Structural basis of the AlgU-MucA cyto interaction and SspB-mediated degradation in Pseudomonas aeruginosa stress response

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Abstract
In Pseudomonas aeruginosa , the sigma factor AlgU functions as a critical stress-responsive regulator governing essential physiological processes. This study assessed algU -dependent phenotypes through targeted gene knockout, including effects on alginate secretion and host cell invasion. Furthermore, an innovative fusion protein strategy addressed complex preparation challenges, enabling the determination of a high-resolution (2.101 Å) structure of the AlgU-MucA cyto complex and revealing an exceptionally low backbone deviation of merely 0.01 Å relative to the reported 6IN7 complex. Structural analysis revealed an extensive mutation-resistant interaction network at the binding interface, stabilized primarily by salt bridges, hydrogen bonds, and hydrophobic interactions. Subsequent mutational validation confirmed that triple or sextuple alanine substitutions at critical interaction sites maintained complex binding integrity while significantly compromising thermal stability, indicating that although dispensable for binding, these residues are indispensable for complex stability. Crucially, pull-down assays identified SspB as the essential adaptor bridging the AlgU-MucA cyto complex to the ClpX/ClpP protease; AlphaFold3 modeling and quantitative BLI measurements reveal that SspB engages AlgU via its N-terminal domain and binds ClpX through its disordered C-terminus, enabling substrate delivery and proteolysis-coupled AlgU release. Collectively, these findings elucidate a dual-safeguard regulatory mechanism: inherent mutation resistance at the binding interface prevents aberrant AlgU activation by common clinical mucA mutations, while SspB-mediated proteolysis ensures stimulus-responsive AlgU liberation. This evolutionary adaptation confers resistance against detrimental AlgU overactivation in P. aeruginosa and provides novel therapeutic targets. IMPORTANCE: Deletion of the algU gene affected the production of pyoverdine, pyocyanin, and alginate in Pseudomonas aeruginosa , as well as its cell invasiveness. By fusing AlgU and MucA cyto , we were able to obtain an AlgU-MucA cyto protein complex with both high yield and purity, and successfully obtained a crystal structure of the AlgU-MucA cyto complex. Structural analysis revealed an extensive and robust network of salt bridges, hydrogen bonds, and charge interactions between the two proteins. Multi-site alanine substitutions (up to sextuple mutations) in interfacial charged clusters of the AlgU-MucA cyto complex fail to disrupt binding but severely impair thermal stability, revealing that these residues are binding-redundant yet stability-essential. Structural modeling and BLI measurements identify SspB as a dual-interface adaptor: its N-terminal domain recognizes the preassembled AlgU-MucA cyto complex (via the AlgU hairpin surface), whereas its flexible C-terminal tail engages ClpX, thereby bridging AlgU-MucA cyto to ClpXP and positioning MucA cyto for proteolysis-coupled AlgU release.
Funding Information
  • National Natural Science Foundation of China (32360046, 82350117, 82260462)
  • Yunnan Provincial Medical Discipline Reserve Talents Support Program (H-2024088)
  • First-Class Discipline Team of Kunming Medical University (2024XKTDYS07, 2024KKTDPY07)
  • Yunnan Provincial Department of Science and Technology Key Research and Development Plan for Social Development Special Projects (202403AC100022)
  • Xingdian Talent Support Program for medical and health personnel of Yunnan Provinces (XDYC-YLWS-2024-0028)
  • Major Science and Technology Special Program of Yunnan Provincial Department of Science and Technology (202302AA310043)
  • National Natural Science Foundation of China (82060426, 82272980, 82360562)
  • The “High-Level Talent and Innovation Team Selection Program” (202405AS350015)
  • Yunnan Provincial Clinical Medicine Center Research Program Fund Project (2024YNLCYXZX0399)

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