The CrdRS (HP1365-HP1364) Two-Component System Is Not Involved in pH-Responsive Gene Regulation in the <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Strains 26695 and G27

The human gastric pathogen Helicobacter pylori is extremely well adapted to the highly acidic conditions encountered in the stomach. The pronounced acid resistance of H. pylori relies mainly on the ammonia-producing enzyme urease. However, urease-independent mechanisms are likely to contribute to acid adaptation. pH-responsive gene regulation in this organism is mediated by a two-component system (HP0166-HP0165) designated ArsRS and the metal-dependent regulators NikR and Fur. Recently, it was reported that another two-component system termed CrdRS (HP1365-HP1364) is required for pH-responsive regulation of the major acid-resistance systems in the H. pylori strain J99. By the analysis of crdRS null mutants of the H. pylori strains 26695 and G27, we show that low pH induction of both the urease and the amidase genes occurs in the absence of crdRS in these strains, suggesting substantial strain-specific differences in the regulation of a major virulence determinant.     

H. pylori CagL-Y58/E59 Prime Higher Integrin α5β1 in Adverse pH Condition to Enhance Hypochlorhydria Vicious Cycle for Gastric Carcinogenesis

Background/Aims

H. pylori CagL amino acid polymorphisms such as Y58/E59 can increase integrin α5β1 expression and gastric cancer risk. Hypochlorhydria during chronic H. pylori infection promotes gastric carcinogenesis. The study test whether CagL-Y58/E59 isolates may regulate integrin α5β1 to translocate CagA via the type IV secretory system even under adverse pH conditions, and whether the integrin α5β1 expression primed by H. pylori is a pH-dependent process involving hypochlorhydria in a vicious cycle to promote gastric carcinogenesis.

Methods

The expressions of integrin α5 and β1, CagA phosphorylation, IL-8, FAK, EGFR, and AKT activation of AGS cells exposed to CagL-Y58/E59 H. pylori, isogenic mutants, and different H. pylori CagL amino acid replacement mutants under different pH values were determined. Differences in the pepsinogen I/II ratio (indirectly indicating gastric acidity) and gastric integrin α5β1 expression were compared among the 172 H. pylori-infected patients with different cancer risks.

Results

Even under adversely low pH condition, H. pylori CagL-Y58/E59 still keep active integrin β1 with stronger binding affinity, CagA translocation, IL-8, FAK, EGFR, and AKT activation than the other mutants (p<0.05). The in vitro assay revealed higher priming of integrin α5β1 by H. pylori under elevated pH as hypochlorhydria (p<0.05). In the H. pylori-infected patients, the gastric integrin α5β1 expressions were higher in those with pepsinogen I/II ratio <6 than in those without (p<0.05).

Conclusions

H. pylori CagL-Y58/E59 prime higher integrin under adverse pH and may involve to enhance hypochlorhydria vicious cycle for gastric carcinogenesis, and thus require an early eradication.     

Helicobacter pylori Colonization Ameliorates Glucose Homeostasis in Mice through a PPAR γ-Dependent Mechanism

Background

There is an inverse secular trend between the incidence of obesity and gastric colonization with Helicobacter pylori, a bacterium that can affect the secretion of gastric hormones that relate to energy homeostasis. H. pylori strains that carry the cag pathogenicity island (PAI) interact more intimately with gastric epithelial cells and trigger more extensive host responses than cag⁻ strains. We hypothesized that gastric colonization with H. pylori strains differing in cag PAI status exert distinct effects on metabolic and inflammatory phenotypes.

Methodology/Principal Findings

To test this hypothesis, we examined metabolic and inflammatory markers in db/db mice and mice with diet-induced obesity experimentally infected with isogenic forms of H. pylori strain 26695: the cag PAI wild-type and its cag PAI mutant strain 99–305. H. pylori colonization decreased fasting blood glucose levels, increased levels of leptin, improved glucose tolerance, and suppressed weight gain. A response found in both wild-type and mutant H. pylori strain-infected mice included decreased white adipose tissue macrophages (ATM) and increased adipose tissue regulatory T cells (Treg) cells. Gene expression analyses demonstrated upregulation of gastric PPAR γ-responsive genes (i.e., CD36 and FABP4) in H. pylori-infected mice. The loss of PPAR γ in immune and epithelial cells in mice impaired the ability of H. pylori to favorably modulate glucose homeostasis and ATM infiltration during high fat feeding.

