Mapping inhibitor binding modes on an active cysteine protease via nuclear magnetic resonance spectroscopy

Biochemistry. 2012 Dec 18;51(50):10087-98. doi: 10.1021/bi301305k. Epub 2012 Dec 10.


Cruzain is a member of the papain/cathepsin L family of cysteine proteases, and the major cysteine protease of the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. We report an autoinduction methodology that provides soluble cruzain in high yields (>30 mg/L in minimal medium). These increased yields provide sufficient quantities of active enzyme for use in nuclear magnetic resonance (NMR)-based ligand mapping. Using circular dichroism and NMR spectroscopy, we also examined the solution-state structural dynamics of the enzyme in complex with a covalently bound vinyl sulfone inhibitor (K777). We report the backbone amide and side chain carbon chemical shift assignments of cruzain in complex with K777. These resonance assignments were used to identify and map residues located in the substrate binding pocket, including the catalytic Cys25 and His162. Selective [(15)N]Cys, [(15)N]His, and [(13)C]Met labeling was performed to quickly assess cruzain-ligand interactions for a set of eight low-molecular weight compounds exhibiting micromolar binding or inhibition. Chemical shift perturbation mapping verified that six of the eight compounds bind to cruzain at the active site. Three different binding modes were delineated for the compounds, namely, covalent, noncovalent, and noninteracting. These results provide examples of how NMR spectroscopy can be used to screen compounds for fast evaluation of enzyme-inhibitor interactions to facilitate lead compound identification and subsequent structural studies.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Catalytic Domain
  • Cysteine Endopeptidases / chemistry*
  • Cysteine Endopeptidases / isolation & purification
  • Cysteine Endopeptidases / metabolism
  • Cysteine Proteinase Inhibitors / pharmacology
  • Dipeptides / pharmacology
  • Nuclear Magnetic Resonance, Biomolecular
  • Phenylalanine / analogs & derivatives
  • Piperazines
  • Protozoan Proteins / antagonists & inhibitors
  • Protozoan Proteins / chemistry*
  • Protozoan Proteins / isolation & purification
  • Protozoan Proteins / metabolism
  • Tosyl Compounds
  • Trypanosoma cruzi / enzymology
  • Vinyl Compounds / pharmacology


  • Cysteine Proteinase Inhibitors
  • Dipeptides
  • Piperazines
  • Protozoan Proteins
  • Tosyl Compounds
  • Vinyl Compounds
  • N-pip-phenylalanine-homophenylalanine-vinyl sulfone phenyl
  • Phenylalanine
  • Cysteine Endopeptidases
  • cruzain, Trypanosoma cruzi