Tugaeva Kristina V, Remeeva Alina, Gushchin Ivan, Cooley Richard B, Sluchanko Nikolai N
A.N. Bach Institute of Biochemistry, Federal Research Center of Biotechnology of the Russian Academy of Sciences, 119071, Moscow, Russia; Department of Biochemistry, School of Biology, M.V. Lomonosov Moscow State University, 119991, Moscow, Russia.
Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, 141701, Dolgoprudny, Russia.
Protein Expr Purif. 2020 Nov;175:105707. doi: 10.1016/j.pep.2020.105707. Epub 2020 Jul 16.
14-3-3 protein isoforms regulate multiple processes in eukaryotes, including apoptosis and cell division. 14-3-3 proteins preferentially recognize phosphorylated unstructured motifs, justifying the protein-peptide binding approach to study 14-3-3/phosphotarget complexes. Tethering of human 14-3-3σ with partner phosphopeptides via a short linker has provided structural information equivalent to the use of synthetic phosphopeptides, simultaneously facilitating purification and crystallization. Nevertheless, the broader applicability to other 14-3-3 isoforms and phosphopeptides was unclear. Here, we designed a novel 14-3-3ζ chimera with a conserved phosphopeptide from BAD, whose complex with 14-3-3 is a gatekeeper of apoptosis regulation. The chimera could be bacterially expressed and purified without affinity tags. Co-expressed PKA efficiently phosphorylates BAD within the chimera and blocks its interaction with a known 14-3-3 phosphotarget, suggesting occupation of the 14-3-3 grooves by the tethered BAD phosphopeptide. Efficient crystallization of the engineered protein suggests suitability of the "chimeric" approach for studies of other relevant 14-3-3 complexes.
14-3-3蛋白异构体调节真核生物中的多种过程，包括细胞凋亡和细胞分裂。14-3-3蛋白优先识别磷酸化的无结构基序，这为研究14-3-3/磷酸化靶点复合物的蛋白质-肽结合方法提供了依据。通过短连接子将人14-3-3σ与伴侣磷酸肽连接起来，所提供的结构信息等同于使用合成磷酸肽，同时便于纯化和结晶。然而，其对其他14-3-3异构体和磷酸肽的更广泛适用性尚不清楚。在此，我们设计了一种新型的14-3-3ζ嵌合体，它带有来自BAD的保守磷酸肽，BAD与14-3-3的复合物是细胞凋亡调控的守门人。该嵌合体可以在没有亲和标签的情况下进行细菌表达和纯化。共表达的蛋白激酶A（PKA）能有效磷酸化嵌合体内的BAD，并阻断其与已知的14-3-3磷酸化靶点的相互作用，这表明连接的BAD磷酸肽占据了14-3-3的凹槽。工程化蛋白的高效结晶表明“嵌合”方法适用于研究其他相关的14-3-3复合物。
