Engineering a protein-protein interface using a computationally designed library
Engineering a protein-protein interface using a computationally designed library
复制标题
DOI：
10.1073/pnas.1006528107
复制
发表时间：
2010-11-09
影响因子：
11.1
通讯作者：
Kuhlman, Brian
中科院分区：
文献类型：
--
作者：
Guntas, Gurkan;Purbeck, Carrie;Kuhlman, Brian
蛋白质设计的计算算法可以对大区域的序列空间进行采样，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要，但对大量序列空间的访问有限。在这里，我们测试，如果这两个传统的替代，但潜在的互补方法可以结合起来，设计一个变体的泛素连接酶E6 AP，将结合到一个非天然的合作伙伴，NEDD 8共轭酶Ubc 12。构建了三个E6 AP文库：（i）天然文库，其中在E6 AP的靶结合表面上的每个位置允许所有20个氨基酸（13个位置），（ii）半定向库，其改变了与原始库中相同的残基位置，但不允许计算预测会使E6 AP不稳定的突变，和（iii）定向文库，其使用对接和序列优化模拟来鉴定预测有利于结合Ubc 12的突变。在用分裂-二氢叶酸还原酶互补测定进行第一轮筛选后，两个定向文库均显示出超过天然文库的> 30倍富集，并且在四轮选择后产生多个紧密结合物（K-d < 100 nM）。用未处理文库进行的四轮选择未能产生K-d低于50 μ M的任何结合物。这些结果表明，蛋白质设计模拟可用于创建富含紧密结合物的定向文库，并且在某些情况下，无需明确的结合计算，就足以计算筛选良好折叠的序列。
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 mu M. 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.