Chen V C, Chao L, Pimenta D C, Bledsoe G, Juliano L, Chao J
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
J Biol Chem. 2001 Jan 12;276(2):1276-84. doi: 10.1074/jbc.M005791200.
Kallistatin is a heparin-binding serine proteinase inhibitor (serpin), which specifically inhibits human tissue kallikrein by forming a covalent complex. The inhibitory activity of kallistatin is blocked upon its binding to heparin. In this study we attempted to locate the heparin-binding site of kallistatin using synthetic peptides derived from its surface regions and by site-directed mutagenesis of basic residues in these surface regions. Two synthetic peptides, containing clusters of positive-charged residues, one derived from the F helix and the other from the region encompassing the H helix and C2 sheet of kallistatin, were used to assess their heparin binding activity. Competition assay analysis showed that the peptide derived from the H helix and C2 sheet displayed higher and specific heparin binding activity. The basic residues in both regions were substituted to generate three kallistatin double mutants K187A/K188A (mutations in the F helix) and K307A/R308A and K312A/K313A (mutations in the region between the H helix and C2 sheet), using a kallistatin P1Arg variant as a scaffold. Analysis of these mutants by heparin-affinity chromatography showed that the heparin binding capacity of the variant K187A/K188A was not altered, whereas the binding capacity of K307A/R308A and K312A/K313A mutants was markedly reduced. Titration analysis with heparin showed that the K312A/K313A mutant has the highest dissociation constant. Like kallistatin, the binding activity of K187A/K188A to tissue kallikrein was blocked by heparin, whereas K307A/R308A and K312A/K313A retained significant binding and inhibitory activities in the presence of heparin. These results indicate that the basic residues, particularly Lys(312)-Lys(313), in the region between the H helix and C2 sheet of kallistatin, comprise a major heparin-binding site responsible for its heparin-suppressed tissue kallikrein binding.
激肽释放酶抑制蛋白是一种肝素结合性丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂），它通过形成共价复合物特异性抑制人组织激肽释放酶。激肽释放酶抑制蛋白与肝素结合后其抑制活性被阻断。在本研究中，我们试图利用源自其表面区域的合成肽并通过对这些表面区域碱性残基进行定点诱变来定位激肽释放酶抑制蛋白的肝素结合位点。使用了两个含有带正电荷残基簇的合成肽，一个源自F螺旋，另一个源自包含激肽释放酶抑制蛋白的H螺旋和C2片层的区域，来评估它们的肝素结合活性。竞争分析表明，源自H螺旋和C2片层的肽表现出更高的特异性肝素结合活性。以激肽释放酶抑制蛋白P1Arg变体为支架，对这两个区域的碱性残基进行取代，生成了三个激肽释放酶抑制蛋白双突变体K187A/K188A（F螺旋中的突变）以及K307A/R308A和K312A/K313A（H螺旋和C2片层之间区域中的突变）。通过肝素亲和色谱对这些突变体进行分析表明，变体K187A/K188A的肝素结合能力未改变，而K307A/R308A和K312A/K313A突变体的结合能力显著降低。用肝素进行滴定分析表明，K312A/K313A突变体具有最高的解离常数。与激肽释放酶抑制蛋白一样，K187A/K188A与组织激肽释放酶抑制蛋白的结合活性被肝素阻断，而K307A/R308A和K312A/K313A在肝素存在下仍保留显著的结合和抑制活性。这些结果表明，激肽释放酶抑制蛋白的H螺旋和C2片层之间区域中的碱性残基，尤其是Lys(312)-Lys(313)，构成了一个主要的肝素结合位点，负责其肝素抑制的组织激肽释放酶结合。
