POROCESO DE VERIFICACION DE HIPOTESIS

The Rvb1/2 complex is a prominent feature of INO80 and there was no structure of an Rvb1/2 within its native complex. The structures of isolated Rvb1/2 could not clarify (i) if Rvb1/2 is a hexamer or a dodecamer; (ii) if the rings are homo- or hetero-oligomeric and (iii) how the rings interact with each other.

The INO80 complex is among the first structure containing the Rvb1/2 in a native context. However, conclusions gained about INO80’s Rvb1/2 are not necessarily applicable to Rvb1/2 in other complexes or in an isolated state, if there is one. In the majorities of isolated structures, Rvb1/2 is dodecameric (Lopez-Perrote et al., 2012; Puri et al., 2007; Torreira et al., 2008), but Rvb1/2 was even be found in other oligomeric states (Cheung et al., 2010; Gorynia et al., 2011; Niewiarowski et al., 2010). Endogenously purified yeast Rvb1/2 in complex with the chaperone Hsp90 has been shown to be hetero-hexameric (Cheung et al., 2010). Rvb1/2 in the native INO80 environment adopts a dodecameric state, comprised of two hexameric rings (Tosi et al., 2013).

The resolution of the isolated Rvb1/2 EM structures was too low to differentiate Rvb1 from Rvb2; consequently the composition of the rings could not directly be abbreviated (Lopez- Perrote et al., 2012; Puri et al., 2007; Torreira et al., 2008). However, antibody labeling suggested that the rings could be composed of one species (Torreira et al., 2008). In addition, there are crystal structures of homo-rings composed of either Rvb1 (Matias et al., 2006) or Rvb2 (Petukhov et al., 2012), and hetero-hexameric rings (Gorynia et al., 2011), but this structures has a deleted domain 2. The distance restraints determined by XL-MS exclusively allow a hetero-hexameric composition of Rvb1/2 in INO80 (Tosi et al., 2013).

The stacking of the rings has been under debate as well. Yeast and human structures from the Llorca lab could easily interpreted as a domain 2-2 stacking (Lopez-Perrote et al., 2012; Torreira et al., 2008), but the stacking could not be unambiguously determined in the structure from the Tsaneva lab (Puri et al., 2007). A following work from the same lab showed that the deletion of domain 2 of Rvb1 destabilizes the dodecamer and they thus concluded that this argues against a domain 2-2 interaction of the rings (Niewiarowski et al., 2010). However, a

complete deletion of the domain 2 resulted in insoluble protein. Therefore Niewiaroski et al., deleted only the external part, the OB-folds and thus leaving the internal part of domain 2, which then can mediate the interaction (Niewiarowski et al., 2010). The volume and shape of the head of INO80 were sufficient to accommodate two rings of Rvb1 (Matias et al., 2006; Tosi et al., 2013). However, the diameter of the head was only ~120 Å. Thus, it was the most compact version compared to any isolated Rvb1/2 structure. Movements in the domain 2 were shown to mediate between the stretched (145 Å) and compact (130 Å) form of human isolated Rvb1/2 (Lopez-Perrote et al., 2012). Our dodecameric model of Rvb1/2 was based on the crystal structure of the hetero-dodecameric Rvb1/2 (Gorynia et al., 2011). In this structure, it was indicated that the two rings form a complex via the internal domain. But the electron densities of Gorynia et al. as well as of Matias et al. showed poor coverage in this region impeding a detailed analysis of interaction interphases (Gorynia et al., 2011; Matias et al., 2006). The compact packing of Rvb1/2 in INO80 would bring the internal domains 2 of the two opposing rings in such a close proximity principally enabling an interaction between them (Tosi et al., 2013). This would also explain why the deletion of the external OB-folds did not split the dodecameric version of Rvb1/2 (Niewiarowski et al., 2010).

In our model, the domain 2 of the same sort (Rvb1 or Rvb2) would interact with each other. This makes it likely that the hetero-hexameric composition promotes a dodecameric complex formation.

The six-fold axis in the head of INO80 enabled a placement of Rvb1/2 (Tosi et al., 2013). However, a closer inspection revealed that it is not a strict six-fold symmetry axis. The main deviation from a stringent six-fold symmetry was found in the region where the neck-body-foot cone is inserted. Such an insertion could be mediated by the insertion loop of Ino80 that was shown to recruit Rvb1/2 to SWR1 (Wu et al., 2005). In theory, if every Rvb1/2 pair in the dodecamer could bind an Ino80 insertion loop, three Ino80s could be inserted resulting in a tripod like structure. However, insertion at one site could influence the symmetry and the EM density indicated that the insertion of Ino80 resulted in an opening of the rings (Tosi et al., 2013). This would generate tension in the rings that may prevent further insertions of Ino80.

A similar hybrid approach as we performed on INO80 was applied on SWR1. SWR1 is more compact and the authors inferred that it contains a single hetero-hexameric ring (Nguyen et al., 2013). Both remodelers belong to the same family of remodelers, but SWR1 is composed of a different sub-set of subunits as INO80. The insertion in the split ATPase is a common structural feature with Ino80, albeit is not conserved on a sequence level. The insertion was shown to recruit the Rvb1/2 complex in SWR1 (Wu et al., 2005). Consistently, different insertion loops could assemble a different kind of Rvb1/2 species: a hexamer in the case of Swr1 and a dodecamer in the case of Ino80. However, non-GraFix treated samples from the study on SWR1 contained particles resembling a two layered Rvb1/2 particle (Nguyen et al., 2013). The author also quantified the intensity of the Rvb1 and 2 from a SDS-PAGE of SWR1 and INO80. The observed stoichiometry was consistent with a hexamer in both complexes. However, a similar quantifications of our highly purified and structural integer INO80 preparation resulted in ratios of 1:5.4:5.5 (Ino80:Rvb1:Rvb2), which is in agreement with the dodecamer observed in the EM map (Tosi et al., 2013). Remarkably, Nguyen et al. also found fractions containing Rvb1/2 and Swr1 with a stoichiometry of 6:6:1 in their glycerol gradient strongly indicating that the purified SWR1 was composed of hexamers and dodecamers (Nguyen et al., 2013). In general, protein staining methods are highly dependent on the amino acid composition, which is why a simple quantification of bands of a SDS-PAGE is not suitable for proper analysis of stoichiometry. Instead structural methods as analytical ultracentrifugation or native MS should be applied.

In Archaea only one form of Rvb1/2 exists indicating that two sorts of Rvb have developed during evolution, maybe by gene duplication. Rvb1 has approximately 12,000 copies per cell, whereas Rvb2 was found with 3,000 molecules (Ghaemmaghami et al., 2003). The higher copy number of Rvb1 suggests that it is also associated in other complexes independent of Rvb2.

In summary, we could show that in the INO80 complex Rvb1/2 form a hetero-dodecamer that is stacked via domains 2.