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Originaltitel:
Kristallographische und biophysikalische Untersuchungen an Serinproteinase-Effektor-Komplexen 
Übersetzter Titel:
Crystallographic and biophysical studies on serine proteinase-effector complexes 
Jahr:
2002 
Dokumenttyp:
Dissertation 
Institution:
Fakultät für Chemie 
Betreuer:
Huber, Robert (Prof. Dr. Dr. h.c.) 
Gutachter:
Buchner, Johannes (Prof. Dr.); Bode, Wolfram (Prof. Dr.) 
Format:
Text 
Sprache:
de 
Fachgebiet:
CHE Chemie 
Stichworte:
Proteinkristallographie; Serinproteinase; Thrombin; Inhibitor; Aktivator 
Übersetzte Stichworte:
Protein crystallography; serine proteinase; thrombin; inhibitor; activator 
Schlagworte (SWD):
Serinproteinasen; Enzyminhibitor; Röntgenstrukturanalyse; Enzymaktivator 
TU-Systematik:
CHE 829d; CHE 828d 
Kurzfassung:
Na-Methyl-Arginin als P1-Rest stellt ein kombiniertes „Prolin- und Arginin-Analogon“ dar und ermöglicht damit stabile Peptidmimetika mit hoher Affinität. Die Bindungsweise eines Thrombininhibitors mit dieser Aminosäure wurde im Rahmen dieser Arbeit untersucht. I-11 wurde mit humanem a-Thrombin kokristallisiert und die Kristallstruktur bis zu einer Auflösung von 1,8 Å bestimmt. Die vorliegende Struktur erklärt die stark reduzierte Reaktivität der Na-Methyl-Arginin–Xaa-Bindung, die auf der „Abstan...    »
 
Übersetzte Kurzfassung:
Bivalent peptidic thrombin inhibitors consisting of an N-terminal d-Cyclohexylalanine–Pro–Na(Me)Arg active-site fragment, a flexible polyglycine linker, and a C-terminal hirugen-like segment directed towards the fibrinogen recognition exosite inhibit thrombin with Ki-values in the picomolar range, remaining stable in buffered solution at pH 7.8 for at least 15 hours. In order to investigate the structural basis accounting for this increased stability, the most potent of these inhibitors, I-11 (Ki=37pM), containing an Na(Me)Arg–Thr bond, was crystallized in complex with human  -thrombin. X-ray data were collected to 1.8 Å resolution and the crystal structure of this complex was determined. The Fourier map displays clear electron density for the N-terminal fragment and for the exosite binding segment. It indicates, however, that in agreement with Edman sequencing the peptide had been cleaved in the crystal, presumably due to the long incubation time of 14 days needed for crystallization and data collection. The N (Me) group is directed toward the carbonyl oxygen of Ser214, pushing the Ser195 Og atom out of its normal site. This structure suggests that upon thrombin binding the scissile peptide bond of the intact peptide and the Ser195 Og are separated from each other, impairing the nucleophilic attack of the Ser195 Og toward the Na(Me)Arg carbonyl group. In the time scale of two weeks, however, cleavage geometries favoured by the crystal allow catalysis at a slow rate. A protein secreted by Staphylococcus aureus, staphylocoagulase, activates mammalian prothrombin without proteolytic cleavage and initiates direct conversion of fibrinogen to fibrin. Here we present crystal structures of human thrombin and prethrombin-2 bound to a fully active fragment of the bacterial cofactor, along with a detailed analysis of prothrombin activation and fibrinogen clotting by the staphylocoagulase-prothrombin complex. Staphylocoagulase consists of two domains of helical bundles, a previously uncharacterized fold distinct from known serine proteinase activators. The N-terminus of the cofactor inserts into the Ile 16-pocket of bound prethrombin-2 and is important for zymogen activation, confirming the molecular sexuality hypothesis. The (pre)thrombin-staphylocoagulase complexes form extended dimers with increased affinity for fibrinogen, indicating that dimerization is critical for fibrinogen clotting. The type-II transmembrane multi-domain serine proteinase MT-SP1/matriptase is highly expressed in many human cancer-derived cell lines and has been implicated in extracellular matrix re-modeling, tumor growth and metastasis. We have expressed the catalytic domain of MT-SP1 and solved the crystal structures of complexes with benzamidine at 1.3 Å and BPTI at 2.9 Å. MT-SP1 exhibits a trypsin-like serine proteinase fold, featuring a unique nine-residue 60 insertion loop that influences interactions with protein substrates. The structure discloses a trypsin-like S1 pocket, a small hydrophobic S2 subsite, and an open negatively charged S4 cavity that favors the binding of basic P3/P4 residues. A complementary charge pattern on the surface opposite the active-site cleft suggests a distinct docking of the preceding LDL-receptor class A domain. The benzamidine crystals possess a freely accessible active site and are hence well suited for soaking small molecules, facilitating the improvement of inhibitors. The crystal structure of the MT-SP1 complex with BPTI serves as a model for hepatocyte growth factor activator inhibitor 1, the physiological inhibitor of MT-SP1, and suggests determinants for the substrate specificity. 
Veröffentlichung:
Universitätsbibliothek der TU München 
Mündliche Prüfung:
19.12.2002 
Dateigröße:
22288856 bytes 
Seiten:
103 
Letzte Änderung:
11.06.2007