Crystallization and crystallographic studies of kallistatin

Lin, Fanghua; Zhou, Aiwu; Wei, Zhenquan

doi:10.1107/s2053230x15012893

Cited by 3 publications

(2 citation statements)

References 23 publications

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“…2.1. Macromolecule production cDNA of CL-AI was cloned into the pSUMO3 expression vector (Addgene) and overexpressed as described previously (Lin et al, 2015). Briefly, the target gene was induced by 0.2 mM isopropyl -d-1-thiogalactopyranoside (IPTG) when the cell density reached an optical density at 600 nm (OD 600 ) of approximately 0.6 in 2ÂYT medium.…”

Section: Methodsmentioning

confidence: 99%

Crystallization and crystallographic studies of a novel chickpea 11S globulin

2022

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Chickpea is a crop that is known as a source of high-quality proteins. CL-AI, which belongs to the 11S globulin and cupin superfamily, was initially identified in chickpea seeds. CL-AI has recently been shown to inhibit various types of α-amylases. To determine its molecular mechanism, the crystal structure of CL-AI was solved at a final resolution of 2.2 Å. Structural analysis indicated that each asymmetric unit contains three molecules with threefold symmetry and a head-to-tail association, and each molecule is divided into an α-chain and a β-chain. CL-AI has high structural similarity to other 11S globulins and canonical metal-dependent enzyme-related cupin proteins, whereas its stimilarity to α-amylase inhibitor from Phaseolus vulgaris is quite low. The structure presented here will provide insight into the function of CL-AI.

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Section: Methodsmentioning

confidence: 99%

Crystallization and crystallographic studies of a novel chickpea 11S globulin

2022

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“…Recombinant kallistatin was prepared from Escherichia coli as previously described [ 38 ]. Briefly, the coding sequence covering residues 45–427 of human kallistatin was cloned in E. coli expression vector pE-SUMO3.…”

Section: Methodsmentioning

confidence: 99%

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

Zheng

et al. 2020

Biomolecules

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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.

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