Crystal structure of the S. solfataricus archaeal exosome reveals conformational flexibility in the RNA-binding ring

Abstract

Background: The exosome complex is an essential RNA 3'-end processing and degradation machinery. In archaeal organisms, the exosome consists of a catalytic ring and an RNA-binding ring, both of which were previously reported to assume three-fold symmetry.

Methodology/principal findings: Here we report an asymmetric 2.9 A Sulfolobus solfataricus archaeal exosome structure in which the three-fold symmetry is broken due to combined rigid body and thermal motions mainly within the RNA-binding ring. Since increased conformational flexibility was also observed in the RNA-binding ring of the related bacterial PNPase, we speculate that this may reflect an evolutionarily conserved mechanism to accommodate diverse RNA substrates for degradation.

Conclusion/significance: This study clearly shows the dynamic structures within the RNA-binding domains, which provides additional insights on mechanism of asymmetric RNA binding and processing.

Figure 1. Purification and activity assay of the purified S. solfataricus full exosome.

(a) SDS-PAGE of the purified S. solfataricus full exosome (left) and the Rrp4-exosome isoform (right). (b) RNase activity assay for the intact S. solfataricus exosome. The exosome is stalled by the HDV ribozyme sequence. (c) Side and top-down view of the 2.9 Å S. solfataricus exosome structure. Gold, trimeric Rrp4 RNA-binding ring; blue/cyan, Rrp41/Rrp42 catalytic ring.

Figure 2. Rrp4 trimeric cap between our S. solfataricus exosome (blue) deviates from perfect three-fold symmetry as compared with the structure by Lorentzen et al (orange).

Subunit F and I considerably deviates (2 to 3 Å at the periphery) from Lorentzen et al previously report when aligning subunit C. Inlets: 2.9 Å experimental electron density map of Rrp4 subunit F contoured at 1.0 σ.

Figure 3. Structure alignment of Rrp4 trimeric cap between our S. solfataricus exosome (red) and that Lorentzen et al previously reported (cyan).

(a) Overall alignment of the 750− aa trimeric cap (r.m.s.d. = 1.5 Å). (b) Alignment of the most flexible Rrp4 subunit (Chain F) by N-ter domain, aa 1–50 (r.m.s.d. = 0.4 Å). (c) Alignment of Chain F subunit by S1 domain, aa 56–126 (r.m.s.d. = 0.3 Å). (d) Alignment of Chain F subunit by KH domain, aa 135–250 (r.m.s.d. = 0.7 Å).

Figure 4. Detecting thermal motions in Rrp4 RNA-binding ring using thermal ellipsoid and B-factor analyses.

(a) Overall analysis of the trimeric cap. (b) According to the thermal ellipsoid analysis, the most thermal flexible Rrp4 subunit is Chain I, but not Chain F, which displays the largest rigidbody motion. TLS sensors were obtained from TLS refinement in Refmac5 (see Methods section for details) and plotted using Raster3D .(c) B-factor comparison of the Rrp4 between our S. solfataricus exosome (left) and that Lorentzen et al previously reported (right). B-factor coloring: blue, 30 and below; red, 100 and above.