Acid-denatured small heat shock protein HdeA from Escherichia coli forms reversible fibrils with an atypical secondary structure

Abstract

The periplasmic small heat shock protein HdeA from Escherichia coli is inactive under normal growth conditions (at pH 7) and activated only when E. coli cells are subjected to a sudden decrease in pH, converting HdeA into an acid-denatured active state. Here, using in vitro fibrillation assays, transmission EM, atomic-force microscopy, and CD analyses, we found that when HdeA is active as a molecular chaperone, it is also capable of forming inactive aggregates that, at first glance, resemble amyloid fibrils. We noted that the molecular chaperone activity of HdeA takes precedence over fibrillogenesis under acidic conditions, as the presence of denatured substrate protein was sufficient to suppress HdeA fibril formation. Further experiments suggested that the secondary structure of HdeA fibrils deviates somewhat from typical amyloid fibrils and contains α-helices. Strikingly, HdeA fibrils that formed at pH 2 were immediately resolubilized by a simple shift to pH 7 and from there could regain molecular chaperone activity upon a return to pH 1. HdeA, therefore, provides an unusual example of a “reversible” form of protein fibrillation with an atypical secondary structure composition. The competition between active assistance of denatured polypeptides (its “molecular chaperone” activity) and the formation of inactive fibrillary deposits (its “fibrillogenic” activity) provides a unique opportunity to probe the relationship among protein function, structure, and aggregation in detail.

Formation of Fibrils by the Periplasmic Molecular Chaperone HdeB from Escherichia coli

“…The specific pH where fibrillation was observed (pH < 3, Figure 1a) corresponded to conditions where the HdeB dimer denatures and dissociates into monomers, and is inactive as a molecular chaperone [5,12]. The pH range at which fibrillation was observed was similar to the conditions where HdeA tended to form fibrils, as determined in a previous study [13]. Functionally, comparing the characteristics of HdeA and HdeB fibrillation is interesting, since evidence suggests that for HdeA, there might be a certain amount of overlap between conformations that are important for molecular chaperone activity and conformations that were prone to form fibrils, resulting in a competitive mechanism of sorts.…”

“…The dynamic and unstable nature of the active acid denatured form of HdeA prompted us to consider if this state could be capable of forming alternate structures under the same low pH conditions. We subsequently performed in vitro experiments which demonstrated that acid denatured HdeA, upon mild shaking, could form regular fibrillar aggregates that were very similar to the amyloid fibrils that could be observed in cells of patients afflicted with neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease [ 13 ]. The fibril form of HdeA, however, displayed two unusual characteristics that are not generally seen in the fibrils associated with the above diseases; first, HdeA fibrils that are formed at low pH readily resolubilize and revert to the original soluble native dimer upon a simple shift to neutral pH, and second, HdeA fibrils retained a significant fraction of α-helical content, suggestive of a partial preservation of the original secondary structure.…”

Stress-Responsive Periplasmic Chaperones in Bacteria

“…In addition, SurA or DegP assists HdeA in refolding acid-denatured alkaline phosphatase in vitro, suggesting that HdeA protects chaperones, such as SurA and DegP, and subsequently enables these chaperones to participate in the refolding of substrate proteins that are dissociated from HdeA following the transition to neutral pH (Zhang et al, 2011). Fibrillation of HdeA at pH 2 has been observed in a recent study, and these fibrils can be resolubilized following a shift to pH 7 (Miyawaki et al, 2019). This unusual reversibility of fibrillation for HdeA suggests this is another pH-dependent regulatory mechanism for HdeA.…”

Amyloidogenic Propensities of Ribosomal S1 Proteins: Bioinformatics Screening and Experimental Checking

“…Ribosomal S1 protein acts as an RNA chaperone to unfold structured mRNA on the ribosome, which was studied at approximately neutral pH values of 7.5 [ 19 ]. Interestingly, changing the pH from neutral (pH 7) to acidic (pH 1–2) can switch “molecular chaperone” activity to “fibrillogenic” activity as shown for the heat shock protein HdeA from E. coli [ 20 ]. The change in pH from 7 to 2 did not activate fibrillation for the S1 protein (unpublished data), but as shown in the study, it affected the fibrillogenesis of peptides from the sequence of S1 from E. coli and T. thermophilus .…”