Conclusions/Significance

Gastric infection with some commensal strains of H. pylori ameliorates glucose homeostasis in mice through a PPAR γ-dependent mechanism and modulates macrophage and Treg cell infiltration into the abdominal white adipose tissue.     

Involvement of Aquaporin 3 in Helicobacter pylori-Related Gastric Diseases

Background

Gastric cancer is one of the most common and lethal malignant cancers worldwide, and numerous epidemiological studies have demonstrated that Helicobacter pylori (H. pylori) infection plays a key role in the development of gastric carcinomas. Our previous studies showed that aquaporin 3 (AQP3) is overexpressed in gastric carcinoma and promotes the migration and proliferation of human gastric carcinoma cells, suggesting that AQP3 may be a potentially important determinant of gastric carcinoma. However, the role of AQP3 in H. pylori carcinogenesis is unknown.

Methods

The AQP3 protein and H. pylori were detected in human gastric tissues by immunohistochemistry and modified Giemsa staining respectively. AQP3 knockdown was obtained by small interfering (si) RNA. Western blot assays and RT-PCR were used to evaluate the change of AQP3 in the human gastric cancer AGS and SGC7901 cell lines after co-culture with H. pylori. Sprague Dawley rats were orally inoculated with H. pylori to establish a rat model colonized by H. pylori.

Results

The present study found that AQP3 expression correlated with H. pylori infection status in gastric cancer tissues and corresponding normal mucosa, and H. pylori co-culture upregulated AQP3 expression in human gastric adenocarcinoma cells in vitro via the extracellular signal-regulated kinase signaling pathway. H. pylori infection also increased AQP3 expression in gastric mucosa colonized by H. pylori in a Sprague Dawley rat model.

Conclusions

These findings provide further information to understand the mechanism of H. pylori carcinogenesis and a potential strategy for the treatment of H. pylori-associated gastric carcinoma.     

Age-Dependent Association among Helicobacter pylori Infection,Serum Pepsinogen Levels and Immune Response of Children to Live Oral Cholera Vaccine CVD 103-HgR

Background

Through its effects on gastric secretion, we hypothesized that Helicobacter pylori infection may influence oral immunization. Accordingly, we examined the association between H. pylori infection, serum pepsinogen (PG) (measures for H. pylori gastritis) and vibriocidal antibody (a correlate of protection) seroconversion following oral immunization with CVD 103-HgR live cholera vaccine among children of different ages.

Methods

Sera from 422 Chilean children who were vaccinated with a single dose of CVD 103-HgR were tested by ELISA for serum IgG antibodies to H. pylori, PG I and PG II levels and antibodies to Shigella flexneri 2a lipopolysaccharide and hepatitis A virus (as markers of low socioeconomic status and exposure to enteric pathogens).

Results

The likelihood of vibriocidal antibody seroconversion following vaccination with CVD 103-HgR was significantly decreased in H. pylori-seropositive children age 6 months to 4 years with PG II>8 µg/L (adjusted OR 0.14 (95% CI 0.03–0.61; P = 0.009), and also in H. pylori seropositives with lower PG II level (adjusted OR 0.34, 95% CI 0.14–0.83; P = 0.017), compared to H. pylori-seronegatives. H. pylori-seropositive children aged 5–9 years with serum PG I>30 µg/L (indicating more severe gastritis) had higher odds of vibriocidal seroconversion than those with lower PG I levels (adjusted OR 4.41, 95%CI 1.26–15.38; P = 0.02). There was no significant association between exposures to S. flexneri 2a or hepatitis A virus and vibriocidal seroconversion.

Conclusions

As H. pylori gastritis progresses with increasing pediatric age in developing country venues, changes in gastric secretion ensue that we believe explain the observed differences in age-related immune responses to immunization with live oral cholera vaccine. The effect of H. pylori and changes of gastric acid secretion on the immunogenicity of various oral vaccines should be studied in different developing, transitional and industrialized country settings.     

Enhanced M1 macrophage polarization in human helicobacter pylori-associated atrophic gastritis and in vaccinated mice

Background

Infection with Helicobacter pylori triggers a chronic gastric inflammation that can progress to atrophy and gastric adenocarcinoma. Polarization of macrophages is a characteristic of both cancer and infection, and may promote progression or resolution of disease. However, the role of macrophages and their polarization during H. pylori infection has not been well defined.

