Reduced global cooperativity is a common feature underlying the amyloidogenicity of pathogenic lysozyme mutations

J Mol Biol. 2005 Feb 25;346(3):773-88. doi: 10.1016/j.jmb.2004.11.020. Epub 2004 Nov 24.

Abstract

One of the 20 or so human amyloid diseases is associated with the deposition in vital organs of full-length mutational variants of the antibacterial protein lysozyme. Here, we report experimental data that permit a detailed comparison to be made of the behaviour of two of these amyloidogenic variants, I56T and D67H, under identical conditions. Hydrogen/deuterium exchange experiments monitored by NMR and mass spectrometry reveal that, despite their different locations and the different effects of the two mutations on the structure of the native state of lysozyme, both mutations cause a cooperative destabilisation of a remarkably similar segment of the structure, comprising in both cases the beta-domain and the adjacent C-helix. As a result, both variant proteins populate transiently a closely similar, partially unstructured intermediate in which the beta-domain and the adjacent C-helix are substantially and simultaneously unfolded, whereas the three remaining alpha-helices that form the core of the alpha-domain still have their native-like structure. We show, in addition, that the binding of a camel antibody fragment, cAb-HuL6, which was raised against wild-type lysozyme, restores to both variant proteins the stability and cooperativity characteristic of the wild-type protein; as a consequence, it inhibits the formation of amyloid fibrils by both variants. These results indicate that the reduction in global cooperativity, and the associated ability to populate transiently a specific, partly unfolded intermediate state under physiologically relevant conditions, is a common feature underlying the behaviour of these two pathogenic mutations. The formation of intermolecular interactions between lysozyme molecules that are in this partially unfolded state is therefore likely to be the fundamental trigger of the aggregation process that ultimately leads to the formation and deposition in tissue of amyloid fibrils.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Amyloidosis, Familial / enzymology
  • Amyloidosis, Familial / genetics
  • Animals
  • Antibodies
  • Camelus
  • Enzyme Stability
  • Humans
  • In Vitro Techniques
  • Kinetics
  • Microscopy, Electron
  • Models, Molecular
  • Muramidase / chemistry*
  • Muramidase / genetics*
  • Muramidase / immunology
  • Muramidase / metabolism
  • Mutation*
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Denaturation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Recombinant Proteins / metabolism
  • Spectrometry, Mass, Electrospray Ionization

Substances

  • Antibodies
  • Recombinant Proteins
  • Muramidase