title: Crystal structure of the human carboxypeptidase N (kininase I) catalytic domain.
abstract: Human carboxypeptidase N (CPN), a member of the CPN/E subfamily of "regulatory" metallo-carboxypeptidases, is an extracellular glycoprotein synthesized in the liver and secreted into the blood, where it controls the activity of vasoactive peptide hormones, growth factors and cytokines by specifically removing C-terminal basic residues. Normally, CPN circulates in blood plasma as a hetero-tetramer consisting of two 83 kDa (CPN2) domains each flanked by a 48 to 55 kDa catalytic (CPN1) domain. We have prepared and crystallized the recombinant C-terminally truncated catalytic domain of human CPN1, and have determined and refined its 2.1 A crystal structure. The structural analysis reveals that CPN1 has a pear-like shape, consisting of a 319 residue N-terminal catalytic domain and an abutting, cylindrically shaped 79 residue C-terminal beta-sandwich transthyretin (TT) domain, more resembling CPD-2 than CPM. Like these other CPN/E members, two surface loops surrounding the active-site groove restrict access to the catalytic center, offering an explanation for why some larger protein carboxypeptidase inhibitors do not inhibit CPN. Modeling of the Pro-Phe-Arg C-terminal end of the natural substrate bradykinin into the active site shows that the S1' pocket of CPN1 might better accommodate P1'-Lys than Arg residues, in agreement with CPN's preference for cleaving off C-terminal Lys residues. Three Thr residues at the distal TT edge of CPN1 are O-linked to N-acetyl glucosamine sugars; equivalent sites in the membrane-anchored CPM are occupied by basic residues probably involved in membrane interaction. In tetrameric CPN, each CPN1 subunit might interact with the central leucine-rich repeat tandem of the cognate CPN2 subunit via a unique hydrophobic surface patch wrapping around the catalytic domain-TT interface, exposing the two active centers.
authors.affiliation: Arbeitsgruppe Proteinaseforschung, Max-Planck-Institut für Biochemie, Am Klopferspitz 18, D-82152 Planegg-Martinsried, Germany.
authors.forename: Cora
authors.initials: C
authors.lastname: Keil
authors.forename: Klaus
authors.initials: K
authors.lastname: Maskos
authors.forename: Manuel
authors.initials: M
authors.lastname: Than
authors.forename: J Todd
authors.initials: JT
authors.lastname: Hoopes
authors.forename: Robert
authors.initials: R
authors.lastname: Huber
authors.forename: Fulong
authors.initials: F
authors.lastname: Tan
authors.forename: Peter A
authors.initials: PA
authors.lastname: Deddish
authors.forename: Ervin G
authors.initials: EG
authors.lastname: Erdös
authors.forename: Randal A
authors.initials: RA
authors.lastname: Skidgel
authors.forename: Wolfram
authors.initials: W
authors.lastname: Bode
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D001920:Bradykinin / Q000737:chemistry; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006801:Humans; D001121:Lysine Carboxypeptidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011228:Prealbumin / Q000737:chemistry; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D011228:Prealbumin; D001121:Lysine Carboxypeptidase; D001920:Bradykinin