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Structural basis and mechanism of the unfolding-induced activation of HdeA, a bacterial acid response chaperone.

ABSTRACT: The role of protein structural disorder in biological functions has gained increasing attention in the past decade. The bacterial acid-resistant chaperone HdeA belongs to a group of "conditionally disordered" proteins, because it is inactive in its well-structured state and becomes activated via an order-to-disorder transition under acid stress. However, the mechanism for unfolding-induced activation remains unclear because of a lack of experimental information on the unfolded state conformation and the chaperone-client interactions. Herein, we used advanced solution NMR methods to characterize the activated-state conformation of HdeA under acidic conditions and identify its client-binding sites. We observed that the structure of activated HdeA becomes largely disordered and exposes two hydrophobic patches essential for client interactions. Furthermore, using the pH-dependent chemical exchange saturation transfer (CEST) NMR method, we identified three acid-sensitive regions that act as structural locks in regulating the exposure of the two client-binding sites during the activation process, revealing a multistep activation mechanism of HdeA's chaperone function at the atomic level. Our results highlight the role of intrinsic protein disorder in chaperone function and the self-inhibitory role of ordered structures under nonstress conditions, offering new insights for improving our understanding of protein structure-function paradigms.

SUBMITTER: Yu XC

PROVIDER: S-EPMC6398119 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The Mechanism of HdeA Unfolding and Chaperone Activation.

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NMR-monitored titration of acid-stress bacterial chaperone HdeA reveals that Asp and Glu charge neutralization produces a loosened dimer structure in preparation for protein unfolding and chaperone activation.

Project description:HdeA is a periplasmic chaperone found in several gram-negative pathogenic bacteria that are linked to millions of cases of dysentery per year worldwide. After the protein becomes activated at low pH, it can bind to other periplasmic proteins, protecting them from aggregation when the bacteria travel through the stomach on their way to colonize the intestines. It has been argued that one of the major driving forces for HdeA activation is the protonation of aspartate and glutamate side chains. The goal for this study, therefore, was to investigate, at the atomic level, the structural impact of this charge neutralization on HdeA during the transition from near-neutral conditions to pH 3.0, in preparation for unfolding and activation of its chaperone capabilities. NMR spectroscopy was used to measure pKa values of Asp and Glu residues and monitor chemical shift changes. Measurements of R2/R1 ratios from relaxation experiments confirm that the protein maintains its dimer structure between pH 6.0 and 3.0. However, calculated correlation times and changes in amide protection from hydrogen/deuterium exchange experiments provide evidence for a loosening of the tertiary and quaternary structures of HdeA; in particular, the data indicate that the dimer structure becomes progressively weakened as the pH decreases. Taken together, these results provide insight into the process by which HdeA is primed to unfold and carry out its chaperone duties below pH 3.0, and it also demonstrates that neutralization of aspartate and glutamate residues is not likely to be the sole trigger for HdeA dissociation and unfolding.

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Project description:HdeA is a small acid-stress chaperone protein with a unique activity profile. At physiological pH, it forms a folded, but inactive, dimer. Below pH 3.0, HdeA unfolds and dissociates into disordered monomers, utilizing exposed hydrophobic patches to bind other unfolded proteins and prevent their irreversible aggregation. In this way, HdeA has a key role in helping pathogenic bacteria survive our acidic stomach and colonize our intestines, facilitating the spread of dysentery. Despite numerous publications on the topic, there remain questions about the mechanism by which HdeA unfolding and activation are triggered. Previous studies usually assessed HdeA unfolding over pH increments that are too far apart to gain fine detail of the process of unfolding and dimer dissociation, and often employed techniques that prevented thorough evaluation of specific regions of the protein. We used a variety of heteronuclear NMR experiments to investigate changes to backbone and side chain structure and dynamics of HdeA at four pHs between 3.0 and 2.0. We found that the long loop in the dimer interface is an early site of initiation of dimer dissociation, and that a molecular "clasp" near the disulfide bond is broken open at low pH as part, or as a trigger, of unfolding; this process also results in the separation of C-terminal helices and exposure of key hydrophobic client binding sites. Our results highlight important regions of HdeA that may have previously been overlooked because they lie too close to the disulfide bond or are thought to be too dynamic in the folded state to influence unfolding processes.

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Project description:The small pH stress-sensing chaperone HdeA helps pathogenic enteric E. coli survive passage through the severely acidic environment of the mammalian stomach. Under stress conditions, HdeA transitions from an inactive folded dimer to a chaperone-active unfolded monomer to prevent the acid-induced aggregation of periplasmic proteins. Here we use a topology-based Gō-like model to delineate the relationship between dimer interface formation and monomer folding and to better understand the structural details of the chaperone activation mechanism. Free energy surfaces show that dimer interface formation and monomer folding proceed concurrently through an on-pathway dimeric intermediate in which one monomer is partially unfolded. The absence of a preexisting fully folded monomer in the proposed binding mechanism is in agreement with HdeA's rapid chaperone response. Binding between unfolded monomers exhibits an enhancement of molecular recognition reminiscent of the fly-casting mechanism. Overall, our simulations further highlight the efficient nature of HdeA's chaperone response and we anticipate that knowledge of a dimeric intermediate will facilitate the interpretation of experimental studies.

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