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 (original) (raw)
Related papers
The Mechanism of HdeA Unfolding and Chaperone Activation
Journal of molecular biology, 2018
HdeA is a periplasmic chaperone that is rapidly activated upon shifting the pH to acidic conditions. This activation is thought to involve monomerization of HdeA. There is evidence that monomerization and partial unfolding allow the chaperone to bind to proteins denatured by low pH, thereby protecting them from aggregation. We analyzed the acid-induced unfolding of HdeA using NMR spectroscopy and fluorescence measurements, and obtained experimental evidence suggesting a complex mechanism in HdeA's acid-induced unfolding pathway, as previously postulated from molecular dynamics simulations. Counterintuitively, dissociation constant measurements show a stabilization of the HdeA dimer upon exposure to mildly acidic conditions. We provide experimental evidence that protonation of Glu37, a glutamate residue embedded in a hydrophobic pocket of HdeA, is important in controlling HdeA stabilization and thus the acid activation of this chaperone. Our data also reveal a sharp transition fr...
Biophysical Chemistry, 2020
HdeA is a small acid-stress chaperone protein found in the periplasm of several pathogenic gramnegative bacteria. In neutral pH environments HdeA is an inactive folded homodimer but when exposed to strong acidic environments it partially unfolds and, once activated, binds to other periplasmic proteins, protecting them from irreversible aggregation. Here we use a combination of hydrogen/deuterium exchange NMR experiments and constant pH molecular dynamics simulations to elucidate the role of HdeA's N-terminus in its activation mechanism. Previous work indicates that the N-terminus is flexible and unprotected at high pH while exhibiting interactions with some HdeA client binding site residues. It, however, becomes partially solvent-protected at pH 2.6-2.8 and then loses protection again at pH 2.0. This protection is not due to the appearance of new secondary structure, but rather increased contacts between N-terminal residues and the Cterminus of the other protomer in the dimer, as well as concurrent loosening of its hold on the client binding site residues, priming HdeA for interactions with periplasmic client proteins. This work also uncovers unusual protonation profiles of some titratable residues and suggests a complex role in chaperone function.
bioRxiv (Cold Spring Harbor Laboratory), 2021
HdeA is an acid-stress chaperone that operates in the periplasm of various strains of pathogenic gramnegative bacteria. Its primary function is to prevent irreversible aggregation of other periplasmic proteins when the bacteria enter the acidic environment of the stomach after contaminated food is ingested; its role is therefore to help the bacteria survive long enough to enter and infect the intestines. The mechanism of operation of HdeA is unusual in that this helical homodimer is inactive when folded at neutral pH but becomes activated at low pH after the dimer dissociates and becomes partially unfolded. Studies with chemical reducing agents have previously suggested that the intramolecular disulfide bond of HdeA aids in the maintenance of some residual structure at low pH; it is believed that this residual structure is important for clustering exposed hydrophobic residues together for the purpose of binding unfolded client proteins. In order to explore its role in HdeA structure and chaperone function we mutated the disulfide as conservatively as possible, swapping cysteines with serines. We found that, although residual structure is diminished at low pH without the disulfide, it is not completely lost; conversely, we found that the mutant is almost completely random coil at pH 6. Aggregation assays showed that mutated HdeA, although less successful as a chaperone than wild type, still maintains a surprising level of function, and is better at maintaining the solubility of malate dehydrogenase than chemically-reduced wild type. These studies highlight that we still have much to learn about residual structure at low pH and the role of disulfide bonds, as well as revealing exciting areas of future research.
Solubilization of Protein Aggregates by the Acid Stress Chaperones HdeA and HdeB
Journal of Biological Chemistry, 2008
The acid stress chaperones HdeA and HdeB of Escherichia coli prevent the aggregation of periplasmic proteins at acidic pH. We show in this report that they also form mixed aggregates with proteins that have failed to be solubilized at acidic pH and allow their subsequent solubilization at neutral pH. HdeA, HdeB, and HdeA and HdeB together display an increasing efficiency for the solubilization of protein aggregates at pH 3. They are less efficient for the solubilization of aggregates at pH 2, whereas HdeB is the most efficient. Increasing amounts of periplasmic proteins draw increasing amounts of chaperone into pellets, suggesting that chaperones co-aggregate with their substrate proteins. We observed a decrease in the size of protein aggregates in the presence of HdeA and HdeB, from very high molecular mass aggregates to 100-5000-kDa species. Moreover, a marked decrease in the exposed hydrophobicity of aggregated proteins in the presence of HdeA and HdeB was revealed by 1,1-bis(4-anilino)naphtalene-5,5-disulfonic acid binding experiments. In vivo, during the recovery at neutral pH of acid stressed bacterial cells, HdeA and HdeB allow the solubilization and renaturation of protein aggregates, including those formed by the maltose receptor MalE, the oligopeptide receptor OppA, and the histidine receptor HisJ. Thus, HdeA and HdeB not only help to maintain proteins in a soluble state during acid treatment, as previously reported, but also assist, both in vitro and in vivo, in the solubilization at neutral pH of mixed protein-chaperone aggregates formed at acidic pH, by decreasing the size of protein aggregates and the exposed hydrophobicity of aggregated proteins.
