Abstract
Grp170 and Hsp110 proteins constitute two evolutionary distinct branches of the Hsp70 family that share the ability to function as nucleotide exchange factors (NEFs) for canonical Hsp70s. Although the NEF mechanism of the cytoplasmic Hsp110s is well understood, little is known regarding the mechanism used by Grp170s in the endoplasmic reticulum. In this study, we compare the yeast Grp170 Lhs1 with the yeast Hsp110 Sse1. We find that residues important for Sse1 NEF activity are conserved in Lhs1 and that mutations in these residues in Lhs1 compromise NEF activity. As previously reported for Sse1, Lhs1 requires ATP to trigger nucleotide exchange in its cognate Hsp70 partner Kar2. Using site-specific cross-linking, we show that the nucleotide-binding domain (NBD) of Lhs1 interacts with the NBD of Kar2 face to face, and that Lhs1 contacts the side of the Kar2 NBD via its protruding C-terminal alpha-helical domain. To directly address the mechanism of nucleotide exchange, we have compared the hydrogen-exchange characteristics of a yeast Hsp70 NBD (Ssa1) in complex with either Sse1 or Lhs1. We find that Lhs1 and Sse1 induce very similar changes in the conformational dynamics in the Hsp70. Thus, our findings demonstrate that despite some differences between Hsp110 and Grp170 proteins, they use a similar mechanism to trigger nucleotide exchange.
Highlights
Minal nucleotide-binding domain (NBD),4 interdomain communication ensures that the C-terminal substrate-binding domain (SBD) exhibits low affinity for substrates, which bind and release with high rates
We hypothesized that Grp170s shared SBD subdomain structure with Hsp110s and tested this hypothesis by employing local instead of global sequence alignments of the SBD sequences of Lhs1 and Sse1
We find that Lhs1 carries a putative flexible loop structure right before the last -sheet of SBD (Fig. 1A, L), a motif shared with Hsp110s that is not present in Hsp70s
Summary
Minal nucleotide-binding domain (NBD),4 interdomain communication ensures that the C-terminal substrate-binding domain (SBD) exhibits low affinity for substrates, which bind and release with high rates. Our in vivo and in vitro mutational analysis suggests that Lhs1 depends on similar residues as Sse1 for its NEF activity. The residual NEF activity of Lhs1–2 (in SBD␣) and Lhs1– 4 (in NBD) is expected if Lhs1, like Sse1, employs a large surface with multiple contact points to interact with the Hsp70 [8].
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