Pih1p-Tah1p Puts a Lid on Hexameric AAA+ ATPases Rvb1/2p

Abstract

The Saccharomyces cerevisiae (Sc) R2TP complex affords an Hsp90-mediated and nucleotide-driven chaperone activity to proteins of small ribonucleoprotein particles (snoRNPs). The current lack of structural information on the ScR2TP complex, however, prevents a mechanistic understanding of this biological process. We characterized the structure of the ScR2TP complex made up of two AAA+ ATPases, Rvb1/2p, and two Hsp90 binding proteins, Tah1p and Pih1p, and its interaction with the snoRNP protein Nop58p by a combination of analytical ultracentrifugation, isothermal titration calorimetry, chemical crosslinking, hydrogen-deuterium exchange, and cryoelectron microscopy methods. We find that Pih1p-Tah1p interacts with Rvb1/2p cooperatively through the nucleotide-sensitive domain of Rvb1/2p. Nop58p further binds Pih1p-Tahp1 on top of the dome-shaped R2TP. Consequently, nucleotide binding releases Pih1p-Tah1p from Rvb1/2p, which offers a mechanism for nucleotide-driven binding and release of snoRNP intermediates.

RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex

“…The N-terminal PIH domain of Pih1p/PIH1D1 binds a CK2-phosphorylation motif on Tel2p/TELO2, mediating recruitment of the TTT complex to R2TP 2 , 3 . Most recently, we have determined the cryo-EM structure of the intact yeast R2TP complex, in which a single Tah1p–Pih1p sub-complex binds a heterohexameric Rvb1–Rvb2 ring 24 , a finding subsequently confirmed by others 25 . In metazoan R2TP, the small (12 kDa) single-TPR domain protein Tah1p is replaced by the much larger (75 kDa) RPAP3/hSpagh whose N-terminal half contains a tandem pair of TPR domains that bind in concert to a single HSP90 dimer 2 .…”

“…3d ). These images suggested that PIH1D1 located close to the RUVBL ring, as in yeast 24 , 25 . Therefore, RPAP3–PIH1D1 and RPAP3–ΔNT–PIH1D1 are sufficient to disrupt the RUVBL1–RUVBL2 dodecamers.…”

“…Nonetheless, the number of RPAP3 molecules per RUVBL ring in vivo, and/or in the context of a larger assembly, cannot be determined by our work. Cryo-EM shows that PIH1D1, with which RPAP3 also interacts, binds asymmetrically to the DII domain-face of the ring in a similar location as in the yeast R2TP complex 24 , 25 . The RPAP3–PIH1D1 sub-complex behaved as an elongated heterodimer in sedimentation velocity experiment, with an estimated average molecular mass of 103,900 ± 320 Da determined by sedimentation equilibrium assays (Supplementary Fig.…”

“…Previously described interactions with RUVBL1–RUVBL2 such as in SWR1 33 , INO80 11 , or yeast R2TP 24 , 25 are mediated by the face of the ring presenting the DII ‘insertion’ domain. Interaction with the opposite ATPase domain face of the RUVBL1–RUVBL2, as seen here with RPAP3-RBD, has not previously been found, although contacts between this face of the RUVBL ring have been observed in crystals of Rvb1/Rvb2 dodecameric complexes 34 .…”

“…In the yeast system, the single TPR domain of Tah1p is part of a well-ordered cluster of domains along with the CS and PIH domains of Pih1p, that lies in the cup formed by the DII domains of the RUVBL ring 24 , 25 . Conformational adaptability in the yeast system is thus limited to the inherent flexibility of the ~30 residues linking the last globular domain of the bound HSP90 to the TPR-binding MEEVD motif at its extreme C-terminus.…”