Crystal Structure and Autoactivation Pathway of the Precursor Form of Human Tripeptidyl-peptidase 1, the Enzyme Deficient in Late Infantile Ceroid Lipofuscinosis

Abstract

Late infantile neuronal ceroid lipofuscinosis is a fatal childhood neurological disorder caused by a deficiency in the lysosomal protease tripeptidyl-peptidase 1 (TPP1). TPP1 represents the only known mammalian member of the S53 family of serine proteases, a group characterized by a subtilisin-like fold, a SerGlu-Asp catalytic triad, and an acidic pH optimum. TPP1 is synthesized as an inactive proenzyme (pro-TPP1) that is proteolytically processed into the active enzyme after exposure to low pH in vitro or targeting to the lysosome in vivo. In this study, we describe an endoglycosidase H-deglycosylated form of TPP1 containing four Asn-linked N-acetylglucosamines that is indistinguishable from fully glycosylated TPP1 in terms of autocatalytic processing of the proform and enzymatic properties of the mature protease. The crystal structure of deglycosylated pro-TPP1 was determined at 1.85 Å resolution. A large 151-residue C-shaped prodomain makes extensive contacts as it wraps around the surface of the catalytic domain with the two domains connected by a 24-residue flexible linker that passes through the substrate-binding groove. The proenzyme structure reveals suboptimal catalytic triad geometry with its propiece linker partially blocking the substrate-binding site, which together serve to prevent premature activation of the protease. Finally, we have identified numerous processing intermediates and propose a structural model that explains the pathway for TPP1 activation in vitro. These data provide new insights into TPP1 function and represent a valuable resource for constructing improved TPP1 variants for treatment of late infantile neuronal ceroid lipofuscinosis.

Functional consequences and rescue potential of pathogenic missense mutations in tripeptidyl peptidase I

“…All three of these residues also provide H-bonds to the adjacent part of the prosegment; thus, those contacts would be affected as well. Cys365 participates in an intramolecular disulfide bond formation with Cys526 [Guhaniyogi et al, 2009;Pal et al, 2009]. Therefore, the assisted folding process of this variant might be prolonged because of the inability of the ER machinery to form a native disulfide bond.…”

“…Most of the mutated aa residues are buried in the structure, however; Arg127, Asn286, Cys365, and Ser475 have substantial surface exposure. The solvent accessibilities of these four residues calculated by PoPMuSic server (http:// babylone.ulb.ac.be/popmusic/index.php) using PDB entry 3EDY) [Guhaniyogi et al, 2009], are 47.6, 25.9, 35.5, and 18.4%, respectively. Theoretical changes in the thermodynamic stability of the mutated proenzymes due to specific substitutions were calculated using ERIS software [Yin et al, 2007] and PDB entry 3EDY with fixed backbone and no prerelaxation settings.…”

“…The effects of mutations on the tertiary structure of TPPI were analyzed on a visual level with the Swiss-PdbViewer [Guex and Peitsch, 1997] using protein structure files PDB 3EDY [Guhaniyogi et al, 2009] and PDB 3EE6 [Pal et al, 2009].…”

“…Mutagenic analyses and inhibition studies identified residues Ser475, Glu272, Asp276, Asp360, and Asp327 as being closely involved in catalytic activity and substrate binding, with Ser475 acting as an active-site nucleophile Oyama et al, 2005;Walus et al, 2005]. Recently, two independent teams of researchers published crystal structures of TPPI proenzyme, which helped refine the mechanisms of activation and tripeptidase activity of the enzyme [Guhaniyogi et al, 2009;Pal et al, 2009].…”

Identification of Interaction Site of Propeptide toward Mature Carboxypeptidase Y (mCPY) Based on the Similarity between Propeptide and CPY Inhibitor (IC)

“…The propeptides are usually removed either by autoprocessing following a drop in pH or by exogenous proteases. 3,4) It has also been reported that propeptides function as intramolecular chaperones and that they play crucial roles in protein folding that guide mature domains to functional conformations. 5,6) They are not part of the mature domain, as they are removed after the completion of protein folding, but they play important roles in the folding processes of functional proteins, because proteins cannot take proper conformations without propeptides.…”