An NMR-based identification of peptide fragments mimicking the interactions of the cathepsin B propeptide.
Selected fragments of the 62-residue proregion (or residues 1p-62p) of the cysteine protease cathepsin B were synthesized and their interactions with cathepsin B studied by use of proton NMR spectroscopy. Peptide fragments 16p-51p and 26p-51p exhibited differential perturbations of their proton resonances in the presence of cathepsin B. These resonance perturbations were lost for the further truncated 36p-51p fragment, but remained in the 26p-43p and 28p-43p peptide fragments. Residues 23p-26p or TWQ25A in the N-terminal 1p-29p fragment did not show cathepsin B-induced resonance perturbations although the same residues had strongly perturbed proton resonances within the 16p-51p peptide. Both the 1p-29p and 36p-51p fragments lack a common set of hydrophobic residues 30p-35p or F30YNVDI35 from the proregion. The presence of residues F30YNVDI35 appears to confer a conformational preference in peptide fragments 16p-51p, 26p-51p, 28p-43p and 26p-43p, but the same residues induce the aggregation of peptides 16p-36p and 1p-36p. The peptide fragment 26p-43p binds to the active site, as indicated by its inhibition of the catalytic activity of cathepsin B. The cathepsin B prosegment can therefore be reduced into smaller, but functional subunits 28p-43p or 26p-43p that retain specific binding interactions with cathepsin B. These results also suggest that residues F30YNVDI35 may constitute an essential element for the selective inhibition of cathepsin B by the full-length cathepsin B proregion.
Yu
Y
Y
Protein Engineering Network of Centres of Excellence, Montreal, Que., Canada.
Vranken
W
W
Goudreau
N
N
de Miguel
E
E
Magny
M C
MC
Mort
J S
JS
Dupras
R
R
Storer
A C
AC
Ni
F
F
D002401:Cathepsin B / Q000737:chemistry*; D003546:Cysteine Endopeptidases / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D018716:Molecular Mimicry; D010446:Peptide Fragments / Q000737:chemistry*; D010843:Pichia / Q000737:chemistry
D005656:Fungal Proteins; D010446:Peptide Fragments; D003546:Cysteine Endopeptidases; D002401:Cathepsin B