Heparin-binding site

Campanella GS, Lee EM, Sun J, Luster AD. CXCR3 and heparin binding sites of the chemokine IP-10 (CXCL10). J Biol Chem 2003 278 17066-74. 

Fromm, J.R., R.E. Hileman, E.E. Caldwell, J.M. Weiler, and R.J. Linhardt. 1997. Pattern and spacing of basic amino acids in heparin binding sites. Arch Biochem Biophys 343 92-100. 

Example 1 Using ACE to Determine the Heparin-Binding Site in Proteins... 

Schematic representation of Subsite Utilization in Thrombin Complexes (after Reference 8). Fibrinogen interacts with three thrombin subsites (here thrombin is represented by a large oval and the interconnected subsites by an irregular three-armed shape). Physiological effectors of thrombin and thrombin inhibitors form distinct interactions at these subsites. Additional subsites, such as the heparin-binding site, exist on the thrombin surface and are not indicated here.

Ritty, T. M., Broekelmann, T. J., Werneck, C. C., and Mecham, R. P. (2003). Fibrillin-1 and -2 contain heparin-binding sites important for matrix deposition and that support cell attachment. Biochem. J. 375, 425-432. 

Fig. 33.1. Structure of DNA aptamers for recognition fibrinogen (A) [15,29] and heparin binding sites (B) [17] of thrombin. The non-canonical hydrogen bonds between guanines are shown by dotted lines. In the case of structure (A), the spacer composed of 15 T chain terminated by thiol group at the end of hydrophobic spacer is shown [34]. (C) Structure of RNA aptamer against fibrinogen binding site of thrombin [23].

The results were explained by two effects (1) dehydration of cations and guanine 06 atomic group and (2) the water uptake upon folding of a single-stranded DNA into a G-quadruplex [29]. It is, however, interesting that DNA aptamer with high affinity to heparin binding site is insensitive to K+ ions [17]. 

Fig. 33.5. The plot of the series resonance frequency of AT-cut crystal covered by neutravidin and with immobilized biotinylated 32-mer DNA aptamer selective to heparin binding site of thrombin as a function of thrombin and HSA concentration, respectively. The fundamental frequency of the crystal was 9MHz. The frequency was determined by HP4395A Network-Spectrum analyzer (Hewlet Packard, Colorado Springs, CO, USA). The crystal was placed in a flow cell developed by Thompson et al. [37] (experiment was performed by I. Grman in M. Thompson laboratory [75]).

Use, for example, 32-mer DNA aptamer modified by biotin at 3 end of the following sequence 3 -BIO-GGG TTT TCA CTT TTG TGG GTT GGA CGG GAT GG - 5. This aptamer has at its 5 end typical motif with high affinity to the heparin-binding site of the thrombin [1], (Please note that aptamer that selectively bind the fibrinogen-binding site of thrombin has a different structure—see Chapter 33 in CD accompanying this book). The aptamer can be purchased from Thermo Electron Corporation, Germany. 

Deficiency of antithrombin predisposes the patient to thrombotic complications. Antithrombin deficiencies can be the result of low protein levels or due to functionally abnormal molecules. Low protein levels can be brought about by reduced synthesis or an increased turnover of the molecule, Functional deficiencies can be brought about by mutations in either the reactive site or heparin binding sites, A number of such mutations have been documented (81,86,87),... 

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,... 

In cells containing low amounts of surface heparans, the toxin does not bind well unless soluble heparin is added (Valdizan et al., 1995). Apparently, the heparin-binding site of the toxin receptor covers the binding site for the toxin until heparin is bound. In normal animal cells surface heparan proteoglycans carry out this function. 

Figure 2 Structure of human pancreatic lipase with indication of the active site buried beneath a mobile Trp 252 surface loop (flap) in order to give access to the substrate and the colipase-binding site faced in the same direction as the active site. The enzyme is glycosylated at Asn 166 (porcine), stabilized at the surface segment by a Ca2+-binding site (partly hidden, marked by an asterisk), and can be anchored by means of the heparin-binding site to heparan sulfate on cell surfaces. (From Ref. 8.)...

Sung, U., O Rear, J.J. and Yurchenco, P.D. (1993) Cell and heparin-binding sites in the distal long arm of laminin identification of active and cryptic sites with recombinant and hybrid glycoprotein. J. Cell Biol. 123 1255-1268. 

A number of other synthetic polyanions that bind to the heparin-binding site of FGF (more appropriately perhaps termed the polyanion-binding site) serve not to protect FGF, but rather to antagonize its activity. The most familiar compound of this type is probably suramin, which prevents... 

Eriksson, A.E., Cousens, L.S. and Matthews, B.W. (1993) Refinement of the structure of human basic fibroblast growth factor at 1.6A resolution and analysis of presumed heparin binding sites by selenate substitution. Protein Sci. 2 1274-1284. 

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