Methodology/Principal Findings

By using a mouse model of infection and gastric biopsies from 29 individuals, we have analyzed macrophage recruitment and polarization during H. pylori infection by flow cytometry and real-time PCR. We found a sequential recruitment of neutrophils, eosinophils and macrophages to the gastric mucosa of infected mice. Gene expression analysis of stomach tissue and sorted macrophages revealed that gastric macrophages were polarized to M1 after H. pylori infection, and this process was substantially accelerated by prior vaccination. Human H. pylori infection was characterized by a mixed M1/M2 polarization of macrophages. However, in H. pylori-associated atrophic gastritis, the expression of inducible nitric oxide synthase was markedly increased compared to uncomplicated gastritis, indicative of an enhanced M1 macrophage polarization in this pre-malignant lesion.

Conclusions/Significance

These results show that vaccination of mice against H. pylori amplifies M1 polarization of gastric macrophages, and that a similar enhanced M1 polarization is present in human H. pylori-induced atrophic gastritis.     

Isocitrate dehydrogenase of Helicobacter pylori potentially induces humoral immune response in subjects with peptic ulcer disease and gastritis

Background

H. pylori causes gastritis and peptic ulcers and is a risk factor for the development of gastric carcinoma. Many of the proteins such as urease, porins, flagellins and toxins such as lipo-polysaccharides have been identified as potential virulence factors which induce proinflammatory reaction. We report immunogenic potentials of isocitrate dehydrogenase (ICD), an important house keeping protein of H. pylori.

Methodology/Principal Findings

Amino acid sequences of H. pylori ICD were subjected to in silico analysis for regions with predictably high antigenic indexes. Also, computational modelling of the H. pylori ICD as juxtaposed to the E. coli ICD was carried out to determine levels of structure similarity and the availability of surface exposed motifs, if any. The icd gene was cloned, expressed and purified to a very high homogeneity. Humoral response directed against H. pylori ICD was detected through an enzyme linked immunosorbent assay (ELISA) in 82 human subjects comprising of 58 patients with H. pylori associated gastritis or ulcer disease and 24 asymptomatic healthy controls. The H. pylori ICD elicited potentially high humoral immune response and revealed high antibody titers in sera corresponding to endoscopically-confirmed gastritis and ulcer disease subjects. However, urea-breath-test negative healthy control samples and asymptomatic control samples did not reveal any detectable immune responses. The ELISA for proinflammatory cytokine IL-8 did not exhibit any significant proinflammatory activity of ICD.

Conclusions/Significance

ICD of H. pylori is an immunogen which interacts with the host immune system subsequent to a possible autolytic-release and thereby significantly elicits humoral responses in individuals with invasive H. pylori infection. However, ICD could not significantly stimulate IL8 induction in a cultured macrophage cell line (THP1) and therefore, may not be a notable proinflammatory agent.     

Functional identification of HugZ, a heme oxygenase from Helicobacter pylori

Background

Iron is recognized as an important trace element, essential for most organisms including pathogenic bacteria. HugZ, a protein related to heme iron utilization, is involved in bacterial acquisition of iron from the host. We previously observed that a hugZ homologue is correlated with the adaptive colonization of Helicobacter pylori (H. pylori), a major gastro-enteric pathogen. However, its exact physiological role remains unclear.

Results

A gene homologous to hugZ, designated hp0318, identified in H. pylori ATCC 26695, exhibits 66% similarity to cj1613c of Campylobacter jejuni NCTC 11168. Soluble 6 × His fused-HugZ protein was expressed in vitro. Hemin-agrose affinity analysis indicated that the recombinant HugZ protein can bind to hemin. Absorption spectroscopy at 411 nm further revealed a heme:HugZ binding ratio of 1:1. Enzymatic assays showed that purified recombinant HugZ protein can degrade hemin into biliverdin and carbon monoxide in the presence of either ascorbic acid or NADPH and cytochrome P450 reductase. The biochemical and enzymatic characteristics agreed closely with those of Campylobacter jejuni Cj1613c protein, implying that hp0318 is a functional member of the HugZ family. A hugZ deletion mutant was obtained by homologous recombination. This mutant strain showed poor growth when hemoglobin was provided as the source of iron, partly because of its failure to utilize hemoglobin efficiently. Real-time quantitative PCR also confirmed that the expression of hugZ was regulated by iron levels.

