The folding pathways of large proteins are complex, with many of them requiring the aid of chaperones and others folding spontaneously. Along the folding pathways, partially folded intermediates are frequently populated; their role in the driving of the folding process is unclear. The structures of these intermediates are generally not amenable to high-resolution structural techniques because of their transient nature. Here we employed single-molecule force measurements to scrutinize the hierarchy of intermediate structures along the folding pathway of the nucleotide binding domain (NBD) of Hsp70 DnaK. DnaK-NBD is a member of the sugar kinase superfamily that includes Hsp70s and the cytoskeletal protein actin. Using optical tweezers, a stable nucleotide-binding competent folding intermediate comprising lobe II residues (183-383) was identified as a critical checkpoint for productive folding. We obtained a structural snapshot of this folding intermediate that shows native-like conformation. To assess the fundamental role of folded lobe II for efficient folding, we turned our attention to yeast mitochondrial NBD, which does not fold without a dedicated chaperone. After replacing the yeast lobe II residues with stable lobe II, the obtained chimeric protein showed native-like ATPase activity and robust folding into the native state, even in the absence of chaperone. In summary, lobe II is a stable nucleotide-binding competent folding nucleus that is the key to time-efficient folding and possibly resembles a common ancestor domain. Our findings provide a conceptual framework for the folding pathways of other members of this protein superfamily.
Single-molecule mechanical experiments
have proven to be ideal
tools for probing the energetics and mechanics of large proteins and
domains. In this paper, we investigate the nucleotide-dependent unfolding
mechanics of the nucleotide-binding domain (NBD) of the Hsp70 chaperone
DnaK. The NBD binds ADP or ATP in the binding cleft formed by lobe
I and lobe II, which consists of two subdomains each. When force is
applied to the termini of the NBD, the observed unfolding forces are
independent of the nucleotide state. In contrast, when force is applied
across the nucleotide-binding pocket, the unfolding forces report
specifically on the nucleotide–phosphate state. In this active,
ligand-responsive pulling geometry, we observed a bifurcation of the
unfolding pathway; the pathway proceeds either through a cooperative
“coupled pathway” or through a noncooperative “uncoupled
pathway”. The partitioning between individual unfolding pathways
can be effectively tuned by mutation or by the nucleotide exchange
factor GrpE, i.e., by the factors affecting the strength of the lobe
I–lobe II interactions within the native NBD. These experiments
provide important insight into the molecular origin of the internal
signaling between the subdomains of the nucleotide-binding domain
of Hsp70 proteins and how signals are efficiently transferred inside
the protein molecule.
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