title: NMR structure determination of tick anticoagulant peptide (TAP).
abstract: Tick anticoagulant peptide (TAP) is a potent and selective 60-amino acid inhibitor of the serine protease Factor Xa (fXa), the penultimate enzyme in the blood coagulation cascade. The structural features of TAP responsible for its remarkable specificity for fXa are unknown, but the binding to its target appears to be unique. The elucidation of the TAP structure may facilitate our understanding of this new mode of serine protease inhibition and could provide a basis for the design of novel fXa inhibitors. Analyses of homo- and heteronuclear two-dimensional NMR spectra (total correlation spectroscopy, nuclear Overhauser effect spectroscopy [NOESY], constant time heteronuclear single quantum correlation spectroscopy [CT-HSQC], and HSQC-NOESY; 600 MHz; 1.5 mM TAP; pH 2.5) of unlabeled, 13C-labeled, and 15N-labeled TAP provided nearly complete 1H sequence-specific resonance assignments. Secondary structural elements were identified by characteristic NOE patterns and D2O amide proton-exchange experiments. A three-dimensional structure of TAP was generated from 412 NOESY-derived distance and 47 dihedral angle constraints. The structural elements of TAP are similar in some respects to those of the Kunitz serine protease inhibitor family, with which TAP shares weak sequence homology. This structure, coupled with previous kinetic and biochemical information, confirms previous suggestions that TAP has a unique mode of binding to fXa.
authors.affiliation: Corvas International, Inc., San Diego, California 92121, USA.
authors.forename: M S
authors.initials: MS
authors.lastname: Lim-Wilby
authors.forename: K
authors.initials: K
authors.lastname: Hallenga
authors.forename: M
authors.initials: M
authors.lastname: de Maeyer
authors.forename: I
authors.initials: I
authors.lastname: Lasters
authors.forename: G P
authors.initials: GP
authors.lastname: Vlasuk
authors.forename: T K
authors.initials: TK
authors.lastname: Brunck
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D007611:Aprotinin / Q000378:metabolism; D060829:Arthropod Proteins; D003903:Deuterium; D015951:Factor Xa / Q000378:metabolism; D065427:Factor Xa Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013987:Ticks / Q000737:chemistry*
chemical_list: D060829:Arthropod Proteins; D065427:Factor Xa Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D015842:Serine Proteinase Inhibitors; C063967:tick anticoagulant peptide; D007611:Aprotinin; D003903:Deuterium; D015951:Factor Xa