The active-site residue Cys-29 is responsible for the neutral-pH inactivation and the refolding barrier of human cathepsin B.
Human cathepsin B, the most abundant lysosomal cysteine protease, has been implicated in a variety of important physiological and pathological processes. It has been known for a long time that like other lysosomal cysteine proteases, cathepsin B becomes inactivated and undergoes irreversible denaturation at neutral or alkaline pH. However, the mechanism of this denaturation process remains mostly unknown up to this day. In the present work, nuclear magnetic resonance spectroscopy was used to characterize the molecular origin of the neutral-pH inactivation and the refolding barrier of human cathepsin B. Two forms of human cathepsin B, the native form with Cys-29 at the active site and a mutant with Cys-29 replaced by Ala, were shown to have well-folded structures at the active and slightly acidic condition of pH 5. Surprisingly, while the native cathepsin B irreversibly unfolds at pH 7.5, the C29A mutant was found to maintain a stable three-dimensional structure at neutral pH conditions. In addition, replacement of Cys-29 by Ala renders the process of the urea denaturation of human cathepsin B completely reversible, in contrast to the opposite behavior of the wild-type cathepsin B. These results are very surprising in that replacement of one single residue, the active-site Cys-29, can eliminate the neutral-pH denaturation and the refolding barrier. We speculate that this finding may have important implications in understanding the process of pH-triggered inactivation commonly observed for most lysosomal cysteine proteases.
Song
J
J
Biomolecular NMR Laboratory and the Montr¿eal Joint Centre for Structural Biology, Biotechnology Research Institute, National Research Council of Canada, Qu¿ebec, H4P 2R2, Montr¿eal, Canada.
Xu
P
P
Xiang
H
H
Su
Z
Z
Storer
A C
AC
Ni
F
F
D002401:Cathepsin B / Q000737:chemistry*; D003545:Cysteine; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D017510:Protein Folding
D002401:Cathepsin B; D003545:Cysteine