A functional calcium-binding site in the metalloprotease domain of ADAMTS13
A functional calcium-binding site in the metalloprotease domain of ADAMTS13
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DOI：
10.1182/blood-2008-03-144683
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发表时间：
2009-01-29
期刊：
影响因子：
20.3
通讯作者：
Lane, David A.
中科院分区：
文献类型：
--
作者：
Gardner, Michelle D.;Chion, Chan K. N. K.;Lane, David A.
ADAMTS 13调节血管性血友病因子（VWF）的多聚体大小。其功能高度依赖于Ca 2+离子。使用底物（VWF 115，VWF残基1554-1668）蛋白水解的ADAMTS 13预孵育不同的Ca 2+浓度的初始速率，高亲和力的功能ADAMTS 13 Ca 2+结合位点建议与K-D（应用程序）80 μ M（+/- 15 μ M）证实了以前报道的研究。当Glu 83或Asp 173（参与ADAMTS 13金属蛋白酶结构域中预测的Ca 2+结合位点的残基）突变为丙氨酸时，底物蛋白水解的Ca 2+依赖性不受影响。因此，我们寻找并鉴定了ADAMTS 13活性位点附近的候选Ca 2+结合位点，可能包括Glu 184、Asp 187和Glu 212。将该位点内的这些残基突变为丙氨酸显著减弱了ADAMTS 13的Ca 2+的K-D（app），并且D187 A和E212 A的V-max也降低至正常的约25%。Asp 187突变体在过量Ca 2+存在下的动力学分析显示特异性常数k（cat）/K-m降低约13倍，这是由K-m和k（cat）的变化造成的。使用血浆纯化的VWF作为底物证实了这些结果。总之，我们的研究结果表明，ADAMTS 13功能的主要影响是通过结合到一个高亲和力的网站附近的活性位点裂缝介导的Ca 2+。（血。2009;113：1149-1157）
ADAMTS13 regulates the multimeric size of von Willebrand factor (VWF). Its function is highly dependent upon Ca2+ ions. Using the initial rates of substrate (VWF115, VWF residues 1554-1668) proteolysis by ADAMTS13 preincubated with varying Ca2+ concentrations, a high-affinity functional ADAMTS13 Ca2+-binding site was suggested with K-D(app) of 80 mu M (+/- 15 mu M) corroborating a previously reported study. When Glu83 or Asp173 (residues involved in a predicted Ca2+-binding site in the ADAMTS13 metalloprotease domain) were mutated to alanine, Ca2+ dependence of proteolysis of the substrate was unaffected. Consequently, we sought and identified a candidate Ca2+-binding site in proximity to the ADAMTS13 active site, potentially comprising Glu184, Asp187, and Glu212. Mutagenesis of these residues within this site to alanine dramatically attenuated the K-D(app) for Ca2+ of ADAMTS13, and for D187A and E212A also reduced the V-max to approximately 25% of normal. Kinetic analysis of the Asp187 mutant in the presence of excess Ca2+ revealed an approximately 13-fold reduction in specificity constant, k(cat)/K-m, contributed by changes in both K-m and k(cat). These results were corroborated using plasma-purified VWF as a substrate. Together, our results demonstrate that a major influence of Ca2+ upon ADAMTS13 function is mediated through binding to a high-affinity site adjacent to its active site cleft. (Blood. 2009;113:1149-1157)