Flexibility and Design: Conformational Heterogeneity along the Evolutionary Trajectory of a Redesigned Ubiquitin

被引:19
|
作者
Biel, Justin T. [1 ,2 ]
Thompson, Michael C. [1 ]
Cunningham, Christian N. [3 ]
Corn, Jacob E. [3 ,4 ]
Fraser, James S. [1 ]
机构
[1] Univ Calif San Francisco, Dept Bioengn & Therapeut Sci, San Francisco, CA 94158 USA
[2] Univ Calif San Francisco, Grad Grp Biophys, San Francisco, CA 94158 USA
[3] Genentech Inc, Dept Early Discovery Biochem, 1 DNA Way, San Francisco, CA 94080 USA
[4] Univ Calif Berkeley, Innovat Genom Initiat, Berkeley, CA 94702 USA
关键词
X-RAY CRYSTALLOGRAPHY; CATION-PI INTERACTION; COMPUTATIONAL DESIGN; HYDROPHOBIC CORE; ENZYME DESIGN; PROTEIN STRUCTURES; BINDING PROTEINS; LIGAND-BINDING; KEMP ELIMINASE; DYNAMICS;
D O I
10.1016/j.str.2017.03.009
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Although protein design has been used to introduce new functions, designed variants generally only function as well as natural proteins after rounds of laboratory evolution. One possibility for this pattern is that designed mutants frequently sample nonfunctional conformations. To test this idea, we exploited advances in multiconformer modeling of room-temperature X-ray data collection on redesigned ubiquitin variants selected for increasing binding affinity to the deubiquitinase USP7. Initial core mutations disrupt natural packing and lead to increased flexibility. Additional, experimentally selected mutations quenched conformational heterogeneity through new stabilizing interactions. Stabilizing interactions, such as cation-pi stacking and ordered waters, which are not included in standard protein design energy functions, can create specific interactions that have long-range effects on flexibility across the protein. Our results suggest that increasing flexibility may be a useful strategy to escape local minima during initial directed evolution and protein design steps when creating new functions.
引用
收藏
页码:739 / +
页数:14
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