Zinc-dependent structural stability of human Sonic hedgehog
Zinc-dependent structural stability of human Sonic hedgehog
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DOI：
10.1021/bi9910068
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发表时间：
1999-11-09
期刊：
影响因子：
2.9
通讯作者：
Baker, DP
中科院分区：
文献类型：
--
作者：
Day, ES;Wen, DY;Baker, DP
通过比较野生型ShhN与突变体的生物物理和功能特性，探讨了锌位点在人Sonic hedgehog（ShhN）N-末端片段中的作用，其中锌配位残基H140、D147和H182或E176通过桥接水分子与金属离子相互作用，突变为丙氨酸。野生型和E176 A突变体蛋白在广泛透析后保留1 mol锌/mol蛋白，而H140 A和D147 A突变体分别仅保留0.03和0.05 mol锌/mol蛋白。在两个活性测定中对野生型和突变体蛋白的测定表明，野生型和E176 A突变体蛋白具有相似的活性，而H140 A和D147 A突变体的活性显著较低。这些测定还表明H140 A和D147 A突变体对蛋白水解敏感。在20或25 ℃下测量的H140 A、D147 A和E176 A突变体的CD、荧光和H-1 NMR光谱与野生型ShhN观察到的光谱非常相似。然而，在37 ℃下的CD测量显示H140 A和D147 A突变体中存在一些结构差异的证据。盐酸胍（GuHCl）变性研究表明，H140 A和D147 A突变体中锌的损失使折叠蛋白质不稳定，其稳定性接近3.5 kcal/mol，与通过EDTA处理从野生型ShhN中去除锌的效果相当。野生型ShhN的热熔融曲线给出了一个单一的解折叠转变，中点T-m类似于59 ℃，而H140 A和D147 A突变体都显示出两个不同的转变，T-m值为37-38和52-54 ℃，与EDTA处理的野生型ShhN观察到的相似。向H140 A和D147 A突变体中添加锌导致对热和GuHCl变性的稳定性部分恢复。这些突变体结合锌的能力使用基于荧光的结合测定来证实，该测定表明它们分别以类似于1.6和类似于15 nM的K-d值结合锌，而野生型ShhN的值小于或等于100 pM。E176 A突变体的特性在所有生物物理和功能测定中与野生型ShhN的特性没有区别，表明该残基对锌结合位点的稳定性没有显著贡献，并且ShhN不需要水解酶活性用于体外生物功能。
The role of the zinc site in the N-terminal fragment of human Sonic hedgehog (ShhN) was explored by comparing the biophysical and functional properties of wild-type ShhN with those of mutants in which the zinc-coordinating residues H140, D147, and H182, or E176 which interacts with the metal ion via a bridging water molecule, were mutated to alanine. The wild-type and E176A mutant proteins retained 1 mol of zinc/mol of protein after extensive dialysis, whereas the H140A and D147A mutants retained only 0.03 and 0.05 mol of zinc/mol of protein, respectively. Assay of the wild-type and mutant proteins in two activity assays indicated that the wild-type and E176A mutant proteins had similar activity, whereas the H140A and D147A mutants were significantly less active. These assays also indicated that the H140A and D147A mutants were susceptible to proteolysis. CD, fluorescence, and H-1 NMR spectra of the H140A, D147A, and E176A mutants measured at 20 or 25 degrees C were very similar to those observed for wild-type ShhN. However, CD measurements at 37 degrees C showed evidence of some structural differences in the H140A and D147A mutants. Guanidine hydrochloride (GuHCl) denaturation studies revealed that the loss of zinc from the H140A and D147A mutants destabilized the folded proteins by similar to 3.5 kcal/mol, comparable to the effect of removing zinc from wild-type ShhN by treatment with EDTA. Thermal melting curves of wild-type ShhN gave a single unfolding transition with a midpoint T-m of similar to 59 degrees C, whereas both the H140A and D147A mutants displayed two distinct transitions with T-m values of 37-38 and 52-54 degrees C, similar to that observed for EDTA-treated wild-type ShhN. Addition of zinc to the H140A and D147A mutants resulted in a partial restoration of stability against thermal and GuHCl denaturation. The ability of these mutants to bind zinc was confirmed using a fluorescence-based binding assay that indicated that they bound zinc with K-d values of similar to 1.6 and similar to 15 nM, respectively, as compared to a value of less than or equal to 100 pM for wild-type ShhN. The properties of the E176A mutant were indistinguishable from those of wild-type ShhN in all biophysical and functional assays, indicating that this residue does not contribute significantly to stabilization of the zinc-binding site and that ShhN does not require hydrolase activity for in vitro biological function.