Lysine 114 of Antithrombin Is of Crucial Importance for the Affinity and Kinetics of Heparin Pentasaccharide Binding

Abstract

Lys114 of the plasma coagulation proteinase inhibitor, antithrombin, has been implicated in binding of the glycosaminoglycan activator, heparin, by previous mutagenesis studies and by the crystal structure of antithrombin in complex with the active pentasaccharide unit of heparin. In the present work, substitution of Lys 114 by Ala or Met was shown to decrease the affinity of antithrombin for heparin and the pentasaccharide by ϳ105 -fold at I 0.15, corresponding to a reduction in binding energy of ϳ50%. The decrease in affinity was due to the loss of two to three ionic interactions, consistent with Lys 114 and at least one other basic residue of the inhibitor binding cooperatively to heparin, as well as to substantial nonionic interactions. The mutation minimally affected the initial, weak binding of the two-step mechanism of pentasaccharide binding to antithrombin but appreciably (>40-fold) decreased the forward rate constant of the conformational change in the second step and greatly (>1000-fold) increased the reverse rate constant of this step. Lys 114 is thus of greater importance for the affinity of heparin binding than any of the other antithrombin residues investigated so far, viz. and Arg 129 to increasing the rate of induction of the activating conformational change, a role presumably exerted by interactions with the nonreducing end trisaccharide unit of the heparin pentasaccharide. However, its major effect, also larger than that of these two residues, is in maintaining antithrombin in the activated state by interactions that most likely involve the reducing end disaccharide unit.

Crystal structures of native and thrombin-complexed heparin cofactor II reveal a multistep allosteric mechanism

“…1d compares the heparin binding sites of AT and HCII, with key residues indicated. The critical residues involved in the tight binding of the heparin pentasaccharide by AT have been demonstrated by both structural and biochemical studies to include Lys-114, Lys-125, and Arg-129, whereas Arg-132, Lys-133, and Lys-136 contribute to the binding of longer heparin chains (8,(29)(30)(31)(32)(33). The conservation in HCII of the key residues involved in the interaction of the pentasaccharide with AT supports a similar mechanism of GAG binding by HCII.…”

The heparin-binding site of antithrombin is crucial for antiangiogenic activity

“…[24][25][26][27] Mutations of Lys125 or Lys114 to Met result in substantial 200-fold and 10 5 -fold reductions in the affinity of native antithrombin for heparin, respectively. 21,22 These mutations reduced the affinity of cleaved antithrombin for heparin as well, as judged from the decreased salt concentrations at which the cleaved mutants eluted from heparin-agarose relative to the cleaved wild-type serpin (Table 1). However, the Lys125 and Lys114 mutations caused comparable losses in the affinity of cleaved antithrombin for heparin, in sharp contrast to the very different heparin affinity losses produced by these mutations in native antithrombin (Table 1).…”

“…23,28 The purity of native and cleaved forms of all antithrombins was assessed by sodium dodecyl sulfate (SDS) and native polyacrylamide gel electrophoresis (PAGE). 21,22 The natural heparin pentasaccharide that specifically binds antithrombin and a modified pentasaccharide with much higher affinity for the serpin 29 (respectively compounds 39 and 92 in Van Boeckel and Petitou 30 ) were generously provided by Dr Maurice Petitou of Sanofi Recherche (Toulouse, France). A full-length heparin of approximately 50 saccharides and containing the pentasaccharide was isolated from commercial heparin by size and antithrombin-affinity fractionation as described.…”

“…20 Recombinant wild-type and K125M, K114M, and R393W variant antithrombins were expressed in baby hamster kidney (BHK) cells and purified as described. [21][22][23] The wild-type, K114M, and R393W variant antithrombins contained an additional N135Q mutation to eliminate glycosylation heterogeneity arising from incomplete glycosylation at Asn135. 24 The corresponding glycoform of K125M antithrombin lacking carbohydrate at N135 was isolated.…”

Disease-causing mutations in the serpin antithrombin reveal a key domain critical for inhibiting protease activities

“…Variants were produced in baculovirus-infected insect cells using the expression system from Invitrogen, as described by the manufacturer. Recombinant antithrombins were purified on a 5-ml Hi-Trap heparin-Sepharose column followed by ion exchange chromatography using a mono Q column, as described previously (33,34). Those fractions containing pure antithrombin were finally desalted, and concentrations were determined by measuring the absorbance at 280 nm using an extinction coefficient of 37,700 M Ϫ1 cm Ϫ1 (35).…”