1adz
THE SOLUTION STRUCTURE OF THE SECOND KUNITZ DOMAIN OF TISSUE FACTOR PATHWAY INHIBITOR, NMR, 30 STRUCTURES
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OverviewOverview
Tissue Factor Pathway Inhibitor (TFPI) is a 36 kDa glycoprotein that helps, maintain haemostasis by inhibiting Factor Xa and the Factor VIIa/Tissue, Factor (TF) complex. TFPI contains three tandemly linked Kunitz inhibitor, domains, of which the second inhibits factor Xa. We have undertaken a, multidisciplinary approach to study the structure and function of the, second Kunitz domain of TFPI, with a view towards the rational design of, factor Xa inhibitors. Amino acid residues 93 to 154 of the mature TFPI, protein, corresponding to the second Kunitz domain (TFPI-kII), were, expressed in Escherichia coli. The protein was purified to near, homogeneity by ion exchange, hydrophobic interaction, and size exclusion, chromatography, respectively. TFPI-kII is a potent factor Xa inhibitor, with a Ki of 1.5 x 10(-10) M, a value that does not differ significantly, from that of intact TFPI. The three-dimensional structure of TFPI-kII in, aqueous solution was determined by 1H nuclear magnetic resonance, spectroscopy (NMR). A set of 30 conformers was calculated with the program, DIANA using 906 distance constraints derived from nuclear Overhauser, effects and 23 dihedral angle constraints. This set, representing the, solution structure of TFPI-kII, has an average root-mean-square deviation, of 0.78 A for the backbone atoms and 1.38 A for all heavy atoms of, residues 1 to 58. The structure of TFPI-kII has also been determined in, complex with porcine trypsin using X-ray crystallographic techniques. The, complex has been solved to a resolution of 2.6 A, with a final R-factor of, 16.2%. Comparison of the NMR derived structure with that of TFPI-kII in, complex with trypsin reveals little divergence of the two structures, with, the exception of residue Tyr17. Superposition of the trypsin:TFPI-kII, complex on factor Xa provides insights into macromolecular determinants, for the inhibition of factor Xa. Complexation would require a degree of, reorganisation of factor Xa residues, in particular of TyrF99, but also, perhaps of the F148-loop. The interaction was further investigated using, restrained molecular dynamics. Electrostatic interactions would appear to, play a major role. The reorganisation of factor Xa is in contrast to the, proposed factor Xa:TAP interaction, where TAP would bind to the "ground, state" structure of factor Xa.
About this StructureAbout this Structure
1ADZ is a Single protein structure of sequence from Homo sapiens. Full crystallographic information is available from OCA.
ReferenceReference
The second Kunitz domain of human tissue factor pathway inhibitor: cloning, structure determination and interaction with factor Xa., Burgering MJ, Orbons LP, van der Doelen A, Mulders J, Theunissen HJ, Grootenhuis PD, Bode W, Huber R, Stubbs MT, J Mol Biol. 1997 Jun 13;269(3):395-407. PMID:9199408
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