Interaction of Kazal-type inhibitor domains with serine proteinases: biochemical and structural studies.
The interaction of domains of the Kazal-type inhibitor protein dipetalin with the serine proteinases thrombin and trypsin is studied. The functional studies of the recombinantly expressed domains (Dip-I+II, Dip-I and Dip-II) allow the dissection of the thrombin inhibitory properties and the identification of Dip-I as a key contributor to thrombin/dipetalin complex stability and its inhibitory potency. Furthermore, Dip-I, but not Dip-II, forms a complex with trypsin resulting in an inhibition of the trypsin activity directed towards protein substrates. The high resolution NMR structure of the Dip-I domain is determined using multi-dimensional heteronuclear NMR spectroscopy. Dip-I exhibits the canonical Kazal-type fold with a central alpha-helix and a short two-stranded antiparallel beta-sheet. Molecular regions essential for inhibitor complex formation with thrombin and trypsin are identified. A comparison with molecular complexes of other Kazal-type thrombin and trypsin inhibitors by molecular modeling shows that the N-terminal segment of Dip-I fulfills the structural prerequisites for inhibitory interactions with either proteinase and explains the capacity of this single Kazal-type domain to interact with different proteinases.
Schlott
Bernhard
B
Institut für Molekulare Biotechnologie e.V., Postfach 100813, D-07708 Jena, Germany.
Wöhnert
Jens
J
Icke
Christian
C
Hartmann
Manfred
M
Ramachandran
Ramadurai
R
Gührs
Karl-Heinz
KH
Glusa
Erika
E
Flemming
Joachim
J
Görlach
Matthias
M
Grosse
Frank
F
Ohlenschläger
Oliver
O
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D066298:In Vitro Techniques; D019476:Insect Proteins / Q000737:chemistry; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics; D013917:Thrombin / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry; D014359:Trypsin Inhibitor, Kazal Pancreatic / Q000737:chemistry / Q000235:genetics
D019476:Insect Proteins; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D015842:Serine Proteinase Inhibitors; C082506:rhodniin protein, Rhodnius; D014359:Trypsin Inhibitor, Kazal Pancreatic; D014357:Trypsin; D013917:Thrombin