Conclusion

These findings provide biochemical and genetic evidence that hugZ (hp0318) encodes a heme oxygenase involved in iron release/uptake in H. pylori.     

Cofactor Requirement of HpyAV Restriction Endonuclease

Background

Helicobacter pylori is the etiologic agent of common gastritis and a risk factor for gastric cancer. It is also one of the richest sources of Type II restriction-modification (R-M) systems in microorganisms.

Principal Findings

We have cloned, expressed and purified a new restriction endonuclease HpyAV from H. pylori strain 26695. We determined the HpyAV DNA recognition sequence and cleavage site as CCTTC 6/5. In addition, we found that HpyAV has a unique metal ion requirement: its cleavage activity is higher with transition metal ions than in Mg⁺⁺. The special metal ion requirement of HpyAV can be attributed to the presence of a HNH catalytic site similar to ColE9 nuclease instead of the canonical PD-X-D/EXK catalytic site found in many other REases. Site-directed mutagenesis was carried out to verify the catalytic residues of HpyAV. Mutation of the conserved metal-binding Asn311 and His320 to alanine eliminated cleavage activity. HpyAV variant H295A displayed approximately 1% of wt activity.

Conclusions/Significance

Some HNH-type endonucleases have unique metal ion cofactor requirement for optimal activities. Homology modeling and site-directed mutagenesis confirmed that HpyAV is a member of the HNH nuclease family. The identification of catalytic residues in HpyAV paved the way for further engineering of the metal binding site. A survey of sequenced microbial genomes uncovered 10 putative R-M systems that show high sequence similarity to the HpyAV system, suggesting lateral transfer of a prototypic HpyAV-like R-M system among these microorganisms.     

The complete methylome of Helicobacter pylori UM032

Background

The genome of the human gastric pathogen Helicobacter pylori encodes a large number of DNA methyltransferases (MTases), some of which are shared among many strains, and others of which are unique to a given strain. The MTases have potential roles in the survival of the bacterium. In this study, we sequenced a Malaysian H. pylori clinical strain, designated UM032, by using a combination of PacBio Single Molecule, Real-Time (SMRT) and Illumina MiSeq next generation sequencing platforms, and used the SMRT data to characterize the set of methylated bases (the methylome).

Results

The N4-methylcytosine and N6-methyladenine modifications detected at single-base resolution using SMRT technology revealed 17 methylated sequence motifs corresponding to one Type I and 16 Type II restriction-modification (R-M) systems. Previously unassigned methylation motifs were now assigned to their respective MTases-coding genes. Furthermore, one gene that appears to be inactive in the H. pylori UM032 genome during normal growth was characterized by cloning.

Conclusion

Consistent with previously-studied H. pylori strains, we show that strain UM032 contains a relatively large number of R-M systems, including some MTase activities with novel specificities. Additional studies are underway to further elucidating the biological significance of the R-M systems in the physiology and pathogenesis of H. pylori.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1585-2) contains supplementary material, which is available to authorized users.     

Using Stool Antigen to Screen for Helicobacter pylori in Immigrants and Refugees from High Prevalence Countries Is Relatively Cost Effective in Reducing the Burden of Gastric Cancer and Peptic Ulceration

Objectives

Refugees and immigrants from developing countries settling in industrialised countries have a high prevalence of Helicobacter pylori (H. pylori). Screening these groups for H. pylori and use of eradication therapy to reduce the future burden of gastric cancer and peptic ulcer disease is not currently recommended in most countries. We investigated whether a screening and eradication approach would be cost effective in high prevalence populations.

Methods

Nine different screening and follow-up strategies for asymptomatic immigrants from high H. pylori prevalence areas were compared with the current approach of no screening. Cost effectiveness comparisons assumed population prevalence''s of H. pylori of 25%, 50% or 75%. The main outcome measure was the net cost for each cancer prevented for each strategy. Total costs of each strategy and net costs including savings from reductions in ulcers and gastric cancer were also calculated.

Results

Stool antigen testing with repeat testing after treatment was the most cost effective approach relative to others, for each prevalence value. The net cost per cancer prevented with this strategy was US$111,800 (assuming 75% prevalence), $132,300 (50%) and $193,900 (25%). A test and treat strategy using stool antigen remained relatively cost effective, even when the prevalence was 25%.

