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Measuring In Vitro ATPase Activity for Enzymatic Characterization
07:38

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Published on: August 23, 2016

IcmF family protein TssM exhibits ATPase activity and energizes type VI secretion

Lay-Sun Ma1, Franz Narberhaus, Erh-Min Lai

  • 1Institute of Plant and Microbial Biology and the Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, Academia Sinica, Taipei 11529, Taiwan.

The Journal of Biological Chemistry
|March 7, 2012
PubMed

Insights

The type VI secretion system protein TssM energizes hemolysin-coregulated protein (Hcp) secretion in Agrobacterium tumefaciens. TssM

Area of Science:

  • Microbiology
  • Molecular Biology
  • Protein Biochemistry

Background:

  • The type VI secretion system (T6SS) is crucial for virulence in many Proteobacteria.
  • TssM, an IcmF family protein, is a conserved T6SS inner membrane component.
  • The function and nucleotide-binding activity of TssM in T6SS remain largely unknown.

Purpose of the Study:

  • To biochemically characterize the TssM protein from Agrobacterium tumefaciens.
  • To elucidate the role of TssM's conserved motifs in its function within the T6SS.
  • To determine how TssM facilitates the secretion of the T6SS hallmark protein Hcp.

Main Methods:

  • Biochemical assays to measure ATP binding and hydrolysis.
  • Site-directed mutagenesis of conserved motifs in TssM.
  • Protein-protein interaction studies (e.g., yeast two-hybrid, co-immunoprecipitation).
  • Protease susceptibility assays to monitor conformational changes.

Main Results:

  • TssM exhibits ATPase activity, essential for powering hemolysin-coregulated protein (Hcp) secretion.
  • Mutations in the Walker A and Walker B motifs of TssM impair ATP binding and hydrolysis.
  • TssM undergoes ATP-induced conformational changes, critical for recruiting Hcp and forming a ternary complex with TssL.
  • ATP hydrolysis by TssM is necessary for mediating Hcp secretion.

Conclusions:

  • TssM acts as an energizer for the T6SS, utilizing ATP hydrolysis to drive Hcp secretion.
  • TssM's ATPase activity and conformational changes are central to assembling the secretion machinery.
  • This study provides the first biochemical evidence for TssM's energizer role in T6SS-mediated Hcp secretion.

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