2011
Escherichia coli and Gram-negative bacteria that live in the human gut must be able to tolerate rapid and large changes in environmental pH. Low pH irreversibly denatures and precipitates many bacterial proteins. While cytoplasmic proteins are well buffered against such swings, periplasmic proteins are not. Instead, it appears that some bacteria utilize chaperone proteins that stabilize periplasmic proteins, preventing their precipitation. Two highly expressed and related proteins, HdeA and HdeB, have been identified as acid-activated chaperones. The structure of HdeA is known and a mechanism for activation has been proposed. In this model, dimeric HdeA dissociates at low pH, and the exposed dimeric interface binds exposed hydrophobic surfaces of acid-denatured proteins, preventing their irreversible aggregation. We now report the structure and biophysical characterization of the HdeB protein. The monomer of HdeB shares a similar structure with HdeA, but its dimeric interface is different in composition and spatial location. We have used fluorescence to study the behavior of HdeB as pH is lowered, and like HdeA, it dissociates to monomers. We have identified one of the key intersubunit interactions that controls pH-induced monomerization. Our analysis identifies a structural interaction within the HdeB monomer that is disrupted as pH is lowered, leading to enhanced structural flexibility.
Structural plasticity of an acid-activated chaperone allows promiscuous substrate binding
Proceedings of the National Academy of Sciences, 2009
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 acti...
Proceedings of the National Academy of Sciences, 2002
PapD is a periplasmic chaperone essential for P pilus formation in pyelonephritic strains of E. coli. It is composed of two domains, each of which contains a tryptophan residue and Trp-128, in the N-and C-terminal domains, respectively). To explore the role of domain-domain interactions during folding, the protein was labeled with 6-fluorotryptophan for use in 19 F-NMR experiments. 19 F-NMR data collected as a function of urea concentration revealed the presence of a resonance caused by Trp-128 that was distinct from either the folded or unfolded resonances. The time course of refolding from urea was monitored by stopped-flow fluorescence, CD, and 19 F-NMR, each method showing multiple kinetic phases. The 19 F-NMR stopped-flow spectra, collected at 70 M of protein with a fluorine cryoprobe, demonstrated that the intermediate was populated early in the folding process (<5 s). The slow disappearance of the intermediate and unfolded resonance occurred at the same rate as the appearance of the native resonances of both domains. The data are consistent with a model in which the C-terminal domain collapses rapidly to an intermediate, whereas the stabilization of the final structure is slow and requires folding of the N-terminal domain with concomitant readjustment of the C-terminal domain structure.
Journal of Biological Chemistry, 2008
Small heat shock proteins (sHSPs) and the related ␣-crystallins are ubiquitous chaperones linked to neurodegenerative diseases, myopathies, and cataract. To better define their mechanism of chaperone action, we used hydrogen/deuterium exchange and mass spectrometry (HXMS) to monitor conformational changes during complex formation between the structurally defined sHSPs, pea PsHsp18.1, and wheat TaHsp16.9, and the heat-denatured model substrates malate dehydrogenase (MDH) and firefly luciferase. Remarkably, we found that even when complexed with substrate, the highly dynamic local structure of the sHSPs, especially in the N-terminal arm (>70% exchange in 5 s), remains unchanged. These results, coupled with sHSP-substrate complex stability, indicate that sHSPs do not adopt new secondary structure when binding substrate and suggest sHSPs are tethered to substrate at multiple sites that are locally dynamic, a feature that likely facilitates recognition and refolding of sHSP-bound substrate by the Hsp70/DnaK chaperone system. Both substrates were found to be stabilized in a partially unfolded state that is observed only in the presence of sHSP. Furthermore, peptide-level HXMS showed MDH was substantially protected in two core regions (residues 95-156 and 228-252), which overlap with the MDH structure protected in the GroEL-bound MDH refolding intermediate. Significantly, despite differences in the size and structure of TaHsp16.9-MDH and PsHsp18.1-MDH complexes, peptide-level HXMS patterns for MDH in both complexes are virtually identical, indicating that stabilized MDH thermal unfolding intermediates are not determined by the identity of the sHSP.
Protein Science, 2008
Amide proton NMR signals from the N-terminal domain of monomeric a-synuclein (aS) are lost when the sample temperature is raised from 10°Cto35°C at pH 7.4. Although the temperature-induced effects have been attributed to conformational exchange caused by an increase in a-helix structure, we show that the loss of signals is due to fast amide proton exchange. At low ionic strength, hydrogen exchange rates are faster for the N-terminal segment of aS than for the acidic C-terminal domain. When the salt concentration is raised to 300 mM, exchange rates increase throughout the protein and become similar for the N-and C-terminal domains. This indicates that the enhanced protection of amide protons from the C-terminal domain at low salt is electrostatic in nature. Ca chemical shift data point to <10% residual a-helix structure at 10°Cand35°C. Conformational exchange contributions to R2 are negligible at both temperatures. In contrast to the situation in vitro, the majority of amide protons are observed at 37°Cin 1 H-15 NHSQCspectraofaS encapsulated within living Escherichia coli cells. Our finding that temperature effects on aS NMR spectra can be explained by hydrogen exchange obviates the need to invoke special cellular factors. The retention of signals is likely due to slowed hydrogen exchange caused by the lowered intracellular pH of high-density E. coli cultures. Taken together, our results emphasize that aS remains predominantly unfolded at physiological temperature and pH-an important conclusion for mechanistic models of the association of aS with membranes and fibrils.