Rudenko_1995_Structure_3_1249

BACKGROUND The human 'protective protein' (HPP) forms a multi-enzyme complex with beta-galactosidase and neuraminidase in the lysosomes, protecting these two glycosidases from degradation. In humans, deficiency of HPP leads to the lysosomal storage disease galactosialidosis. Proteolytic cleavage of the precursor form of HPP involves removal of a 2 kDa excision peptide and results in a carboxypeptidase activity. The physiological relevance of this activity is, as yet, unknown. RESULTS: The crystal structure of the 108 kDa dimer of the precursor HPP has been elucidated by making extensive use of twofold density averaging. The monomer consists of a 'core' domain and a 'cap' domain. Comparison with the distantly related wheat serine carboxypeptidase dimer shows that the two subunits in the HPP dimer differ by 15 degrees in mutual orientation. Also, the helical subdomain forming part of the cap domains is very different. In addition, the HPP precursor cap domain contains a 'maturation' subdomain of 49 residues which fills the active-site cleft. Merely removing the 'excision' peptide located in the maturation subdomain does not render the catalytic triad solvent accessible.
CONCLUSIONS: The activation mechanism of HPP is unique among proteases with known structure. It differs from the serine proteases in that the active site is performed in the zymogen, but is blocked by a maturation subdomain. In contrast to the zinc metalloproteases and aspartic proteases, the chain segment physically rendering the catalytic triad solvent inaccessible in HPP is not cleaved off to form the active enzyme. The activation must be a multi-step process involving removal of the excision peptide and major conformational changes of the maturation subdomain, whereas the conformation of the enzymatic machinery is probably almost, or completely, unaffected.