Heparin Blocks the Inhibition of Tissue Kallikrein 1 by Kallistatin through Electrostatic Repulsion

Abstract

Kallistatin, also known as SERPINA4, has been implicated in the regulation of blood pressure and angiogenesis, due to its specific inhibition of tissue kallikrein 1 (KLK1) and/or by its heparin binding ability. The binding of heparin on kallistatin has been shown to block the inhibition of KLK1 by kallistatin but the detailed molecular mechanism underlying this blockade is unclear. Here we solved the crystal structures of human kallistatin and its complex with heparin at 1.9 and 1.8 Å resolution, respectively. The structures show that kallistatin has a conserved serpin fold and undergoes typical stressed-to-relaxed conformational changes upon reactive loop cleavage. Structural analysis and mutagenesis studies show that the heparin binding site of kallistatin is located on a surface with positive electrostatic potential near a unique protruded 310 helix between helix H and strand 2 of β-sheet C. Heparin binding on this site would prevent KLK1 from docking onto kallistatin due to the electrostatic repulsion between heparin and the negatively charged surface of KLK1, thus blocking the inhibition of KLK1 by kallistatin. Replacement of the acidic exosite 1 residues of KLK1 with basic amino acids as in thrombin resulted in accelerated inhibition. Taken together, these data indicate that heparin controls the specificity of kallistatin, such that kinin generation by KLK1 within the microcirculation will be locally protected by the binding of kallistatin to the heparin-like glycosaminoglycans of the endothelium.

DNA accelerates the protease inhibition of a bacterial serpin chloropin

“…Heparin could bind across the positively charged surface area of chloropin around residues K126, K127, K268, and K269 to bridge chloropin and thrombin together and promote their interaction ( Figure 2B ). Therefore, the cofactor binding site of chloropin is distinctly different to those of antithrombin, PCI, kallistatin or vaspin ( Jin et al, 1997 ; Li et al, 2007 ; Ulbricht et al, 2017 ; Ma et al, 2020 ).…”

“…The effect of low molecular weight heparin (LMWH) on the interactions between chloropin and thrombin was similarly assessed as previously described ( Wei et al, 2016 ; Ma et al, 2020 ). A total of 1 µM chloropin was incubated with different concentrations of LMWH (0, 0.005, 0.025, 0.05, 0.25, 0.5, 1, and 2.5 mg/mL) for 5 min at room temperature, and then mixed with 100 nM thrombin and the residual thrombin activity was measured using 0.2 mM S-2238 at different time intervals.…”

“…The relative heparin binding affinities of recombinant chloropin variants were assessed by a heparin-Sepharose column ( Wei et al, 2016 ; Ma et al, 2020 ). About 500 µg of protein was loaded onto a 1 mL HiTrap heparin column pre-equilibrated with 10 mM Tris-HCl, pH 7.4, 1mM EDTA, and eluted with a 20-column volume of 100–500 mM NaCl gradient.…”

Screening candidate diagnostic biomarkers for diabetic kidney disease

“…SERPINA4, also known as Kallistatin (KS), is an inhibitor of tissue kallikrein and also inhibits the Wnt signaling pathway. Kallistatin mainly expressed in liver and broadly expressed in other tissues, such as the eye, kidney, heart, and blood vessels 25 . Studies show that the KKS system plays interesting roles in DKD.…”

“…Kallistatin mainly expressed in liver and broadly expressed in other tissues, such as the eye, kidney, heart, and blood vessels. 25 Studies show that the KKS system plays interesting roles in DKD.…”