To validate the FEM1B–CDK5R1 interface, we generated several CDK5R1 mutants and examined their FEM1B binding affinities by ITC. The binding data show that R-1K, R-1A and L-3A abolished or reduced the FEM1B binding, underscoring the important roles of R−1 and L−3.
为了验证FEM1B和CDK5R1的作用界面，我们生成了几个CDK5R1的突变体，并且通过ITC检测他和FEM1B结合的亲和力。结合数据显示，R-1K, R-1A,和L-3A消除或减少了FEM1B的结合，强调了R-1和L-3的重要作用
While the G-4A mutant decreased the binding affinity by 11-fold (Kd of 66 versus 6.0 μM), G-4V disrupt the binding (Supplementary Table 3), suggesting that any bulky residue at the −4 position is disfavored because of potential steric clash with the FEM1B Y153 (Fig. 4c).
虽然G-4A突变将结合亲和力降低了11倍，但G-4A破坏了结合，表明由于与FEM1B y153的潜在空间位阻的冲突，-4的位的任何大块残基都是不理想的
Additionally, L-6A, L-7A and the double mutant L-5A/R-8A decrease the FEM1B binding affinity by roughly 3–7.5-fold (Kd of 18–45 versus 6.0 μM) (Supplementary Table 3), confirming the contribution of the residues upstream of −4GLDR−1 in FEM1B binding. Next, we generated several FEM1B mutants and examine their CDK5R1 binding affinities by ITC.
此外，L-6A，L-7A，和L-5A/R-8A的双突变体将FEM1B的结合亲和力降低了大约3-7.5倍，证实了-4GLDR-1上游残基在FEM1B结合中的贡献。接下来，我们生成了几个FEM1B的突变体，通过ITC来检查它们与CDK5R1结合的亲和力。
F130A and the double mutant D82A/D131A abolished the binding, whereas another double mutant Y163A/F193A reduced the binding affinity by >50-fold (Kd > 300 versus 6.0μM) (Supplementary Table 3), further validating the FEM1B–CDK5R1 interface.
F130A和D82A/D131A的双突变体消除了结合，而另一个突变体Y164A/F193A将亲和力降低了50倍，进一步验证了FEM1B/CDK5R1的结合界面
Collectively, our data indicate that FEM1B prefers the Arg/C-degron ending with -G-L-X-R and interaction requires an additional upstream sequence. We also solved the structure of FEM1C fused with the C-degron of CDK5R1 at a 2.35 Å resolution (Supplementary Table 2 and Extended Data Figs. 2g and 3e).
总的来说，我们的数据表明FEM1B更喜欢以GLXR结尾的C末端，并且相互作用需要额外的上有序列。我们还分析了CDK5R1的C末端残基融合FEM1C的结构。
In the structure, CDK5R1 binds to FEM1C in a way similar to that in the FEM1B–CDK5R1 structure, with its R−1 and Leu−3 positioned into the p1 and p2 pockets, respectively (Extended Data Fig. 5g,). Mutagenesis and ITC assay also confirmed the essential role of R−1 in FEM1C binding (Supplementary Table 3)
在这个结构中，CDK5R1以类似于FEM1B-CDK5R1结构的方式与FEM1C结合。其 R-1 和 L-3 分别位于 p1 和 p2 口袋中。突变形成和 ITC 测定也证实了 R-1 在 FEM1C 结合中的重要作用
