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Mechanistic insights into anti-adaptor mediated regulation of proteolysis

Thursday, September 15, 2016 — Poster Session III

3:30 p.m. – 5:00 p.m.
FAES Terrace
NCI
MOLBIO-6

Authors

  • A Tripathi
  • A Battesti
  • A Kravats
  • J Hoskins
  • S Wickner
  • GX Shaw
  • J Gan
  • P Subburaman
  • YN Zhou
  • X Ji
  • S Gottesman

Abstract

Regulated proteolysis of proteins in all organisms plays a very crucial role during growth, development and stress. RpoS is an RNA polymerase sigma factor, required for activation of stress specific genes in bacteria. The energy-dependent ClpXP proteolysis machinery, in collaboration with RssB, an adaptor, tightly regulates the amount of RpoS. Various stress-specific anti-adaptor proteins regulate the RssB-mediated proteolysis of RpoS. Here we report structural and functional characterization of IraP, an anti-adapter induced in response to phosphate starvation. We determined crystal structure of IraP at 2.35 Å resolution. The structure shows IraP is a dimer in which N-terminal halves form a parallel helix-helix pair. No electron density was observed beyond Gln42, indicating that C-terminal halves are disordered. By structure-guided mutational studies, we identify two groups of residues in IraP. One region is important for direct interaction with RssB, whereas other is required for IraP dimerization. Mutations in the first region, lead to loss of IraP interaction with RssB and decreased ability of IraP to stabilize RpoS. Loss of dimerization had modest effects on IraP activity, suggesting that dimerization is not essential for IraP activity. Mutations of residues in the C-terminal half affected IraP dimerization, indicating that the flexible C-terminal half helps in maintaining the overall structure of IraP. Our data is consistent with a model in which IraP stabilizes RpoS by direct binding to RssB. An understanding of anti-adaptors provides the possibility of designing molecules that disrupt their function, and thus interfere with the ability of pathogens to adapt to stress.

Category: Molecular Biology and Biochemistry