Crystal structure of human procathepsin X: a cysteine protease with the proregion covalently linked to the active site cysteine.
Human cathepsin X is one of many proteins discovered in recent years through the mining of sequence databases. Its sequence shows clear homology to cysteine proteases from the papain family, containing the characteristic residue patterns, including the active site. However, the proregion of cathepsin X is only 38 residues long, the shortest among papain-like enzymes, and the cathepsin X sequence has an atypical insertion in the regions proximal to the active site. This protein was recently expressed and partially characterized biochemically. Unlike most other cysteine proteases from the papain family, procathepsin X is incapable of autoprocessing in vitro but can be processed under reducing conditions by exogenous cathepsin L. Atypically, the mature enzyme is primarily a carboxypeptidase and has extremely poor endopeptidase activity. We have determined the three-dimensional structure of the procathepsin X at 1.7 A resolution. The overall structure of the mature enzyme is characteristic for enzymes of the papain superfamily, but contains several novel features. Most interestingly, the short proregion binds to the enzyme with the aid of a covalent bond between the cysteine residue in the proregion (Cys10p) and the active site cysteine residue (Cys31). This is the first example of a zymogen in which the inhibition of enzyme's proteolytic activity by the proregion is achieved through a reversible covalent modification of the active site nucleophile. Such mode of binding requires less contact area between the proregion and the enzyme than observed in other procathepsins, and no auxiliary binding site on the enzyme surface is used. A three-residue insertion in a highly conserved region, just prior to the active site cysteine residue, confers a significantly different shape on the S' subsites, compared to other proteases from papain family. The 3D structure provides an explanation for the rather unusual carboxypeptidase activity of this enzyme and confirms the predictions based on homology modeling. Another long insertion in the cathepsin X amino acid sequence forms a beta-hairpin pointing away from the active site. This insertion, thought to be an equivalent of cathepsin B occluding loop, is located on the side of the protein, distant from the substrate binding site.
Sivaraman
J
J
Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montréal, Québec, H4P 2R2, Canada.
Nägler
D K
DK
Zhang
R
R
Ménard
R
R
Cygler
M
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D056657:Cathepsin K; D002403:Cathepsins / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray / Q000379:methods; D003545:Cysteine; D004792:Enzyme Precursors / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D004792:Enzyme Precursors; D011498:Protein Precursors; D011994:Recombinant Proteins; D002403:Cathepsins; C516795:procathepsin X, human; C534130:CTSK protein, human; D056657:Cathepsin K; D003545:Cysteine