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The complete chaperone cycle of the prototypical Hsp70 chaperone, DnaK, has been worked on for over a decade and controversies still exist as to the order of events in the functional chaperone cycle (Genevaux et al., 2007; Swain et al., 2007). Armed with a multitude of conformational sensors positioned on the mitochondrial Hsp70, Ssc1, we were able to monitor the principal modes of conformational changes of Ssc1 in real time and also elucidate the order of events that lead to a productive chaperone cycle.

ATP-bound state is known to be the substrate acceptor state of Hsp70s with low affinity but high exchange rate for substrate proteins (Wittung-Stafshede et al., 2003; Zhu et al., 1996). In this work we were able to present convincing evidence that Ssc1, a bona-fide member of the Hsp70 group of chaperones, exist in a domain docked and lid open state in the ATP-bound form as hypothesized from numerous biochemical and biophysical evidences (Schmid et al., 1994; Swain et al., 2007; Zhu et al., 1996) or from the crystal structure of Hsc70 or Hsp110 (Jiang et al., 2005; Liu and Hendrickson, 2007), an Hsp70 homolog. This form is extremely homogeneous without any evidence of heterogeneity in the distances as probed by single-molecule fluorescence spectroscopy. On the contrary,

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the ADP bound form was found to be more heterogenous in respect to the interdomain distance and distance of lid domain to the base of the PBD.

Substrate was found to efficiently bind to Ssc1 in the presence of its J-domain co- chaperone Mdj1 reconfirming the previous reports on the essentiality of J-domain proteins in assisting substrate binding (Karzai and McMacken, 1996; Laufen et al., 1999; Liberek et al., 1991; Wittung-Stafshede et al., 2003). The binding of the substrate peptide, P5, to Ssc1 in presence of Mdj1 led to the formation of a lid-closed and domain undocked structure. Binding of Mdj1 to Ssc1-ATP complex led to the transient formation of lid-closed, domain-undocked conformation, followed by the release of Mdj1 to form a heterogeneous population of Ssc1 molecules which was extremely similar to the conformational distribution of Ssc1-ADP complex, suggesting the possible effect of Mdj1 on Ssc1 conformation to be driven solely by ATP-hydrolysis. Bimodal distribution of the inter-domain distance of Ssc1 for Ssc1/ADP indicates that Ssc1 might be a dynamic molecule in this state, fluctuating between the domain-docked and the undocked state. Contrary to observations made with DnaK (Chang et al., 2008; Swain et al., 2007), we found that that ADP binding to Ssc1 is not able to efficiently undock the domains or lock the lid onto the base of the PBD which was affected efficiently only upon substrate binding. This indicates that substrate binding to Ssc1 is essential to bring about conformational changes and is corroborative with previous studies that examined the effect of substrate binding to isolated domains of DnaK (Tanaka et al., 2005).

Even though it is well understood that binding of J-domain co-chaperone is essential for substrate capture by Hsp70s, the timing of the exit of this J-domain protein in the chaperone cycle is unknown. We were able to develop a FRET based sensor for Mdj1- Ssc1 interaction and reproduce the reported observation that J-domain co-chaperones bind much faster to Hsp70s in the ATP-bound state than in the ADP-bound state (Mayer et al., 1999). Using the same sensors we observed that Mdj1 does not dissociate from the Ssc1/P5/Mdj1/ATP complex soon after hydrolysis of ATP. Even though Mdj1 was found to dissociate from Ssc1/P5, the off-rate measured was extremely slow to account for the functional chaperone cycle. Interestingly, it was observed that substrate can modulate the interaction between Ssc1 and Mdj1, with Mdj1 having a higher off-rate from Ssc1 in presence of the substrate Mge1, the bona fide nucleotide exchange factor of

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Ssc1, could bind to Ssc1 only in the ADP bound state of Ssc1. It was also found that after the exogenous ATP was depleted, Mge1 could form a quaternary complex, Ssc1/P5/Mdj1/Mge1, which dissociated upon addition of ATP. This is consistent with a model of the cycle in which the cycle ends with the binding of the exchange factor to the ternary complex of Hsp70, substrate and J-domain co-chaperone.

Exchange factor assisted ATP-binding to Hsp70 is known to restart the cycle by releasing bound substrates (Brehmer et al., 2001; Harrison et al., 1997), however the sequence of release of the exchange factor and the substrate is not well studied. We were able to show that addition of ATP to Ssc1/P5/Mdj1/Mge1 leads to an instantaneous release of Mge1 from the complex followed by lid opening and domain-docking. This further leads to the dissociation of Mdj1 and P5 from Ssc1. In this work we were able to discern the dissociation of the J-domain protein from Hsp70 in a time resolved manner and show that contrary to some previously held convention (Genevaux et al., 2007), the J-domain co- chaperone dissociates from the Hsp70 at the end of the cycle simultaneous to substrate dissociation. This sequence of events restarts the cycle of Ssc1 which comes back to the substrate-acceptor state. The re-binding rate of a substrate would be dependent on the association rates and the concentrations of Ssc1, substrate and Mdj1. The rebinding rate in turn would dictate the time scale a substrate remains free in solution to be partitioned into folding competent state. Highly hydrophobic stretches, present at high concentrations, would have an inherent tendency to aggregate and since the Ssc1-binding rate for these would also be high, they would spend lesser time unbound from Ssc1 in an aggregation competent state. Based on all the results obtained, a comprehensive model for Ssc1 chaperone cycle in the mitochondrial matrix is summarized (Fig.4.1).

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In this study the comparison between DnaK and Ssc1 reveals interesting new insights into the conformational differences between the two chaperones. In presence of ADP, the conformation of Ssc1 is highly heterogeneous implying that the domain connectivity might be dynamic under these conditions, whereas in the case of DnaK the domains are fully disjoined. Residual domain connectivity in Ssc1-ADP state suggests that although ATP is needed in DnaK to facilitate physical contact and hence allosteric signaling between the two domains, it might not be an essential feature in Ssc1 and in general of the Hsp70 chaperones.

Figure 4.1: A comprehensive model of the Ssc1 chaperone cycle in the mitochondrial matrix. (1).

Ssc1 in the ATP-bound state (domain-docked, lid-open state) interacts with the substrate via its PBD with low affinity which becomes effective only in presence of Mdj1. (2). Binding of Mdj1 and substrate and acceleration of hydrolysis of ATP by Mdj1 and substrate closes the PBD leading to high affinity binding of the substrate forming a ternary complex of Ssc1/Mdj1/substrate. (3). Mge1 can bind to this ternary complex in absence of an exogenous ATP. (4). Addition of ATP to the quaternary complex leads to instantaneous exchange of ADP with the ATP in the NBD of Ssc1 with release of Mge1. (5 & 6).

Subsequently, ATP induced conformational changes of Ssc1 take place followed by near simultaneous dissociation of substrate and Mdj1 from Ssc1 in the ATP-state which starts a new cycle.

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