NMR structural studies of human cystatin C dimers and monomers

Research output: Contribution to journalArticle

Abstract

Human cystatin C undergoes dimerization before unfolding. Dimerization leads to a complete loss of its activity as a cysteine proteinase inhibitor. A similar process of dimerization has been observed in cells, and may be related to the amyloid formation seen for the L68Q variant of the protein. Dimerization is barrier controlled, and no dimer/monomer interconversion can be observed at physiological conditions. As a consequence, very stable, “trapped” dimers can be easily separated from monomers. A study of the structural aspects of cystatin C dimer formation was undertaken using NMR spectroscopy. The monomer/dimer model was verified by (pulse field gradient NMR) self-diffusion molecular mass measurements. Complete backbone resonance assignments and secondary structure determination were obtained for the monomer using data from triple resonance experiments performed on 13C/15N doubly labeled protein. A marked similarity of the cystatin C secondary structure to that of chicken cystatin was observed. Using uniformly and amino-acid-specific 15N-enriched protein, backbone NH signals were assigned for cystatin C in its dimeric state. Comparison of 1H-15N correlation NMR spectra of the monomer and dimer shows that the three-dimensional structure remains unchanged in the dimer and that only local perturbations occur. These are localized to the amino acid residues comprising the cysteine proteinase binding site. Such a mode of dimerization readily explains the complete loss of the inhibitory activity in the dimer. The NMR results also demonstrate that the dimer is symmetric.

Details

Authors
  • I Ekiel
  • Magnus Abrahamson
  • DB Fulton
  • P Lindahl
  • AC Storer
  • W Levadoux
  • M Lafrance
  • S Labelle
  • Y Pomerleau
  • D Groleau
  • L LeSauteur
  • K Gehring
Organisations
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Pharmacology and Toxicology
  • Medicinal Chemistry

Keywords

  • cystatin C, protein self-association, NMR spectroscopy, amyloidosis, protein folding
Original languageEnglish
Pages (from-to)266-277
JournalJournal of Molecular Biology
Volume271
Issue number2
Publication statusPublished - 1997
Publication categoryResearch
Peer-reviewedYes