Dimerization of a 5-kDa domain defines the architecture of the 5-MDa gammaproteobacterial pyruvate dehydrogenase complex.

The Escherichia coli pyruvate dehydrogenase complex (PDHc) is a ~5 MDa assembly of the catalytic subunits pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). The PDHc core is a cubic complex of eight E2 homotrimers. Homodimers of the peripheral subunits E1 and E3 associate with the core by binding to the peripheral subunit binding domain (PSBD) of E2. Previous reports indicated that 12 E1 dimers and 6 E3 dimers bind to the 24-meric E2 core. Using an assembly arrested E2 homotrimer (E23), we show that two of the three PSBDs in the E23 dimerize, that each PSBD dimer cooperatively binds two E1 dimers, and that E3 dimers only bind to the unpaired PSBD in E23. This mechanism is preserved in wild-type PDHc, with an E1 dimer:E2 monomer:E3 dimer stoichiometry of 16:24:8. The conserved PSBD dimer interface indicates that PSBD dimerization is the previously unrecognized architectural determinant of gammaproteobacterial PDHc megacomplexes.

An Active, Ligand-Responsive Pulling Geometry Reports on Internal Signaling between Subdomains of the DnaK Nucleotide-Binding Domain in Single-Molecule Mechanical Experiments.

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.

A folding nucleus and minimal ATP binding domain of Hsp70 identified by single-molecule force spectroscopy.

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 Escherichia coli 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 en route 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 E. coli 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.