Conserved amphiphilic feature is essential for periplasmic chaperone HdeA to support acid resistance in enteric bacteria

The extremely acidic environment of the mammalian stomach (pH 1-3) represents a stressful challenge for enteric pathogenic bacteria, including Escherichia coli, Shigella and Brucella. The hdeA (hns-dependent expression A) gene was found to be crucial for the survival of these enteric bacteria under extremely low pH conditions. We recently demonstrated that HdeA is able to exhibit chaperone-like activity exclusively within the stomach pH range by transforming from a well-folded conformation at higher pH values (above pH 3) into an unfolded conformation at extremely low pH values (below pH 3). This study was performed to characterize the action mechanisms and underlying specific structural features for HdeA to function in this unfolded conformation. In the present study, we demonstrate that the conserved 'amphiphilic' feature of HdeA, i.e. the exposure of the conserved hydrophobic region and highly charged terminal regions, is essential for exhibiting chaperone-like activity under extremely low pH conditions. Mutations that disrupt this amphiphilic feature markedly reduced the chaperone-like activity of HdeA. The results also strongly suggest that this acid-induced chaperone-like activity of HdeA is crucial for acid resistance of the enteric bacteria. Moreover, our new understanding of this amphiphilic structural feature of HdeA helps to better interpret how this unfolded (disordered) conformation could be functionally active.     

A genetically incorporated crosslinker reveals chaperone cooperation in acid resistance

Acid chaperones are essential factors in preserving the protein homeostasis for enteric pathogens to survive in the extremely acidic mammalian stomach (pH 1-3). The client proteins of these chaperones remain largely unknown, primarily because of the exceeding difficulty of determining protein-protein interactions under low-pH conditions. We developed a genetically encoded, highly efficient protein photocrosslinking probe, which enabled us to profile the in vivo substrates of a major acid-protection chaperone, HdeA, in Escherichia coli periplasm. Among the identified HdeA client proteins, the periplasmic chaperones DegP and SurA were initially found to be protected by HdeA at a low pH, but they subsequently facilitated the HdeA-mediated acid recovery of other client proteins. This unique, ATP-independent chaperone cooperation in the ATP-deprived E. coli periplasm may support the acid resistance of enteric bacteria. The crosslinker would be valuable in unveiling the physiological interaction partners of any given protein and thus their functions under normal and stress conditions.     

Solubilization of protein aggregates by the acid stress chaperones HdeA and HdeB

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.     

The Mechanism of HdeA Unfolding and Chaperone Activation

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 from folded dimer to unfolded monomer between pH 3 and pH 2, and suggest the existence of a low-populated, partially folded intermediate that could assist in chaperone activation or function. Overall, this study provides a detailed experimental investigation into the mechanism by which HdeA unfolds and activates.     

分子伴侣HdeA与HdeB的作用机制

分子伴侣能够与其他蛋白质的不稳定构象相结合并使其稳定．它的功能之一是能够帮助蛋白质进行正确的折叠与组装．最新研究发现,在肠道致病菌的周质空间中存在着酸性条件下能帮助周质蛋白复性的分子伴侣HdeA和HdeB．HdeA在极端酸性的胃部环境中由二聚体迅速解离成具有伴侣活性的单体,HdeA单体能够和变性的底物蛋白结合防止它们酸诱导聚集,从而保护肠道致病菌安全到达肠道．本文对肠道致病菌的耐酸机制进行了总结,最后对 HdeA和HdeB作用机制的研究近况进行综述,最后对HdeA和HdeB以后的研究方向进行了展望．     

Escherichia coli HdeB is an acid stress chaperone

We cloned, expressed, and purified the hdeB gene product, which belongs to the hdeAB acid stress operon. We extracted HdeB from bacteria by the osmotic-shock procedure and purified it to homogeneity by ion-exchange chromatography and hydroxyapatite chromatography. Its identity was confirmed by mass spectrometry analysis. HdeB has a molecular mass of 10 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which matches its expected molecular mass. We purified the acid stress chaperone HdeA in parallel in order to compare the two chaperones. The hdeA and hdeB mutants both display reduced viability upon acid stress, and only the HdeA/HdeB expression plasmid can restore their viability to close to the wild-type level, suggesting that both proteins are required for optimal protection of the bacterial periplasm against acid stress. Periplasmic extracts from both mutants aggregate at acidic pH, suggesting that HdeA and HdeB are required for protein solubilization. At pH 2, the aggregation of periplasmic extracts is prevented by the addition of HdeA, as previously reported, but is only slightly reduced by HdeB. At pH 3, however, HdeB is more efficient than HdeA in preventing periplasmic-protein aggregation. The solubilization of several model substrate proteins at acidic pH supports the hypothesis that, in vitro, HdeA plays a major role in protein solubilization at pH 2 and that both proteins are involved in protein solubilization at pH 3. Like HdeA, HdeB exposes hydrophobic surfaces at acidic pH, in accordance with the appearance of its chaperone properties at acidic pH. HdeB, like HdeA, dissociates from dimers at neutral pH into monomers at acidic pHs, but its dissociation is complete at pH 3 whereas that of HdeA is complete at a more acidic pH. Thus, we can conclude that Escherichia coli possesses two acid stress chaperones that prevent periplasmic-protein aggregation at acidic pH.     

