Cysteine modifiers suggest an allosteric inhibitory site on the CAL PDZ domain

Biosci Rep. 2018 Jul 6;38(4):BSR20180231. doi: 10.1042/BSR20180231. Print 2018 Aug 31.


Protein-protein interactions have become attractive targets for both experimental and therapeutic interventions. The PSD-95/Dlg1/ZO-1 (PDZ) domain is found in a large family of eukaryotic scaffold proteins that plays important roles in intracellular trafficking and localization of many target proteins. Here, we seek inhibitors of the PDZ protein that facilitates post-endocytic degradation of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR): the CFTR-associated ligand (CAL). We develop and validate biochemical screens and identify methyl-3,4-dephostatin (MD) and its analog ethyl-3,4-dephostatin (ED) as CAL PDZ inhibitors. Depending on conditions, MD can bind either covalently or non-covalently. Crystallographic and NMR data confirm that MD attacks a pocket at a site distinct from the canonical peptide-binding groove, and suggests an allosteric connection between target residue Cys319 and the conserved Leu291 in the GLGI motif. MD and ED thus appear to represent the first examples of small-molecule allosteric regulation of PDZ:peptide affinity. Their mechanism of action may exploit the known conformational plasticity of the PDZ domains and suggests that allosteric modulation may represent a strategy for targeting of this family of protein-protein binding modules.

Keywords: NMR spectroscopy; X-ray crystallography; covalent allostery; high-throughput screen; protein-protein interactions.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Allosteric Regulation / drug effects
  • Allosteric Site / drug effects*
  • Carrier Proteins / antagonists & inhibitors
  • Carrier Proteins / chemistry
  • Carrier Proteins / metabolism*
  • Crystallography, X-Ray
  • Cysteine / chemistry
  • Cysteine / metabolism
  • Golgi Matrix Proteins
  • Humans
  • Hydroquinones / chemistry*
  • Hydroquinones / pharmacology*
  • Membrane Proteins / antagonists & inhibitors
  • Membrane Proteins / chemistry
  • Membrane Proteins / metabolism*
  • Membrane Transport Proteins
  • Methylation
  • Molecular Docking Simulation
  • Nuclear Magnetic Resonance, Biomolecular
  • PDZ Domains / drug effects*


  • Adaptor Proteins, Signal Transducing
  • Carrier Proteins
  • GOPC protein, human
  • Golgi Matrix Proteins
  • Hydroquinones
  • Membrane Proteins
  • Membrane Transport Proteins
  • ethyl-3,4-dephostatin
  • Cysteine