Structure of the complex of the antistasin-type inhibitor bdellastasin with trypsin and modelling of the bdellastasin-microplasmin system.
The serine proteinase plasmin is, together with tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), involved in the dissolution of blood clots in a fibrin-dependent manner. Moreover, plasmin plays a key role in a variety of other activation cascades such as the activation of metalloproteinases, and has also been implicated in wound healing, pathogen invasion, cancer invasion and metastasis. The leech-derived (Hirudo medicinalis) antistasin-type inhibitor bdellastasin represents a specific inhibitor of trypsin and plasmin and thus offers a unique opportunity to evaluate the concept of plasmin inhibition. The complexes formed between bdellastasin and bovine as well as porcine beta-trypsin have been crystallised in a monoclinic and a tetragonal crystal form, containing six molecules and one molecule per asymmetric unit, respectively. Both structures have been solved and refined to 3.3 A and 2.8 A resolution. Bdellastasin turns out to have an antistasin-like fold exhibiting a bis-domainal structure like the tissue kallikrein inhibitor hirustasin. The interaction between bdellastasin and trypsin is restricted to the C-terminal subdomain of bdellastasin, particularly to its primary binding loop, comprising residues Asp30-Glu38. The reactive site of bdellastasin differs from other antistasin-type inhibitors of trypsin-like proteinases, exhibiting a lysine residue instead of an arginine residue at P1. A model of the bdellastasin-microplasmin complex has been created based on the X-ray structures. Our modelling studies indicate that both trypsin and microplasmin recognise bdellastasin by interactions which are characteristic for canonically binding proteinase inhibitors. On the basis of our three-dimensional structures, and in comparison with the tissue-kallikrein-bound and free hirustasin and the antistasin structures, we postulate that the binding of the inhibitors toward trypsin and plasmin is accompanied by a switch of the primary binding loop segment P5-P3. Moreover, in the factor Xa inhibitor antistasin, the core of the molecule would prevent an equivalent rotation of the P3 residue, making exosite interactions of antistasin with factor Xa imperative. Furthermore, Arg32 of antistasin would clash with Arg175 of plasmin, thus impairing a favourable antistasin-plasmin interaction and explaining its specificity.
Rester
U
U
Abteilung Strukturforschung, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, Martinsried, 82152, Germany. rester@biochem.mpg.de
Bode
W
W
Moser
M
M
Parry
M A
MA
Huber
R
R
Auerswald
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D018360:Crystallography, X-Ray; D065427:Factor Xa Inhibitors; D005341:Fibrinolysin / Q000737:chemistry*; D006860:Hydrogen Bonding; D019476:Insect Proteins; D007447:Invertebrate Hormones / Q000737:chemistry*; D007865:Leeches / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D011485:Protein Binding; D016415:Sequence Alignment; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics; D013552:Swine; D014357:Trypsin / Q000737:chemistry*
D065427:Factor Xa Inhibitors; D019476:Insect Proteins; D007447:Invertebrate Hormones; D010446:Peptide Fragments; D015842:Serine Proteinase Inhibitors; C400221:bdellastasin; C053155:antistasin; C054561:microplasmin; D014357:Trypsin; D005341:Fibrinolysin