The protein phosphatase 5 (PP5) is normally recruited to its substrates by the molecular chaperones, heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90). This interaction requires the tetratricopeptide repeat (TPR) domain of PP5, which binds to an EEVD motif at the extreme C termini of cytosolic Hsp70 and Hsp90 isoforms. In addition to bringing PP5 into proximity with chaperone-bound substrates, this interaction also relieves autoinhibition in PP5’s catalytic domain, promoting its phosphatase activity. To better understand the molecular determinants of this process, we screened a large, pentapeptide library for binding to PP5. This screen identified the amino acid preferences at each position, which we validated by showing that the optimal sequences bind 4- to 7-fold tighter than the natural EEVD motifs and stimulate PP5’s enzymatic activity. The enhanced affinity for PP5’s TPR domain was confirmed using a protein-adaptive differential scanning fluorimetry assay. Using this increased knowledge of structure-activity relationships, we re-examined affinity proteomics results to look for potential EEVD-like motifs in the C termini of known PP5-binding partners. This search identified elongator acetyltransferase complex subunit 1 (IKBKAP) as a putative partner, and indeed, we found that its C-terminal sequence, LSLLD, binds directly to PP5’s TPR domain . Consistent with this idea, mutation of elongator acetyltransferase complex subunit 1’s terminal aspartate was sufficient to interrupt the interaction with PP5 and in cells. Together, these findings reveal the sequence preferences of PP5’s TPR domain and expand the scope of PP5’s functions to include chaperone-independent complexes.

中文翻译：

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蛋白磷酸酶 5 (PP5) 结合决定簇的探索揭示了一种不依赖分子伴侣的激活机制


蛋白磷酸酶 5 (PP5) 通常由分子伴侣、热休克蛋白 70 (Hsp70) 和热休克蛋白 90 (Hsp90) 募集至其底物。这种相互作用需要 PP5 的四肽重复 (TPR) 结构域，该结构域与胞质 Hsp70 和 Hsp90 异构体 C 末端的 EEVD 基序结合。除了使 PP5 与分子伴侣结合的底物接近之外，这种相互作用还减轻了 PP5 催化结构域的自身抑制，促进其磷酸酶活性。为了更好地了解该过程的分子决定因素，我们筛选了一个大型五肽库以与 PP5 结合。该筛选确定了每个位置的氨基酸偏好，我们通过显示最佳序列的结合比天然 EEVD 基序紧密 4 至 7 倍并刺激 PP5 的酶活性来验证这一点。使用蛋白质自适应差示扫描荧光测定法证实了对 PP5 TPR 结构域的亲和力增强。利用对结构-活性关系的了解，我们重新检查了亲和蛋白质组学结果，以在已知 PP5 结合伴侣的 C 末端寻找潜在的 EEVD 样基序。这项搜索将延伸乙酰转移酶复合体亚基 1 (IKBKAP) 确定为假定的伙伴，事实上，我们发现其 C 端序列 LSLLD 直接与 PP5 的 TPR 结构域结合。与这一想法一致，延伸乙酰转移酶复合物亚基 1 末端天冬氨酸的突变足以中断与 PP5 和细胞内的相互作用。总之，这些发现揭示了 PP5 TPR 结构域的序列偏好，并扩展了 PP5 的功能范围，包括不依赖分子伴侣的复合物。