The Archaeal Exosome: Identification and Quantification of Site-Specific Motions That Correlate with Cap and RNA Binding**

Large molecular machines perform many cellular processes and it is of fundamental interest to understand how these enzyme complexes work in detail. In this regard, not only an accurate description of the static three-dimensional (3D) structure is required, but also a description of how these machines change their structure over time.

. Architecture of the exosome complex. Rrp42 is displayed as a cartoon (green), Rrp41 is shown in gray, Rrp4 in yellow and Csl4 in brown. The displayed structures are based on PDB-entries 2BR2 (exosome core) [1] , 2JEA (exosome Rrp4 complex) [2] and 3M7N (exosome Csl4 complex, displayed structure is a homology model based on the archaeoglobus fulgidus structure of the complex). [3] The exosome core (Rrp41:Rrp42) can interact with substrate RNA and degrade this in a processive manner in the 3' to 5' direction. During this process, the exosome does not release the substrate. We thus expect that cap proteins will not be recruited to the processing exosome core: substrate RNA complex. It should be noted that the amounts of cap-free exosome (Rrp41:Rrp42) is expected to be very low in a cellular context as cap proteins, Rrp41 and Rrp42 are present in similar relative amounts. [4] The interaction between the free exosome and the cap proteins (Csl4 or Rrp4) is very tight. After formation of the exosome:cap complex RNA substrate can be recruited and degraded; the cap proteins will remain bound to the exosome core during this process.
V162 I220 I220 I220 I220 Figure S3. The cap proteins change the dynamics in the exosome core. MQ dispersion profiles observed for Ile 13, 19, 27 and 220. Note that the y-axis has the same range for all graphs for a specific residue to allow for direct comparison of the data. (A) Profiles in the exosome core (identical to Fig 2B in the main text). Blue and red correspond to state A and B respectively. See main text for details. The structure of the exosome core is indicated on the right. (B) Profiles in the exosome-Rrp4 complex. Note that only one state is present in the spectra. The structure of the exosome-Rrp4 complex is shown on the right. (C) Profiles in the exosome-Csl4 complex. Note that only one state is present in the spectra.   Figure S4. Predicted versus measured chemical shifts for all assigned isoleucine residues. The chemical shifts were predicted using shiftx2 [6] using the free exosome complex (2BR2) or the exosome-Rrp4 complex (2JE6) from which Rrp4 was removed as input. The methyl groups that show two conformations have been labeled; the Pearson R correlation coefficient is indicated. A red drawn line indicates the best fit between the predicted and measured shifts (y=x+A), where A corrects for an (potential) offset in chemical shift referencing. Note that none of the correlations is significant, most likely due to the large inaccuracies in the predicted values. S5 Figure S5. "State A mutant" (N9A) exosome complex. Location of N9 in the exosome complex. N9 is remote from the interaction with the cap structure. Mutations in this residue do thus not change the interaction between the exosome core and the cap directly, but rather indirectly through changes in exosome dynamics.
The identification of the N9A mutant was inspired by the spectra of the assignment mutants (Table S1), where we noticed that the relative intensities of the two sets of peaks varied. This indicated that the equilibrium between the two states could be modified. We then systematically mutated residues that were close, but not directly in the capinteraction-helix and monitored the state A: state B peak ratio. In this process we identified that the N9A mutation yielded only a single set of resonances.

Rrp42
Rrp42 N9  In addition to the interaction between the exosome complex and the Rrp4 cap we performed experiments to probe for the interaction between the exosome complex and the reduced Rrp4 cap (that lacks one of the domains; See Figure   S7). Unfortunately, this protein interacted unspecifically with the sensorchip surface, which resulted in a strong signal from the reference cell. As a consequence, we were not able to extract any reliable interaction data for the exosome: reduced Rrp4 cap complex.

Figure S7
To move the Rrp4 binding affinity into a range where one can discriminate cap binding between WT and "state A mutant" exosome, we deleted one of the three domains from the cap structure (A). This reduced Rrp4 cap contains the domains that interacts with Rrp42 (the S1 and KH domains) but lacks the domain (the NTD; N-terminal domain) that interacts with Rrp41. We then used this reduced Rrp4 cap structure to probe for the interactions with the WT and "state A mutant" exosome. In NMR chemical shift titrations (B), where we added the unlabeled exosome to 15 N-labeled reduced cap, we observed a faster decrease in resonance intensity upon addition of the "state A mutant" exosome than upon addition of the WT exosome (C). This implies that the "state A mutant" has a higher affinity for the cap than the WT exosome and establishes that state A plays an important role in the interaction with the Rrp4 cap structure.  (C) Dependence of reduced Rrp4 peak intensities on the molar excess of exosome (blue-cyan scale) or "state A mutant" exosome (red-yellow scale). Four Rrp4 residues that contact Rrp42 are selected. The signals decrease more rapidly upon addition of the "state A mutant" due to the tighter interaction. Note that the decrease in peak intensity is largely due to fast relaxation in the high molecular weight complex that is formed, preventing accurate extraction of binding constants from the NMR data. After addition of a high excess of (WT or "state A") exosome the spectra of the reduced cap are no longer visible due to the formation of a large complex.      Table S1: Assigned chemical shifts for Rrp42 as a monomer and in the exosome core. 1 Leu and Val methyl groups were not stereo-specifically assigned. 2 A and B refer to the states A and B in the exosome core. 3 The Rrp42 monomer only displays one state. 4 Indicated if a point mutation was made to assign (or check the assignment of) the residue.