3-DIMENSIONAL STRUCTURE OF THE HUMAN PROTECTIVE PROTEIN - STRUCTURE OF THE PRECURSOR FORM SUGGESTS A COMPLEX ACTIVATION MECHANISM

The human 'protective protein' (HPP) forms a multi-enzyme complex with P-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 cleava ge of the precursor form of HPP involves removal of a 2 kDa excision p eptide and results in a carboxypeptidase activity. The physiological r elevance of this activity is, as yet, unknown. Results: The crystal st ructure of the 108 kDa dimer of the precursor HPP has been elucidated by making extensive use of twofold density averaging. The monomer cons ists of a 'core' domain and a 'cap' domain. Comparison with the distan tly related wheat serine carboxypeptidase dimer shows that the two sub units in the HPP dimer differ by 15 degrees in mutual orientation. Als o, the helical subdomain forming part of the cap domains is very diffe rent. In addition, the HPP precursor cap domain contains a 'maturation ' subdomain of 49 residues which fills the active-site cleft. Merely r emoving the 'excision' peptide located in the maturation subdomain doe s not render the catalytic triad solvent accessible. Conclusions: The activation mechanism of HPP is unique among proteases with known struc ture. It differs from the serine proteases in that the active site is preformed in the zymogen, but is blocked by a maturation subdomain. In contrast to the zinc metalloproteases and aspartic proteases, the cha in segment physically rendering the catalytic triad solvent inaccessib le in HPP is not cleaved off to form the active enzyme. The activation must be a multi-step process involving removal of the excision peptid e and major conformational changes of the maturation subdomain, wherea s the conformation of the enzymatic machinery is probably almost, or c ompletely, unaffected.