Protein refolding by pH-triggered chaperone binding and release.

Molecular chaperones are typically either adenosine triphosphate (ATP) dependent or rely heavily on their ATP-dependent chaperone counterparts in order to promote protein folding. This presents a challenge to chaperones that are localized to ATP-deficient cellular compartments. Here we describe a mechanism by which the pH-regulated acid stress chaperone HdeA is capable of independently facilitating the refolding of acid-denatured proteins in the bacterial periplasm, which lacks both ATP and ATP-dependent chaperone machines. Our results are consistent with a model in which HdeA stably binds substrates at low pH, thereby preventing their irreversible aggregation. pH neutralization subsequently triggers the slow release of substrate proteins from HdeA, keeping the concentration of aggregation-sensitive intermediates below the threshold where they begin to aggregate. This provides a straightforward and ATP-independent mechanism that allows HdeA to facilitate protein refolding. Unlike previously characterized chaperones, HdeA appears to facilitate protein folding by using a single substrate binding-release cycle. This cycle is entirely regulated by the external environment and is therefore energy-neutral for the bacteria.

Structural plasticity of an acid-activated chaperone allows promiscuous substrate binding.

HdeA has been shown to prevent acid-induced aggregation of proteins. With a mass of only 9.7 kDa, HdeA is one of the smallest chaperones known. Unlike other molecular chaperones, which are typically complex, multimeric ATP-dependent machines, HdeA is known to undergo an acid-induced dimer to monomer transition and functions at low pH as a disordered monomer without the need for energy factors. Thus, HdeA must possess features that allow it to bind substrates and regulate substrate affinity in a small and energy-independent package. To understand better how HdeA accomplishes this, we studied the conformational changes that accompany a shift to low pH and substrate binding. We find that the acid-induced partial unfolding and monomerization that lead to HdeA activation occur very rapidly (k >3.5 s(-1)). Activation exposes the hydrophobic dimer interface, which we found to be critical for substrate binding. We show by intramolecular FRET that the partially unfolded character of active HdeA allows the chaperone to adopt different conformations as required for the recognition and high-affinity binding of different substrate proteins. These efficient adaptations help to explain how a very small protein is rapidly activated and can bind a broad range of substrate proteins in a purely pH-regulated manner.

Kinetic characterization of the disulfide bond-forming enzyme DsbB.

DsbB is an integral membrane protein responsible for the de novo synthesis of disulfide bonds in Escherichia coli and many other prokaryotes. In the process of transferring electrons from DsbA to a tightly bound ubiquinone cofactor, DsbB undergoes an unusual spectral transition at approximately 510 nm. We have utilized this spectral transition to study the kinetic cycle of DsbB in detail using stopped flow methods. We show that upon mixing of Dsb-B(ox) and DsbA(red), there is a rapid increase in absorbance at 510 nm (giving rise to a purple solution), followed by two slower decay phases. The rate of the initial phase is highly dependent upon DsbA concentration (k(1) approximately 5 x 10(5) M(-1) s(-1)), suggesting this phase reflects the rate of DsbA binding. The rates of the subsequent decay phases are independent of DsbA concentration (k(2) approximately 2 s(-1); k(3) approximately 0.3 s(-1)), indicative of intramolecular reaction steps. Absorbance measurements at 275 nm suggest that k(2) and k(3) are associated with steps of quinone reduction. The rate of DsbA oxidation was found to be the same as the rate of quinone reduction, suggestive of a highly concerted reaction. The concerted nature of the reaction may explain why previous efforts to dissect the reaction mechanism of DsbB by examining individual pairs of cysteines yielded seemingly paradoxical results. Order of mixing experiments showed that the quinone must be pre-bound to DsbB to observe the purple intermediate as well as for efficient quinone reduction. These results are consistent with a kinetic model for DsbB action in which DsbA binding is followed by a rapid disulfide exchange event. This is followed by quinone reduction, which is rate-limiting in the overall reaction cycle.

Structural determinants of HscA peptide-binding specificity.

The Hsp70-class molecular chaperone HscA interacts specifically with a conserved (99)LPPVK(103) motif of the iron-sulfur cluster scaffold protein IscU. We used a cellulose-bound peptide array to perform single-site saturation substitution of peptide residues corresponding to Glu(98)-Ile(104) of IscU to determine positional amino acid requirements for recognition by HscA. Two mutant chaperone forms, HscA(F426A) with a DnaK-like arch structure and HscA(M433V) with a DnaK-like substrate-binding pocket, were also studied. Wild-type HscA and HscA(F426A) exhibited a strict preference for proline in the central peptide position (ELPPVKI), whereas HscA(M433V) bound a peptide containing a Pro-->Leu substitution at this location (ELPLVKI). Contributions of Phe(426) and Met(433) to HscA peptide specificity were further tested in solution using a fluorescence-based peptide-binding assay. Bimane-labeled HscA and HscA(F426A) bound ELPPVKI peptides with higher affinity than leucine-substituted peptides, whereas HscA(M433V) favored binding of ELPLVKI peptides. Fluorescence-binding studies were also carried out with derivatives of the peptide NRLLLTG, a model substrate for DnaK. HscA and HscA(F426A) bound NRLLLTG peptides weakly, whereas HscA(M433V) bound NRLLLTG peptides with higher affinity than IscU-derived peptides ELPPVKI and ELPLVKI. These results suggest that the specificity of HscA for the LPPVK recognition sequence is determined in part by steric obstruction of the hydrophobic binding pocket by Met(433) and that substitution with the Val(433) sidechain imparts a broader, more DnaK-like, substrate recognition pattern.