R2TP is a highly conserved chaperone complex formed by two AAA+ ATPases, RUVBL1 and RUVBL2, that associate with PIH1D1 and RPAP3 proteins. R2TP acts in promoting macromolecular complex formation. Here, we establish the principles of R2TP assembly. Three distinct RUVBL1/2-based complexes are identified: R2TP, RUVBL1/2-RPAP3 (R2T), and RUVBL1/2-PIH1D1 (R2P). Interestingly, we find that PIH1D1 does not bind to RUVBL1/RUVBL2 in R2TP and does not function as a nucleotide exchange factor; instead, RPAP3 is found to be the central subunit coordinating R2TP architecture and linking PIH1D1 and RUVBL1/2. We also report that RPAP3 contains an intrinsically disordered N-terminal domain mediating interactions with substrates whose sequences are primarily enriched for Armadillo repeat domains and other helical-type domains. Our work provides a clear and consistent model of R2TP complex structure and gives important insights into how a chaperone machine concerned with assembly of folded proteins into multisubunit complexes might work.

R2TP 是一种高度保守的伴侣复合物，由与 PIH1D1 和 RPAP3 蛋白相关的两个 AAA+ ATP 酶 RUVBL1 和 RUVBL2 形成。R2TP 起到促进大分子复合物形成的作用。在这里，我们建立了 R2TP 组装的原则。确定了三种不同的基于 RUVBL1/2 的复合物：R2TP、RUVBL1/2-RPAP3 (R2T) 和 RUVBL1/2-PIH1D1 (R2P)。有趣的是，我们发现 PIH1D1 不与 R2TP 中的 RUVBL1/RUVBL2 结合，并且不充当核苷酸交换因子；相反，发现 RPAP3 是协调 R2TP 架构并连接 PIH1D1 和 RUVBL1/2 的中央亚基。我们还报告说，RPAP3 包含一个本质上无序的 N 端域介导与底物的相互作用，其序列主要富集犰狳重复域和其他螺旋型域。