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Related Experiment Videos

Mutations in p97/VCP induce unfolding activity.

Andrea Rothballer1, Nikolay Tzvetkov, Peter Zwickl

  • 1Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, 82152 Martinsried, Germany.

FEBS Letters
|March 10, 2007
PubMed
Summary
This summary is machine-generated.

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Introducing aromatic residues into the D1 pore of p97/VCP (valosin-containing protein) enables its protein unfolding activity. Deleting the N domain is also crucial for this activity in vitro.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Protein Science

Background:

  • Two-domain AAA+ ATPases are crucial cellular machines.
  • Bacterial Clp and archaeal VAT proteins possess aromatic residues in their D1 pore, essential for protein unfolding.
  • Eukaryotic p97/VCP proteins lack aromatic residues in the D1 pore, and their in vitro unfolding activity is unproven.

Purpose of the Study:

  • To investigate if introducing aromatic residues into the p97/VCP D1 pore can confer protein unfolding activity.
  • To determine the role of the N domain in p97/VCP's potential unfoldase function.

Main Methods:

  • Site-directed mutagenesis was used to replace aliphatic residues (leucine, alanine) with aromatic tyrosine residues in the D1 pore of full-length p97 and a truncated p97 lacking the N domain (p97DeltaN).

Related Experiment Videos

  • In vitro protein unfolding assays were performed using the model substrate YFP-ssrA to assess the activity of wild-type and mutant p97 proteins.

    • Main Results:

    • Mutant p97DeltaN variants with one or two tyrosine residues in the D1 pore demonstrated in vitro unfolding activity against the model substrate.
    • Full-length p97 with aromatic pore residues and wild-type p97 or p97DeltaN did not exhibit unfolding activity.
    • N-domain deletion and introduction of a single tyrosine residue into the D1 pore were both necessary for p97's in vitro unfoldase activity.

    Conclusions:

    • p97/VCP (valosin-containing protein) possesses latent protein unfolding capabilities.
    • The presence of aromatic residues in the D1 pore and the absence of the N domain are critical for activating p97's in vitro unfoldase activity.
    • This study provides a mechanistic insight into AAA+ ATPase function and potential therapeutic targets.