Activation Of HdeA Research Articles

Characterizations of the Interactions between Escherichia coli Periplasmic Chaperone HdeA and Its Native Substrates during Acid Stress.

The bacterial acid-resistant chaperone HdeA is a "conditionally disordered" protein that functions at low pH when it undergoes a transition from a well-folded dimer to an unfolded monomer. The dimer dissociation and unfolding processes result in exposure of hydrophobic surfaces that allows binding to a broad range of client proteins. To fully elucidate the chaperone mechanism of HdeA, it is crucial to understand how the activated HdeA interacts with its native substrates during acid stress. Herein, we present a nuclear magnetic resonance study of the pH-dependent HdeA-substrate interactions. Our results show that the activation of HdeA is not only induced by acidification but also regulated by the presence of unfolded substrates. The variable extent of unfolding of substrates differentially regulates the HdeA-substrate interaction, and the binding further affects the HdeA conformation. Finally, we show that HdeA binds its substrates heterogeneously, and the "amphiphilic" model for HdeA-substrate interaction is discussed.

HdeB Functions as an Acid-protective Chaperone in Bacteria

Enteric bacteria such as Escherichia coli utilize various acid response systems to counteract the acidic environment of the mammalian stomach. To protect their periplasmic proteome against rapid acid-mediated damage, bacteria contain the acid-activated periplasmic chaperones HdeA and HdeB. Activation of HdeA at pH 2 was shown to correlate with its acid-induced dissociation into partially unfolded monomers. In contrast, HdeB, which has high structural similarities to HdeA, shows negligible chaperone activity at pH 2 and only modest chaperone activity at pH 3. These results raised intriguing questions concerning the physiological role of HdeB in bacteria, its activation mechanism, and the structural requirements for its function as a molecular chaperone. In this study, we conducted structural and biochemical studies that revealed that HdeB indeed works as an effective molecular chaperone. However, in contrast to HdeA, whose chaperone function is optimal at pH 2, the chaperone function of HdeB is optimal at pH 4, at which HdeB is still fully dimeric and largely folded. NMR, analytical ultracentrifugation, and fluorescence studies suggest that the highly dynamic nature of HdeB at pH 4 alleviates the need for monomerization and partial unfolding. Once activated, HdeB binds various unfolding client proteins, prevents their aggregation, and supports their refolding upon subsequent neutralization. Overexpression of HdeA promotes bacterial survival at pH 2 and 3, whereas overexpression of HdeB positively affects bacterial growth at pH 4. These studies demonstrate how two structurally homologous proteins with seemingly identical in vivo functions have evolved to provide bacteria with the means for surviving a range of acidic protein-unfolding conditions.

Structural Studies on the Activation and Substrate Binding of a Conditionally Disordered Acid-Activated Chaperone

HdeA is a 9.7kDa chaperone that is inactive at neutral pH but becomes activated and disordered by shifts to low pH. The chaperone is required for bacterial pathogens to resist the acid-mediated protein aggregation that would otherwise occur in the human gut. We sought to identify which of HdeA's acid-titratable residues were the key players in its activation. Using constant pH molecular dynamics calculations and site-specific mutagenesis, we identified several residues involved in HdeA activation and have isolated variants that are destabilized and constitutively active at even neutral pH. These mutants help us to understand how pH-driven changes in HdeA flexibility drive activation.One of the pressing problems in chaperone biology is how chaperones interact with a multitude of client proteins to facilitate their folding. HdeA's size and accessibility to NMR also makes it ideal for monitoring the structural changes that take place in this chaperone upon activation and upon client binding. Our predicted NMR structure of HdeA at neutral pH is a folded protein and very similar the crystal structures previously solved for the HdeA dimer. Upon shift to low pH however, HdeA simultaneously becomes active as a chaperone and acquires a largely disordered conformation. We have obtained structural information that gives us insights into HdeA's activation process and client binding. The use of small NMR accessible client proteins raises the exciting possibility of monitoring the structural changes that take place within the client upon interaction with the chaperone and in doing so gaining insight into the fundamental question of what chaperones do to their clients to facilitate folding.