Dissecting the microscopic steps of the cyclophilin A enzymatic cycle on the biological HIV-1 capsid substrate by NMR

J Mol Biol. 2010 Nov 12;403(5):723-38. doi: 10.1016/j.jmb.2010.08.001. Epub 2010 Aug 12.


Peptidyl-prolyl isomerases (PPIases) are emerging as key regulators of many diverse biological processes. Elucidating the role of PPIase activity in vivo has been challenging because mutagenesis of active-site residues not only reduces the catalytic activity of these enzymes but also dramatically affects substrate binding. Employing the cyclophilin A PPIase together with its biologically relevant and natively folded substrate, the N-terminal domain of the human immunodeficiency virus type 1 capsid (CA(N)) protein, we demonstrate here how to dissect residue-specific contributions to PPIase catalysis versus substrate binding utilizing NMR spectroscopy. Surprisingly, a number of cyclophilin A active-site mutants previously assumed to be strongly diminished in activity toward biological substrates based only on a peptide assay catalyze the human immunodeficiency virus capsid with wild-type activity but with a change in the rate-limiting step of the enzymatic cycle. The results illustrate that a quantitative analysis of catalysis using the biological substrates is critical when interpreting the effects of PPIase mutations in biological assays.

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

  • Capsid Proteins / chemistry
  • Capsid Proteins / genetics
  • Capsid Proteins / metabolism*
  • Catalytic Domain / genetics
  • Cyclophilin A / chemistry*
  • Cyclophilin A / genetics
  • Cyclophilin A / metabolism*
  • HIV-1 / genetics
  • HIV-1 / metabolism*
  • Humans
  • In Vitro Techniques
  • Kinetics
  • Models, Molecular
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Substrate Specificity


  • Capsid Proteins
  • Mutant Proteins
  • Recombinant Proteins
  • Cyclophilin A