Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260, USA.
Proc Natl Acad Sci U S A. 2010 Nov 9;107(45):19296-301. doi: 10.1073/pnas.1006528107. Epub 2010 Oct 25.
Computational algorithms for protein design can sample large regions of sequence space, but suffer from undersampling of conformational space and energy function inaccuracies. Experimental screening of combinatorial protein libraries avoids the need for accurate energy functions, but has limited access to vast amounts of sequence space. Here, we test if these two traditionally alternative, but potentially complementary approaches can be combined to design a variant of the ubiquitin-ligase E6AP that will bind to a nonnatural partner, the NEDD8-conjugating enzyme Ubc12. Three E6AP libraries were constructed: (i) a naive library in which all 20 amino acids were allowed at every position on the target-binding surface of E6AP (13 positions), (ii) a semidirected library that varied the same residue positions as in the naive library but disallowed mutations computationally predicted to destabilize E6AP, and (iii) a directed library that used docking and sequence optimization simulations to identify mutations predicted to be favorable for binding Ubc12. Both of the directed libraries showed > 30-fold enrichment over the naive library after the first round of screening with a split-dihydrofolate reductase complementation assay and produced multiple tight binders (K(d) < 100 nM) after four rounds of selection. Four rounds of selection with the naive library failed to produce any binders with K(d)'s lower than 50 μM. These results indicate that protein design simulations can be used to create directed libraries that are enriched in tight binders and that in some cases it is sufficient to computationally screen for well-folded sequences without explicit binding calculations.
计算蛋白质设计的算法可以对序列空间的大片区域进行采样，但存在构象空间采样不足和能量函数不准确的问题。组合蛋白质文库的实验筛选避免了对精确能量函数的需求，但对大量序列空间的访问有限。在这里，我们测试了这两种传统上替代但潜在互补的方法是否可以结合使用，以设计一种能够与非天然伴侣 NEDD8 连接酶 Ubc12 结合的泛素连接酶 E6AP 变体。构建了三个 E6AP 文库：（i）在 E6AP 的靶结合表面（13 个位置）上允许所有 20 种氨基酸的原始文库，（ii）在原始文库中变化相同残基位置但不允许计算预测不稳定 E6AP 的突变的半定向文库，以及（iii）使用对接和序列优化模拟识别预测对结合 Ubc12 有利的突变的定向文库。在使用分割二氢叶酸还原酶互补测定法进行第一轮筛选后，定向文库的富集度均高于原始文库 30 倍以上，并且在经过四轮选择后产生了多个紧密结合物（K(d) < 100 nM）。经过四轮原始文库的选择，没有产生 K(d)低于 50 μM 的任何结合物。这些结果表明，蛋白质设计模拟可用于创建定向文库，该文库富含紧密结合物，并且在某些情况下，无需进行显式结合计算即可通过计算筛选出折叠良好的序列。
