Discussion of HX-MS data

This study revealed new insights into both, the conformational dynamics and the complex interface of RAC. Although Zuo and Ssz are members of the Hsp40 and Hsp70 chaperone family, respectively, they display a number of atypical features compared with their canonical homologs. In particular, the formation of a stable Hps70/40 complex is unique and was yet not understood.

The subunits Ssz and Zuo were found to be highly dynamic individually, but are strongly stabilized upon complex formation.

Ssz is stabilized both, by ATP binding and by Zuo binding. Complex formation affects mostly the SBD whereas binding of ATP to Ssz leads to a compaction of the ATPase domain. This suggests that the respective ligands affect distinct and different regions within Ssz. ATP stabilized the ATPase domain of Ssz when uncomplexed and also when in complex with Zuo. This was not the case for Zuo; ATP binding in Ssz seems not to be recognized by Zuo. Therefore distinct functions of the binding of Zuo and ATP to Ssz can be concluded.

The pattern of nucleotide-induced HX protection in Ssz differs from those observed for classical Hsp70s, such as DnaK (Rist et al., 2006; Andreasson et al., 2008a,b). The

3.2. HX-MS analysis of conformational alterations in RAC upon complex formation stabilization of the ATPase domain due to ATP binding is more extensive in Ssz and

restricted almost exclusively to lobe II of the N-terminal domain of Ssz. Deprotection of segments in the potential substrate binding domain does not occur. Indeed Ssz does not show conformational changes which are characteristic for canonical Hsp70 chaperones upon nucleotide binding. This is in agreement with other findings which indicate that Ssz is distinct from other classical Hsp70 chaperone: (I) its substrate binding domain is shorter than that of other family members (Gautschi et al., 2001), pointing towards a somewhat different functionality, (II) so far no substrate binding could be shown (Huang et al., 2005) and (III) Ssz does not efficiently hydrolyze ATP (Conz et al., 2007). Instead, ATP binding of Ssz appears to be transient, showing a stabilizing effect on Ssz only for short HX incubation times up to 2 minutes. However, upon complex formation, the stabilizing effect of ATP on Ssz is noticeably prolonged and persists even after 2 h of HX incubation. This could be either due to a longer remaining of ATP in the binding pocket, triggered by some minor conformational changes within Ssz upon complex formation. Alternatively, the conformation might be locked by ATP binding upon complex formation and persists even after release of ATP from the complex. In both cases, ATP binding is important to reach a maximal compaction of the complex. However, it still remains unclear whether the nucleotide induced stabilization is of any importance. It was not possible to specifically test whether ATP binding is required for complex formation, because Ssz was too unstable in a nucleotide-free state. However, recent findings showed that a Ssz mutant deficient in ATP binding can complement the phenotype of ssz∆ cells in vivo, suggesting that the assembly of a functional RAC can occur even in the absence of bound nucleotide (Conz et al., 2007).

Binding of Zuo to Ssz resulted in major structural and dynamic changes in most of the SBD of Ssz. The observed stabilization might result from (I) direct involvement of this region in the complex interface or (II) from conformational changes induced indirectly by complex formation or both. Both scenarios suggest an important role of this domain in complex formation. This is in contrast to classical Hsp70 chaperones, where the SBD is dedicated to substrate binding. Another example of such diver-gence was reported for yeast Sse. Although Sse shares structural elements with Hsp70 chaperones, it functions as a nucleotide exchange factor and uses its extended SBD to embrace its partner Hsp70 (Schuermann et al., 2008; Polier et al., 2008).

Besides the major structural changes in the SBD of Ssz, a second region (segments 171-179 and 209-216 of the ATPase domain) was affected by Zuo binding. Sequence alignments with known Hsp70 structures showed that these segments are located at the intersection of lobe I and lobe II. Lobe Ia is thought to be involved in docking the SBD onto the ATPase domain in Hsp70 chaperones. Therefore it could be possible, (I) that binding of Zuo to the SBD of Ssz induces interdomain docking or (II) Zuo not only directly contacts the SBD of Ssz, but the ATPase domain as well. These interactions could be responsible for the changed ATP binding behavior of Ssz in complex as compared to Ssz alone. Binding of Zuo might induce a conformational change within Ssz which prevents release of ATP or might lock the conformation of the ATP bound state of Ssz even in the absence of the nucleotide.

Zuotin is stabilized by Ssz binding only, addition of ATP had no effect. A pro-nounced stabilization in the N-terminal region encompassing aa 1 - 52 was found, which appeared to be extremely flexible in uncomplexed Zuo. The N-terminal exten-sion (aa 1 - 102) is a specific feature of Zuotin-like proteins, supporting the assumption that this part is involved in complex formation. However, a second part within the N-terminus, spanning aa 53 - 79 remains surprisingly flexible, even upon complex formation. This implies a linker like function, disconnecting the complex formation binding site from the functional J-domain and thereby providing a high flexibility for the J-domain to successfully stimulate the ATPase activity of Ssb. A second region within Zuo was affected by complex formation. Segments belonging to the J-domain (aa 99 - 168) of Zuo showed an increase in dynamics upon complex formation. The J-domain is responsible for interaction with Ssb and stimulation of its ATPase activity.

Zuo alone only marginally stimulates Ssb’s ATPase activity (Huang et al., 2005), only in complex with Ssz, Zuo can fully stimulate the ATPase activity of Ssb. Thus it is likely that the increase in dynamics of the J-domain upon complex formation is part of an activation process of Zuo.