Metal binding to sites engineered in proteins can provide an increase in their stability and facilitate new functions. Besides the sites introduced in purpose, sometimes they are present accidentally as a consequence of the expression system used to produce the protein. This happens with the copper- and nickel-binding (ATCUN) motif generated by the amino-terminal residues Gly-Ser-His. This ATCUN motif is fortuitously present in many proteins, but how it affects the structural and biophysical characterization of the proteins has not been studied. In this work, we have compared the structure and biophysical properties of a small modular domain, the SH3 domain of the c-Src tyrosine kinase, cloned with and without an ATCUN motif at the N terminus. At pH 7.0, the SH3 domain with the ATCUN motif binds nickel with a binding constant K_a = 28.0 ± 3.0 mM⁻¹. The formation of the nickel complex increases the thermal and chemical stability of the SH3 domain. A comparison of the crystal structures of the SH3 domain with and without the ATCUN motif shows that the binding of nickel does not affect the overall structure of the SH3 domain. In all crystal structures analyzed, residues Gly-Ser-His in complex with Ni²⁺ show a square planar geometry. The CD visible spectrum of the nickel complex shows that this geometry is also present in the solution. Therefore, our results not only show that the ATCUN motif might influence the biophysical properties of the protein, but also points to an advantageous stabilization of the protein with potential biotechnological applications.

中文翻译：

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工程化的ATCUN基序对c-Src酪氨酸激酶SH3结构域的结构和生物物理特性的影响。

金属与蛋白质工程化位点的结合可以增加其稳定性并促进新功能。除了故意引入的位点外，有时由于用于产生蛋白质的表达系统而偶然出现。这是由氨基末端残基Gly-Ser-His生成的铜和镍结合（ATCUN）基序发生的。幸运的是，这种ATCUN基序存在于许多蛋白质中，但是尚未研究它如何影响蛋白质的结构和生物物理特性。在这项工作中，我们比较了一个小的模块化结构域，即c-Src酪氨酸激酶的SH3结构域的结构和生物物理特性，该结构域在N末端带有和不带有ATCUN基序。在pH 7.0时，具有ATCUN基序的SH3结构域以结合常数结合镍K _a  ＝ 28.0±3.0mM ^-1。镍络合物的形成增加了SH3域的热稳定性和化学稳定性。具有和不具有ATCUN基序的SH3结构域的晶体结构的比较表明，镍的结合不影响SH3结构域的整体结构。在所有分析的晶体结构中，与Ni ²⁺络合的残基Gly-Ser-His显示出正方形的平面几何形状。镍络合物的CD可见光谱表明，溶液中也存在这种几何形状。因此，我们的结果不仅表明ATCUN基序可能会影响蛋白质的生物物理特性，而且还指出了潜在生物技术应用对蛋白质的有利稳定作用。