Kunitz domain

Table 17.1 Phage-optimized sequences of Kunitz domain libraries primary binding loop...

How do the mutations identified by phage display improve binding specificity There is as yet no direct stmctural information on the phage-selected inhibitors however they can be modeled using data from the crystal structures of other Kunitz domains bound to serine proteinases. These studies lead to the conclusion that the mutations identified by phage display improve binding specificity by maximizing complementarity between the... 

Dennis, M.S., Herzka, A., Lazarus, R.A. Potent and selective Kunitz domain inhibitors of plasma kallikrein designed by phage display. /. Biol. Chem. 270 25411-25417, 1995. 

Tissue factor pathway inhibitor (TFPI), a 42-kDa protein with three Kunitz domains, is a potent inhibitor of coagulation. It inhibits tissue factor-factor Vila complex upon binding to the active site of Kunitz domain one. Factor Xa is inhibited upon binding to the active site of the second Kunitz domain of TFPI (27). 

Fig. 6. Mechanism of inhibition of tissue factor pathway inhibitor (TFPI). Kunitz domain 1 (Dl) inhibits TF-VIIa complex. Domain 2 (D2) inhibits Xa.

Dennis, M S. and Lazarus, R. A. (1994) Kunitz domain inhibitors of tissue factor-factor Vila II Potent and specific inhibitors by competitive phage selection J. Biol. Chem. 269, 22,137—22,144. 

Enjyoji K, Miyaya X Kamikubo Y Kato H, Effect of heparin on the inhibition of factor Xa by tissue factor pathway inhibitor a segment, Gly 212-Phe 243, of the third Kunitz domain is a heparin binding site. Biochemistry 1995 34 5725-5735,... 

Bik inhibits the trypsin serine proteases through binding of either of its two Kunitz domains. Depending on the serine protease and the Kunitz domain involved, dissociation constants (K ) range from 0.03 to 800 pM [6, 28]. Bik fragmentation and glycation also effect strength and specificity of inhibition. For example, trypsin, chymotrypsin, kallikrein, plasmin, elastase, and cathepsin are inhibited at a A) of 0.03-3 pM, whereas Factors IXa, Xa, XIa, and Xlla are less inhibited with a A) of 15-800 pM. Protease inhibition is observed with both Kunitz domains except for Factors IXa and Xa that... 

Fig. 2. Glycoside chains and the Kunitz domains. Amino acid and glycoside sequences for the two Kunitz-binding domains of Bik are shown. C-C across a chain indicates a cross-link. Predicted fragmentation points for Bik due to trypsin exposure are shown by lines with black dots. The aprotinin-matching peptides of each Kunitz domain are shown by a darkened circle. The first peptide of aprotinin is indicated as Aprol. The X-ray structure-predicted touch points between Bik and trypsin are marked. The predicted PI peptide of the active inhibitory site sequence is indicated as well.

Xu Y, Carr PD, Guss JM, Ollis DL. The crystal structure of bikunin from the inter-a-inhibitor complex A serine protease inhibitor with two kunitz domains. J Mol Biol 1998 276 955-966. 

In subsequent investigations by the author [1] and Ley [2] the Step 4 product was used as a conjugate for the granulocyte colony stimulating factor and Kunitz domain polypeptides, respectively. 

If thrombin and factor Xa, the major activated blood coagulation factors (Fig. 11.6), escape into healthy blood vessels, blood clots will develop and occlude capillaries throughout the body. Direct inhibition of these activated enzymes in the blood flow utilizes serine protease inhibitors, of which there are two common types a Kunitz inhibitor and a serpin. The former possess a Kunitz domain, a convex antiparallel (1-sheet that exactly fits into the concave active site of a serine protease, directly blocking it (lock and key mechanism). By contrast, serpins undergo complex interactions with other proteins to cause conformational changes that bait and block the catalytic action (Fig. 11.12 shows the bait). Table 11.3 fists the major coagulation inhibitors and cofactors, their targets and mechanisms of action. 

Possesses a glycosaminoglycan binding site bSerpin domain Kunitz domain... 

Tissue factor pathway inhibitor (TFPI) is a Kunitz type inhibitor composed of three Kunitz domains. The N-terminal Kunitz domain binds to and inhibits the VHa-TF complex that activates factor X (Fig. 11.6a), whereas the downstream Kunitz domain binds directly to factor Xa and inhibits it strongly and permanently. No function has yet been demonstrated for the third Kunitz domain. The C-terminal region of TFPI is basic and remains attached to endothelial cell surfaces where it inhibits inadvertent factor Xa activation. Studies in mice indicate that knocking out the gene for TFPI stops fetal development. Indeed, clotting diseases such as a stroke and heart attacks are associated with mutations of AITH, HCII and ZPI, but not of TFPI. Like TF (Sect. 11.3.1), diminished TFPI activity may be incompatible with life. 

Human plasma kallikrein and human Variants of the first Kunitz domain of 85... 

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