Conclusions

H. pylori screening and eradication can be an effective strategy for reducing rates of gastric cancer and peptic ulcers in high prevalence populations and our data suggest that use of stool antigen testing is the most cost effective approach.     

Antiadhesive Properties of Abelmoschus esculentus (Okra) Immature Fruit Extract against Helicobacter pylori Adhesion

Background

Traditional Asian and African medicine use immature okra fruits (Abelmoschus esculentus) as mucilaginous food to combat gastritis. Its effectiveness is due to polysaccharides that inhibit the adhesion of Helicobacter pylori to stomach tissue. The present study investigates the antiadhesive effect in mechanistic detail.

Methodology

A standardized aqueous fresh extract (Okra FE) from immature okra fruits was used for a quantitative in vitro adhesion assay with FITC-labled H. pylori J99, 2 clinical isolates, AGS cells, and fluorescence-activated cell sorting. Bacterial adhesins affected by FE were pinpointed using a dot-blot overlay assay with immobilized Lewis^b, sialyl-Lewis^a, H-1, laminin, and fibronectin. ¹²⁵I-radiolabeled Okra FE polymer served for binding studies to different H. pylori strains and interaction experiments with BabA and SabA. Iron nanoparticles with different coatings were used to investigate the influence of the charge-dependence of an interaction on the H. pylori surface.

Principal findings

Okra FE dose-dependently (0.2 to 2 mg/mL) inhibited H. pylori binding to AGS cells. FE inhibited the adhesive binding of membrane proteins BabA, SabA, and HpA to its specific ligands. Radiolabeled compounds from FE bound non-specifically to different strains of H. pylori, as well as to BabA/SabA deficient mutants, indicating an interaction with a still-unknown membrane structure in the vicinity of the adhesins. The binding depended on the charge of the inhibitors. Okra FE did not lead to subsequent feedback regulation or increased expression of adhesins or virulence factors.

Conclusion

Non-specific interactions between high molecular compounds from okra fruits and the H. pylori surface lead to strong antiadhesive effects.     

Sialylation of Campylobacter jejuni lipo-oligosaccharides: impact on phagocytosis and cytokine production in mice

Background

Guillain-Barré syndrome (GBS) is a post-infectious polyradiculoneuropathy, frequently associated with antecedent Campylobacter jejuni (C. jejuni) infection. The presence of sialic acid on C. jejuni lipo-oligosaccharide (LOS) is considered a risk factor for development of GBS as it crucially determines the structural homology between LOS and gangliosides, explaining the induction of cross-reactive neurotoxic antibodies. Sialylated C. jejuni are recognised by TLR4 and sialoadhesin; however, the functional implications of these interactions in vivo are unknown.

Methodology/Principal Findings

In this study we investigated the effects of bacterial sialylation on phagocytosis and cytokine secretion by mouse myeloid cells in vitro and in vivo. Using fluorescently labelled GM1a/GD1a ganglioside-mimicking C. jejuni strains and corresponding (Cst-II-mutant) control strains lacking sialic acid, we show that sialylated C. jejuni was more efficiently phagocytosed in vitro by BM-MΦ, but not by BM-DC. In addition, LOS sialylation increased the production of IL-10, IL-6 and IFN-β by both BM-MΦ and BM-DC. Subsequent in vivo experiments revealed that sialylation augmented the deposition of fluorescent bacteria in splenic DC, but not macrophages. In addition, sialylation significantly amplified the production of type I interferons, which was independent of pDC.

Conclusions/Significance

These results identify novel immune stimulatory effects of C. jejuni sialylation, which may be important in inducing cross-reactive humoral responses that cause GBS.     

TLR4 and TLR2 expression in biopsy specimens from antral and corporal stomach zones in Helicobacter pylori infections

Background:

It is not yet known which types of Toll-like receptors (TLRs) are most effective in Helicobacter pylori (H. pylori) recognition. It is also not known which gastric zones have the most prominent roles in TLR-mediated bacterial recognition. The aim of this work was to analyze the expression of TLR2 and TLR4 in biopsy specimens from H. pylori-infected patients.

