Hodel A, Kautz R A, Fox R O
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511, USA.
Protein Sci. 1995 Mar;4(3):484-95. doi: 10.1002/pro.5560040315.
Staphylococcal nuclease is found in two folded conformations that differ in the isomerization of the Lys 116-Pro 117 peptide bond, resulting in two different conformations of the residue 112-117 loop. The cis form is favored over the trans with an occupancy of 90%. Previous mutagenesis studies have shown that when Lys 116 is replaced by glycine, a trans conformation is stabilized relative to the cis conformation by the release of steric strain in the trans form. However, when Lys 116 is replaced with alanine, the resulting variant protein is identical to the wild-type protein in its structure and in the dominance of the cis configuration. The results of these studies suggested that any nuclease variant with a non-glycine residue at position 116 should also favor the cis form because of steric requirements of the beta-carbon at this position. In this report, we present a structural analysis of four nuclease variants with substitutions at position 116. Two variants, K116E and K116M, follow the "beta-carbon" hypothesis by favoring the cis form. Furthermore, the crystal structure of K116E is nearly identical to that of the wild-type protein. Two additional variants, K116D and K116N, provide exceptions to this simple "beta-carbon" rule in that the trans conformation is stabilized relative to the cis configuration by these substitutions. Crystallographic data indicate that this stabilization is effected through the addition of tertiary interactions between the side chain of position 116 with the surrounding protein and water structure. The detailed trans conformation of the K116D variant appears to be similar to the trans conformation observed in the K116G variant, suggesting that these two mutations stabilize the same conformation but through different mechanisms.
葡萄球菌核酸酶存在两种折叠构象，它们在赖氨酸116 - 脯氨酸117肽键的异构化方面存在差异，导致112 - 117位残基环出现两种不同构象。顺式构象比反式构象更受青睐，占有率为90%。先前的诱变研究表明，当赖氨酸116被甘氨酸取代时，反式构象相对于顺式构象因反式形式空间位阻的释放而得到稳定。然而，当赖氨酸116被丙氨酸取代时，所得变体蛋白在结构和顺式构型的优势方面与野生型蛋白相同。这些研究结果表明，由于该位置β-碳的空间要求，任何在116位具有非甘氨酸残基的核酸酶变体也应更倾向于顺式构象。在本报告中，我们对在116位进行取代的四种核酸酶变体进行了结构分析。两种变体K116E和K116M，由于更倾向于顺式构象而符合“β-碳”假说。此外，K116E的晶体结构与野生型蛋白的晶体结构几乎相同。另外两种变体K116D和K116N则是这一简单“β-碳”规则的例外，因为这些取代使反式构象相对于顺式构象得到稳定。晶体学数据表明，这种稳定作用是通过116位侧链与周围蛋白质和水结构之间增加的三级相互作用实现的。K116D变体的详细反式构象似乎与K116G变体中观察到的反式构象相似，这表明这两种突变通过不同机制稳定了相同的构象。