Products of the Escherichia coli acid fitness island attenuate metabolite stress at extremely low pH and mediate a cell density-dependent acid resistance

Escherichia coli has an ability, rare among the Enterobacteriaceae, to survive extreme acid stress under various host (e.g., human stomach) and nonhost (e.g., apple cider) conditions. Previous microarray studies have exposed a cluster of 12 genes at 79 centisomes collectively called an acid fitness island (AFI). Four AFI genes, gadA, gadX, gadW, and gadE, were already known to be involved in an acid resistance system that consumes an intracellular proton through the decarboxylation of glutamic acid. However, roles for the other eight AFI gene products were either unknown or subject to conflicting findings. Two new aspects of acid resistance are described that require participation of five of the remaining eight AFI genes. YhiF (a putative regulatory protein), lipoprotein Slp, and the periplasmic chaperone HdeA protected E. coli from organic acid metabolites produced during fermentation once the external pH was reduced to pH 2.5. HdeA appears to handle protein damage caused when protonated organic acids diffuse into the cell and dissociate, thereby decreasing internal pH. In contrast, YhiF- and Slp-dependent systems appear to counter the effects of the organic acids themselves, specifically succinate, lactate, and formate, but not acetate. A second phenomenon was defined by two other AFI genes, yhiD and hdeD, encoding putative membrane proteins. These proteins participate in an acid resistance mechanism exhibited only at high cell densities (>10(8) CFU per ml). Density-dependent acid resistance does not require any demonstrable secreted factor and may involve cell contact-dependent activation. These findings further define the complex physiology of E. coli acid resistance.     

Evolutionary silence of the acid chaperone protein HdeB in enterohemorrhagic Escherichia coli O157:H7

The periplasmic chaperones HdeA and HdeB are known to be important for cell survival at low pH (pH < 3) in Escherichia coli and Shigella spp. Here we investigated the roles of HdeA and HdeB in the survival of various enterohemorrhagic E. coli (EHEC) following exposure to pH 2.0. Similar to K-12 strains, the acid protections conferred by HdeA and HdeB in EHEC O145 were significant: loss of HdeA and HdeB led to over 100- to 1,000-fold reductions in acid survival, depending on the growth condition of prechallenge cells. However, this protection was much less in E. coli O157:H7 strains. Deletion of hdeB did not affect the acid survival of cells, and deletion of hdeA led to less than a 5-fold decrease in survival. Sequence analysis of the hdeAB operon revealed a point mutation at the putative start codon of the hdeB gene in all 26 E. coli O157:H7 strains analyzed, which shifted the ATG start codon to ATA. This mutation correlated with the lack of HdeB in E. coli O157:H7; however, the plasmid-borne O157-hdeB was able to restore partially the acid resistance in an E. coli O145ΔhdeAB mutant, suggesting the potential function of O157-HdeB as an acid chaperone. We conclude that E. coli O157:H7 strains have evolved acid survival strategies independent of the HdeA/B chaperones and are more acid resistant than nonpathogenic K-12 for cells grown under nonfavorable culturing conditions such as in Luria-Bertani no-salt broth at 28°C. These results suggest a divergent evolution of acid resistance mechanisms within E. coli.     

底物对变性酶的修复作用

 兎肌肌酸激酶被LDS变性后,底物能够诱导变性酶使其活力和构象得到部分恢复。变性程度不同的酶,构象和活力的恢复程度也不同:低浓度LDS变性酶,恢复程度较高;反之亦然。活力的恢复与构象的恢复两者呈对应关系。底物修复作用的pH以8.2为好。底物修复作用受其它蛋白质(例如BSA)存在的影响。等速电泳结果表明,BSA能竞争性结合LDS-酶复合物的LDS,使酶成为游离酶。变性酶先与BSA保温再加底物所得的活力恢复,大约是变性酶与含BSA的底物保温所得活力的10倍。这一结果似表明LDS变性酶仍能结合底物;被结合的底物还能使变性酶的构象发生变化。     

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.     

pH-dependent catabolic protein expression during anaerobic growth of Escherichia coli K-12