Methods:

Thirty-eight patients with gastrointestinal disorders were divided into four groups in this study. The groups were: (A) H. pylori infection and peptic ulcer (n=15), (B) peptic ulcer only (n=5), (C) H. pylori infection only (n=10) and (D) control, with neither H. pylori infection nor peptic ulcer (n=8). Biopsy specimens from sites of redness or atrophic mucosa from gastric antrum and body in patients with gastritis were collected. RNAs from the antrum and body specimens were isolated. TLR2 and TLR4 mRNA expression was assessed by RT-PCR and quantified as densitometric ratios of TLR2 and TLR4/β-actin mRNA.

Results:

In the antral zones of H. pylori-infected patients (Groups A and C) TLR2 and TLR4 expression was significantly greater than in uninfected patients (Groups B and D) regardless of peptic ulcers (p < 0.05). In the gastric body samples TLR2 expression was significantly greater in Group C (H. pylori infection only) than in Group B (peptic ulcer only) and TLR4 expression was significantly greater in group A (H. pylori infection and peptic ulcer) than in Group B (peptic ulcer only) (p < 0.05). No significant differences in expression of TLR4 and TLR2 were observed between samples from the antrum and body in same groups.

Conclusions:

We conclude that H. pylori infection leads to significant increase in TLR2 and TLR4 molecules expression in antral region related to the control group. Considering the stimulatory effect of H. pylori on TLRs expression in the gastric tissue, we assume that colonization of H. pylori infection might occurs more in the gastric antral region than in the gastric body.Key Words: Helicobacter pylori, Toll-like receptors, TLR4; TLR2, Peptic ulcer     

New implications on genomic adaptation derived from the Helicobacter pylori genome comparison

Background

Helicobacter pylori has a reduced genome and lives in a tough environment for long-term persistence. It evolved with its particular characteristics for biological adaptation. Because several H. pylori genome sequences are available, comparative analysis could help to better understand genomic adaptation of this particular bacterium.

Principal Findings

We analyzed nine H. pylori genomes with emphasis on microevolution from a different perspective. Inversion was an important factor to shape the genome structure. Illegitimate recombination not only led to genomic inversion but also inverted fragment duplication, both of which contributed to the creation of new genes and gene family, and further, homological recombination contributed to events of inversion. Based on the information of genomic rearrangement, the first genome scaffold structure of H. pylori last common ancestor was produced. The core genome consists of 1186 genes, of which 22 genes could particularly adapt to human stomach niche. H. pylori contains high proportion of pseudogenes whose genesis was principally caused by homopolynucleotide (HPN) mutations. Such mutations are reversible and facilitate the control of gene expression through the change of DNA structure. The reversible mutations and a quasi-panmictic feature could allow such genes or gene fragments frequently transferred within or between populations. Hence, pseudogenes could be a reservoir of adaptation materials and the HPN mutations could be favorable to H. pylori adaptation, leading to HPN accumulation on the genomes, which corresponds to a special feature of Helicobacter species: extremely high HPN composition of genome.

Conclusion

Our research demonstrated that both genome content and structure of H. pylori have been highly adapted to its particular life style.     

Histidine hydrogen-deuterium exchange mass spectrometry for probing the microenvironment of histidine residues in dihydrofolate reductase

Background

Histidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C₂-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK _a values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR), an enzyme proposed to undergo multiple conformational changes during catalysis.

Methodology/Principal Findings

Using His-HDX-MS, the pK _a values and the half-lives (t _1/2) of HDX reactions of five histidine residues of apo-DHFR, DHFR in complex with methotrexate (DHFR-MTX), DHFR in complex with MTX and NADPH (DHFR-MTX-NADPH), and DHFR in complex with folate and NADP⁺ (DHFR-folate-NADP⁺) were determined. The results showed that the two parameters (pK _a and t _1/2) are sensitive to the changes of the microenvironment around the histidine residues. Although four of the five histidine residues are located far from the active site, ligand binding affected their pK _a, t _1/2 or both. This is consistent with previous observations of ligand binding-induced distal conformational changes on DHFR. Most of the observed pK _a and t _1/2 changes could be rationalized using the X-ray structures of apo-DHFR, DHFR-MTX-NADPH, and DHFR-folate-NADP⁺. The availability of the neutron diffraction structure of DHFR-MTX enabled us to compare the protonation states of histidine imidazole rings.

Conclusions/Significance

Our results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins.