The Hsp60 Protein of Helicobacter Pylori Exhibits Chaperone and Atpase Activities at Elevated Temperatures

Article The Hsp60 Protein of Helicobacter Pylori Exhibits Chaperone and ATPase Activities at Elevated Temperatures

Jose A. Mendoza * , Julian L. Ignacio and Christopher M. Buckley

Department of Chemistry and Biochemistry, California State University, San Marcos, CA 92096, USA; [email protected] (J.L.I.); [email protected] (C.M.B.) * Correspondence: [email protected]

Abstract: The heat-shock protein, Hsp60, is one of the most abundant proteins in Helicobacter pylori. Given its sequence homology to the Escherichia coli Hsp60 or GroEL, Hsp60 from H. pylori would be expected to function as a molecular chaperone in this organism. H. pylori is a type of bacteria that grows on the gastric epithelium, where the pH can ﬂuctuate between neutral and 4.5, and the intracellular pH can be as low as 5.0. We previously showed that Hsp60 functions as a chaperone under acidic conditions. However, no reports have been made on the ability of Hsp60 to function as a molecular chaperone under other stressful conditions, such as heat stress or elevated temperatures. We report here that Hsp60 could suppress the heat-induced aggregation of the enzymes rhodanese, malate dehydrogenase, citrate synthase, and lactate dehydrogenase. Moreover, Hsp60 was found to have a potassium and magnesium-dependent ATPase activity that was stimulated at elevated temperatures. Although, Hsp60 was found to bind GTP, the hydrolysis of this nucleotide could not be observed. Our results show that Hsp60 from H. pylori can function as a molecular chaperone under conditions of heat stress.

Citation: Mendoza, J.A.; Ignacio, J.L.; Keywords: Hsp60; molecular chaperone; protein aggregation; heat stress Buckley, C.M. The Hsp60 Protein of Helicobacter Pylori Exhibits Chaperone and ATPase Activities at Elevated Temperatures. BioChem 2021, 1. Introduction 1, 19–25. https://doi.org/ Helicobacter pylori is a Gram-negative, microaerophilic bacterium present in the stom- 10.3390/biochem1010002 ach of approximately half of the human population [1]. Chronic infection by this mi- croorganism can, in certain individuals, give rise to gastric and duodenal ulcers, gastric Academic Editor: Yehia Mechref adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma [2,3]. H. pylori sur- vives transient exposure to extreme acid prior to adherence and growth on the gastric Received: 2 March 2021 epithelium, where the pH can ﬂuctuate between neutral and 4.5, and the intracellular pH Accepted: 30 March 2021 Published: 3 April 2021 can be as low as 5.0 [4,5]. Under neutral and moderately acidic conditions, the heat-shock protein Hsp60 is one of the most abundant proteins in H. pylori.[4,6]. Given its sequence Escherichia coli H. pylori Publisher’s Note: MDPI stays neutral homology to the Hsp60 or GroEL [7], Hsp60 from would be ex- with regard to jurisdictional claims in pected to function as a molecular chaperone in this organism. The molecular chaperones published maps and institutional afﬁl- are a class of proteins that have been shown to facilitate the folding of nascent polypeptides, iations. activation of other proteins, and protection of native proteins against the effects of heat, and oxidative and acid stress [8–13]. The co-expression of H. pylori urease, Hsp60, and Hsp10 in E. coli was shown to substantially increase the activity of urease [14], which suggested that urease activity was protected by the heat-shock proteins. Furthermore, we previously demonstrated that Hsp60 from H. pylori had a chaperone activity under Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. acidic conditions [15]. No reports, however, have been made on the potential chaperone This article is an open access article activity of Hsp60 from H. pylori under heat-shock conditions. It is well known that protein distributed under the terms and denaturation can be induced at elevated temperatures and molecular chaperones, such conditions of the Creative Commons as Hsp60 may be needed to stabilize them against heat-induced aggregation. This study Attribution (CC BY) license (https:// was performed to determine if Hsp60 could function as a molecular chaperone at elevated creativecommons.org/licenses/by/ temperatures. The enzymes rhodanese, malate dehydrogenase (MDH), citrate synthase 4.0/). (CS), and lactate dehydrogenase (LDH) were used as substrate proteins for Hsp60 given

BioChem 2021, 1, 19–25. https://doi.org/10.3390/biochem1010002 https://www.mdpi.com/journal/biochem BioChem 2021, 1 20

their propensity to aggregate at elevated temperatures and because they were previously used as protein models to investigate the function of other heat-shock proteins [16–19]. Here, we report that Hsp60 prevented the heat-induced aggregation of these enzymes at elevated temperatures. The aggregation of Hsp60 alone was not observed under these conditions. It is also reported here that Hsp60 was able to hydrolyze ATP in a potassium and magnesium-dependent manner. Furthermore, Hsp60’s ATPase activity was found to be highly stimulated at elevated temperatures. Thus, our results show that Hsp60 from H. pylori can function as a molecular chaperone with an increased ability to hydrolyze ATP under conditions of heat stress.

