The low pH environment of the human stomach, which generally ranges from pH 1 to 3, acts as a defensive barrier against infection of the intestines by pathogenic bacteria. The acidity of the stomach can cause aggregation of periplasmic proteins in gram-negative bacteria, effectively preventing normal bacterial function and often resulting in cell death.[1, 2] When the low-pH obstacle fails the resulting gastrointestinal colonization produces dysentery, a condition characterized by hemorrhagic diarrhea estimated to be responsible for the deaths of at least 800,000 children per year.
The vulnerability of the periplasmic proteins in bacteria results from their open exposure to the environment: porins on the outer surface of the organism permit small molecules, including protons, to flow into the periplasmic space.[4, 5] Proteins in this region are therefore rapidly exposed to the low pH of the stomach, frequently resulting in protein unfolding and aggregation. Some pathogenic bacteria (including Escherichia coli, Shigella flexneri and Brucella abortus) have evolved an effective mechanism to prevent this aggregation by expressing the small chaperone protein HdeA. This 9.7 kDa protein, containing four helices (A–D) per monomer (Fig. 1), exists as an inactive dimer in environments above pH 3.0. The dimer interface is held together primarily by hydrophobic interactions from residues located on helix B. It has been suggested that the loop region between helices B and C, containing negatively charged glutamate and aspartate residues, also help to stabilize the dimer at higher pH values by forming salt bridges with lysines on the opposing monomer.
A very acidic environment (pH < 3), such as that found in the stomach, results in the protonation of the sixteen Glu and Asp residues spread throughout HdeA, thereby eliminating many of the electrostatic interactions with lysines that maintain the folded dimer structure. This is believed to trigger exposure of the hydrophobic interface, which ultimately leads to the formation of an unfolded, active monomer capable of associating with other periplasmic proteins via hydrophobic interactions. Positively charged Lys residues, situated mostly at the N- and C-termini of the chaperone, allow for the HdeA-substrate complex to maintain solubility.[1, 2, 8] The transition between inactive dimer and active monomer has been characterized as ATP-independent, fast, and reversible. An increase in pH upon bacterial entry into the gastrointestinal tract has been shown to result in the slow release of the substrate proteins and the refolding of HdeA to its inactive dimer conformation.
Researchers are motivated to characterize the mechanism by which HdeA becomes activated in order to improve targeting for vaccines or other therapeutics that can disable its activities and thereby weaken the infectivity of pathogenic bacteria. While several studies have been published that investigate biophysical properties of HdeA using a variety of techniques,[1, 7-10] and although a crystal structure of the folded dimer is available, there is still a lack of detailed atomic-level information about HdeA activity, and a deficiency in the number of pH values used to collect data. NMR spectroscopy is an ideal choice for studying the transition of HdeA from folded, inactive dimer at neutral pH to unfolded, active monomer at low pH: NMR permits detailed characterization of the protein with atomic resolution, and the solution state conditions are exquisitely sensitive to disruptions within the protein, even with small step changes in pH.
It has already been mentioned that HdeA activation depends, in part, on the protonation of Asp and Glu residues with increasing acidity; until now, the pKas for these residues had been estimated through simulation. There is a question, however, about whether this charge neutralization is the primary trigger for protein unfolding and activation, or whether it is only one of several contributing factors. The overall goal of these studies is to better describe the extent of the structural impact of neutralizing the Asp and Glu charges with lowered pH, and how it contributes to priming the protein for activation of its chaperone activities.
In this article we present experimental characterization of those pKa values as part of a titration experiment between pH 6.0 and 3.0 that also followed the backbone chemical shift changes at each amino acid in the sequence. Total correlation spectroscopy (TOCSY) experiments were used to obtain pKas for all eleven aspartate and four out of five glutamate residues, and 1H-15N HSQC experiments were recorded to monitor backbone chemical shift changes. Considering that molecular motions often play significant roles in protein functionality, we were also interested in monitoring HdeA backbone dynamics to gain insight into the conformational and flexibility changes that take place due to Asp and Glu charge neutralization. Hydrogen/deuterium exchange and backbone relaxation experiments were recorded to describe these motions. Our studies have confirmed that HdeA maintains its dimer structure down to pH 3.0; notably, however, the structure becomes increasingly loosened as the pH is lowered and may participate in transient opening of the dimer prior to dissociation. We have also found that, although protonation of the aspartate and glutamate side chains heavily influence the changes in HdeA conformation and flexibility that take place in this preactivation state, it is also apparent that those influences are not sufficient to single-handedly initiate the full activation of HdeA into an unfolded monomeric state. Overall, these investigations of the correlation between ionization state, conformational changes and internal motions in the pH range 6.0–3.0 are critical to advance our understanding of the steps that are required to prime HdeA for unfolding, dissociation, and ultimate activation below pH 3.0. The design of future therapeutics will likely target the folded state of the protein; understanding the range of conformations available to HdeA between pH 6.0 and 3.0 may therefore be critical to this design process.