Cooperative binding of the cationic porphyrin tris-t4 enhances catalytic activity of 20s proteasome unveiling a complex distribution of functional states

Anna Maria Santoro, Alessandro D’urso, Alessandra Cunsolo, Danilo Milardi, Roberto Purrello, Diego Sbardella, Grazia R. Tundo, Donatella Diana, Roberto Fattorusso, Antonio Di Dato, Antonella Paladino, Marco Persico, Massimo Coletta, Caterina Fattorusso

Research output: Contribution to journalArticlepeer-review

Abstract

The present study provides new evidence that cationic porphyrins may be considered as tunable platforms to interfere with the structural “key code” present on the 20S proteasome α-rings and, by consequence, with its catalytic activity. Here, we describe the functional and conformational effects on the 20S proteasome induced by the cooperative binding of the tri-cationic 5-(phenyl)-10,15,20-(tri N-methyl-4-pyridyl) porphyrin (Tris-T4). Our integrated kinetic, NMR, and in silico analysis allowed us to disclose a complex effect on the 20S catalytic activity depending on substrate/porphyrin concentration. The analysis of the kinetic data shows that Tris-T4 shifts the relative populations of the multiple interconverting 20S proteasome conformations leading to an increase in substrate hydrolysis by an allosteric pathway. Based on our Tris-T4/h20S interaction model, Tris-T4 is able to affect gating dynamics and substrate hydrolysis by binding to an array of negatively charged and hydrophobic residues present on the protein surface involved in the 20S molecular activation by the regulatory proteins (RPs). Accordingly, despite the fact that Tris-T4 also binds to the α3ΔN mutant, allosteric modulation is not observed since the molecular mechanism connecting gate dynamics with substrate hydrolysis is impaired. We envisage that the dynamic view of the 20S conformational equilibria, activated through cooperative Tris-T4 binding, may work as a simplified model for a better understanding of the intricate network of 20S conformational/functional states that may be mobilized by exogenous ligands, paving the way for the development of a new generation of proteasome allosteric modulators.

Original languageEnglish
Article number7190
Pages (from-to)1-26
Number of pages26
JournalInternational Journal of Molecular Sciences
Volume21
Issue number19
DOIs
Publication statusPublished - Oct 1 2020

Keywords

  • 20S proteasome
  • Allosteric modulator
  • Conformational/functional equilibria
  • Integrated interaction model
  • Kinetic analysis
  • Molecular dynamics simulation
  • NMR studies

ASJC Scopus subject areas

  • Catalysis
  • Molecular Biology
  • Spectroscopy
  • Computer Science Applications
  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Inorganic Chemistry

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