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Conformational Insights into the Control of CNF1 Toxin Activity by Peptidyl-Prolyl Isomerization: A Molecular

Eléa Paillares1,2, Maud Marechal1, Léa Swistak1,2

  • 1Unité des Toxines Bactériennes, UMR CNRS 2001, Institut Pasteur, 75015 Paris, France.

International Journal of Molecular Sciences
|September 28, 2021
PubMed

Insights

Cytotoxic necrotizing factor 1 (CNF1) toxin activates Rho GTPases by deamidating a glutamine residue. Proline mutations in CNF1 disrupt its catalytic activity by altering protein structure and dynamics.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • Cytotoxic necrotizing factor 1 (CNF1) is a toxin from uropathogenic *Escherichia coli* that activates Rho GTPases.
  • CNF1 catalyzes the deamidation of a critical glutamine residue in the switch II (SWII) region of Rho GTPases.
  • Crystallographic structures reveal unusual *cis* conformations for peptidyl-prolyl imide bonds (X-Pro) at positions P768 and P968 in the CNF1 catalytic domain (CNF1CD).

Purpose of the Study:

  • To investigate the role of P768 and P968 proline residues in CNF1CD activity and mechanism.
  • To elucidate how peptidyl-prolyl *cis-trans* isomerization influences the catalytic cleft and substrate interaction.
  • To understand the long-distance effects of isomerization on the CNF1 active site and its implications for target modification.

Main Methods:

  • Site-directed mutagenesis of proline residues (P768G, P968G) in CNF1CD.
  • In vitro deamidase activity assays.
  • Cellular functional assays using RhoA.
  • Molecular dynamics simulations.
  • Protein-peptide docking simulations.

Main Results:

  • Mutation of P768 or P968 to glycine abrogated CNF1CD in vitro deamidase activity.
  • Mutant CNF1 forms retained functionality on RhoA in cellular assays.
  • Molecular dynamics and docking revealed that P768 isomerization enlarges the catalytic cleft, facilitating SWII peptide binding.
  • P768 *cis-trans* isomerization promotes an active orientation of the catalytic cysteine C866.

Conclusions:

  • Peptidyl-prolyl *cis-trans* isomerizations of P768 and P968 are crucial for CNF1 catalytic activity.
  • Isomerization of P768 significantly impacts the CNF1 active site structure, dynamics, and substrate interaction.
  • These findings highlight the importance of long-distance conformational changes in enzyme catalysis and have implications for understanding CNF1's mechanism of action.
Keywords:
CNF1Escherichia coliX-Pro imide bonddeamidasemolecular dynamicspeptide dockingpeptidyl prolyl cis-trans isomerization

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