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

Lina Ma,Zhenquan Wei,Robin W Carrell,Zimei Shu,Ying Zheng,Yinbiao Sun,Jiawei Wu,Aiwu Zhou

doi:10.3390/biom10060828

Lina Ma, Zhenquan Wei + Show 6 more

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https://doi.org/10.3390/biom10060828

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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.

Highlights

Human kallistatin was first identified and characterized as a kallikrein binding protein [1,2]
The closely migrated doublet bands of kallistatin in lane 2 reflects some of the Recombinant human kallistatin (rKAL) being cleaved by kallikrein 1 (KLK1) during inhibition, which is consistent with the stoichiometry of inhibition (SI) measurement (Table 2)
The inhibition of KLK1 by rKAL was quantitatively assessed by an enzymatic assay using a KLK1’s fluorescent substrate. rKAL almost completely inhibited the KLK1 activity in the absence of heparin, but this inhibition is significantly reduced by the presence of 0.5 mg/mL of high or low molecular weight heparins with ≈ 60%–80% of KLK1’s activity remaining after 30 min incubation (Figure 1B)

Summary

Introduction

Human kallistatin was first identified and characterized as a kallikrein binding protein [1,2]. At the acyl-enzyme intermediate stage the serpin undergoes a rapid stressed-to-relaxed (S-to-R) conformational change where the reactive center loop is fully inserted into the central beta-sheet of the serpin resulting in translocation of the protease and distortion in the catalytic triad of the protease [7,9]. The protease specificity of a serpin is largely determined by its reactive loop sequence, especially the P1 residue, in some cases, residues which do not belong to the reactive center loop can critically affect specificity [9,11] This appears to be so with kallistatin which unusually has a P2-P1-P1’ sequence of Phe-Phe-Ser targeting P1 Arg-preferred KLK1 [12,13]

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