP protease, yet the interactions underlying such a conformational change are not understood due to lack of structural information.…”

Selective adaptor dependent protein degradation in bacteria

“…Binding of the YjbH adaptor is required for Spx degradation by ClpXP in gram-positive bacteria, but YjbH does not bind ClpX well on its own. Here, YjbH binding unveils a C-terminal degron in Spx that is in turn recognized by ClpXP [43]. The RssB adaptor binds RpoS directly, but its weak binding to ClpX suggests that priming of the substrate by the adaptor is needed for protease recognition [38,44].…”

Regulated Proteolysis in Bacteria:Caulobacter

“…For example, the YjbH adaptor enhances ClpXP degradation of Spx, a transcriptional regulator in Bacillus subtilis (40). However, YjbH does not directly interact with ClpX (17), but binds to the C-terminus region of Spx to induce a conformational change which reveals a degron for ClpX recognition (18). Similarly the RssB adaptor promotes degradation of RpoS in E. coli by binding RpoS and promoting ClpX recognition, but RssB alone appears to bind poorly to ClpX (54; 116; 132).…”