During aerobic growth of Escherichia coli, expression of catabolic enzymes and envelope and periplasmic proteins is regulated by pH. Additional modes of pH regulation were revealed under anaerobiosis. E. coli K-12 strain W3110 was cultured anaerobically in broth medium buffered at pH 5.5 or 8.5 for protein identification on proteomic two-dimensional gels. A total of 32 proteins from anaerobic cultures show pH-dependent expression, and only four of these proteins (DsbA, TnaA, GatY, and HdeA) showed pH regulation in aerated cultures. The levels of 19 proteins were elevated at the high pH; these proteins included metabolic enzymes (DhaKLM, GapA, TnaA, HisC, and HisD), periplasmic proteins (ProX, OppA, DegQ, MalB, and MglB), and stress proteins (DsbA, Tig, and UspA). High-pH induction of the glycolytic enzymes DhaKLM and GapA suggested that there was increased fermentation to acids, which helped neutralize alkalinity. Reporter lac fusion constructs showed base induction of sdaA encoding serine deaminase under anaerobiosis; in addition, the glutamate decarboxylase genes gadA and gadB were induced at the high pH anaerobically but not with aeration. This result is consistent with the hypothesis that there is a connection between the gad system and GabT metabolism of 4-aminobutanoate. On the other hand, 13 other proteins were induced by acid; these proteins included metabolic enzymes (GatY and AckA), periplasmic proteins (TolC, HdeA, and OmpA), and redox enzymes (GuaB, HmpA, and Lpd). The acid induction of NikA (nickel transporter) is of interest because E. coli requires nickel for anaerobic fermentation. The position of the NikA spot coincided with the position of a small unidentified spot whose induction in aerobic cultures was reported previously; thus, NikA appeared to be induced slightly by acid during aeration but showed stronger induction under anaerobic conditions. Overall, anaerobic growth revealed several more pH-regulated proteins; in particular, anaerobiosis enabled induction of several additional catabolic enzymes and sugar transporters at the high pH, at which production of fermentation acids may be advantageous for the cell.     

Salt bridges regulate both dimer formation and monomeric flexibility in HdeB and may have a role in periplasmic chaperone function

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.     

Role of electrostatic repulsion in the acidic molten globule of cytochrome c.

The molten globule has been assumed to be a major intermediate state of protein folding. To extend our understanding of protein folding it is important to elucidate the thermodynamic mechanism of conformational stability of the molten globule. To clarify the role of electrostatic charge repulsion in the stability of the acidic molten globule state, we prepared a series of acetylated horse ferricytochrome c species with various degrees of charge repulsion. On the basis of circular dichroism measurement, we show that the stability of the acidic molten globule is determined by a balance of electrostatic repulsions between positive residues, which favor the extended conformation, and the opposing forces, which stabilize the molten globule. These results provide a clear example of charge repulsions producing unfolding of the compact protein structure, and suggest that the reversibly denatured conformation of ferricytochrome c under physiological conditions (i.e. neutral pH, ambient temperature and no denaturant) is the molten globule.     

Structural and thermodynamic consequences of removal of a conserved disulfide bond from equine beta-lactoglobulin

A disulfide bond between cysteine 66 and cysteine 160 of equine beta-lactoglobulin was removed by substituting cysteine residues with alanine. This disulfide bond is conserved across the lipocalin family. The conformation and stability of the disulfide-deleted mutant protein was investigated by circular dichroism. The mutant protein assumes a native-like structure under physiological conditions and assumes a helix-rich molten globule structure at acid pH or at moderate concentrations of urea as the wild-type protein does. The urea-induced unfolding experiment shows that the stability of the native conformation was reduced but that of the molten globule intermediate is not significantly changed at pH 4 by removal of the disulfide bond. On the other hand, the molten globule at acid pH was destabilized by removal of the disulfide bond. This difference in the stabilizing effect of the disulfide bond was interpreted by the effect of the disulfide in keeping the molecule compact against the electrostatic repulsion at acid pH. In contrast to the wild-type protein, the circular dichroism spectrum in the molten globule state at acid pH depends on anion concentration, suggesting that the expansion of the molecule through electrostatic repulsion induces alpha-helices as observed in the cold denatured state of the wild-type protein.     

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

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 pK_a 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.     

Chaperone-dependent mechanisms for acid resistance in enteric bacteria

The extremely acidic environment of the mammalian stomach not only serves to facilitate food digestion but also acts as a natural barrier against infections of food-borne pathogens. Many pathogenic bacteria, such as enterohemorrhagic Escherichia coli, can breach this host defense and cause severe diseases. These pathogens have evolved multiple intricate strategies to overcome the bactericidal activity of acids. In particular, recent studies have uncovered the central roles of two periplasmic chaperones, HdeA and HdeB, in protecting enteric bacteria from extremely acidic conditions. Here, we review recent advances in the understanding of the acid resistance mechanisms of Gram-negative bacteria and focus on the mechanisms of HdeA and HdeB in preventing acid-induced protein aggregation and facilitating protein refolding following pH neutralization.     

分子伴侣HdeA与底物蛋白SurA作用机制的模拟研究

分子伴侣HdeA与底物蛋白间的相互作用可帮助底物蛋白复性,这是肠道致病菌得以在酸性环境中幸存的重要原因之一.为探究HdeA发挥伴侣活性的作用机制,本研究采用分子对接和分子动力学的方法,模拟了HdeA与底物蛋白SurA间的相互作用,计算了二者的结合自由能.通过分析HdeA-SurA复合物体系的作用模式、氢键作用以及能量分解的结果,确定了HdeA与底物蛋白SurA结合时发挥重要作用的关键氨基酸残基.该研究结果为以后采用实验手段探究HdeA与底物蛋白之间的作用提供了重要的理论参考,同时为今后设计与开发HdeA的抑制剂提供了理论指导依据.     

Nonprotein amino acid furanomycin, unlike isoleucine in chemical structure, is charged to isoleucine tRNA by isoleucyl-tRNA synthetase and incorporated into protein

Nonprotein amino acid furanomycin was found to bind with Escherichia coli isoleucyl-tRNA synthetase (IleRS) almost as tightly as the substrate L-isoleucine. The conformation of furanomycin bound to the enzyme was determined by NMR analyses including the transferred nuclear Overhauser effect method. The conformation of IleRS-bound furanomycin was similar to that of L-isoleucine, although the chemical structure of furanomycin is unlike that of L-isoleucine. By E. coli IleRS, E. coli tRNAIle was charged with furanomycin as efficiently as with L-isoleucine. Furthermore, furanomycyl-tRNAIle was bound to polypeptide chain elongation factor Tu as tightly as isoleucyl-tRNAIle. Furanomycin was found to be incorporated into beta-lactamase precursor by in vitro protein biosynthesis. A newly designed amino acid will probably be incorporated into proteins, provided that the new amino acid takes a similar conformation as a protein-constituting amino acid in the active site of an aminoacyl-tRNA synthetase.     

Crystal structure and solution NMR studies of Lys48-linked tetraubiquitin at neutral pH

Ubiquitin modification of proteins is used as a signal in many cellular processes. Lysine side-chains can be modified by a single ubiquitin or by a polyubiquitin chain, which is defined by an isopeptide bond between the C terminus of one ubiquitin and a specific lysine in a neighboring ubiquitin. Polyubiquitin conformations that result from different lysine linkages presumably differentiate their roles and ability to bind specific targets and enzymes. However, conflicting results have been obtained regarding the precise conformation of Lys48-linked tetraubiquitin. We report the crystal structure of Lys48-linked tetraubiquitin at near-neutral pH. The two tetraubiquitin complexes in the asymmetric unit show the complete connectivity of the chain and the molecular details of the interactions. This tetraubiquitin conformation is consistent with our NMR data as well as with previous studies of diubiquitin and tetraubiquitin in solution at neutral pH. The structure provides a basis for understanding Lys48-linked polyubiquitin recognition under physiological conditions.     

Association of fibronectin with carboxy-group-modified proteins in vitro

Treatment of human immunoglobulin G, albumin and fibronectin with water-soluble carbodi-imide at pH4.75 in the presence of glycine ethyl ester resulted in an avid binding of ¹²⁵I-labelled native fibrinectin to the modified proteins. Succinoylation, reduction and alkylation or heat-denaturation had no such effect. In affinity chromatography under physiological conditions, serum was depleted of fibronectin when run through columns of the carbodi-imide-treated proteins coupled to agarose. Fractions eluted from such columns with urea were enriched in fibronectin. The binding of radiolabelled fibronectin to the carbodi-imide-treated proteins was inhibited by unlabelled fibronectin in relatively low concentrations, but also by albumin in higher concentrations. Heat-denatured albumin inhibited at concentrations approx. 10–30 times lower than native albumin. The binding reaction had a pH optimum of 6–8. It was inhibited at high ionic strength and in the presence of urea. Anionic detergents inhibited at millimolar concentrations, but non-ionic detergents did not inhibit the binding reaction. The results were interpreted as showing that: (1) fibronectin is capable of binding to itself, to immunoglobulin G and to albumin after a reduction of the negative surface charge of these proteins, and may have a general ability to bind such modified proteins; (2) this binding can take place under physiological conditions; (3) carboxy-group-modified proteins selectively bind fibronectin from serum. This novel binding phenomenon could be important in terms of the opsonin function of circulatory fibronectin. We propose that fibronectin may recognize modified (denatured) proteins and mediate their uptake by the reticuloendothelial system.