2. Materials and Methods All the reagents used here were of analytical grade. IPTG was purchased from Gold Biotechnology and benzonase nuclease from Novagen. The protease inhibitor cocktail, rhodanese, MDH, CS, and LDH were purchased from Sigma Co. The H. pylori Hsp60 gene was synthesized by GenScript (Piscataway, NJ, USA) and inserted into the expression vector pET-22b (+). The resulting plasmid pET-Hsp60 was transformed into competent E. coli BL21 (DE3) cells using ampicillin resistance for selection. The expressed protein was purified using His GraviTrap chromatography (GE Healthcare, Chicago, IL, USA) followed by dialysis. Purification of Hsp60 was confirmed by SDS-PAGE [20] and its concentration determined by using a Bradford assay (BioRad, California, CA, USA). Protein aggregation assay: the aggregation of rhodanese, MDH, CS, and LDH was monitored by using light scattering. Protein (1 µM) samples were incubated in 50 mM Tris-HCl (pH 7.5). Measurement of the light scattering of each enzyme, alone or in the presence of Hsp60 (0.5–2 µM), was made with a Fluoromax-3 spectrophotometer using an excitation wavelength of 350 nm. The light scattered intensity at 90◦ was recorded at the same wavelength. The appropriate blanks were subtracted. ATPase activity assay: Hsp60 (1 µM) was incubated in a reaction mixture containing ◦ 50 mM Tris-HCl (pH 7.5), 1 mM KCl, 1 mM MgCl2, and 250 µM ATP at 25 C. Every 5 min, aliquots were removed and the released phosphate was detected using a Malachite Green Assay Kit (Cayman, UK) by measuring the absorbance at 620 nm. All the experiments were independently performed twice (n = 2), and the results are the average of both. Error bars were used to show the range or variability of the data.

3. Results and Discussion 3.1. Hsp60 Reduces Protein Thermal Aggregation Given the sequence homology of H. pylori Hsp60 to the E. coli Hsp60 protein or GroEL [7,21], Hsp60 from H. pylori would be expected to function as a molecular chaperone. Here, we tested the ability of Hsp60 from H. pylori to prevent the heat-induced aggregation of several heat-sensitive enzymes. Thus, to test the ability of Hsp60 to prevent the aggregation of these enzymes, each enzyme was incubated at 48 ◦C in the absence or presence of Hsp60, and the samples light scattering was measured after 60 min. As shown in Figure1, Hsp60 was able to reduce aggregation of the tested enzyme, resulting in decreased scattered light intensities in the case of rhodanese (96.2%), MDH (91.9%), CS (94.2%), and LDH (99.9%). The potential aggregation of Hsp60 alone was monitored by incubating the protein at the same temperature and measuring the sample light scattering with time. No signiﬁcant changes in light scattering were detected (not shown) that would indicate either Hsp60 aggregation or disassembly by an increase or decrease in the light scattering, respectively. BioChem 2021, 1, FOR PEER REVIEW 3 of 8

molecular chaperone with an increased ability to hydrolyze ATP under conditions of heat stress.

2. Materials and Methods All the reagents used here were of analytical grade. IPTG was purchased from Gold Biotechnology and benzonase nuclease from Novagen. The protease inhibitor cocktail, rhodanese, MDH, CS, and LDH were purchased from Sigma Co. The H. pylori Hsp60 gene was synthesized by GenScript (Piscataway, NJ, USA) and inserted into the expression vector pET-22b (+). The resulting plasmid pET-Hsp60 was transformed into competent E. coli BL21 (DE3) cells using ampicillin resistance for selection. The expressed protein was purified using His GraviTrap chromatography (GE Healthcare, Chicago, IL, USA) followed by dialysis. Purification of Hsp60 was confirmed by SDS-PAGE [20] and its concentration determined by using a Bradford assay (BioRad, California, CA, USA). Protein aggregation assay: the aggregation of rhodanese, MDH, CS, and LDH was monitored by using light scattering. Protein (1 μM) samples were incubated in 50 mM Tris-HCl (pH 7.5). Measurement of the light scattering of each enzyme, alone or in the presence of Hsp60 (0.5–2 μM), was made with a Fluoromax-3 spectrophotometer using an excitation wavelength of 350 nm. The light scattered intensity at 90° was recorded at the same wavelength. The appropriate blanks were subtracted. ATPase activity assay: Hsp60 (1 μM) was incubated in a reaction mixture containing 50 mM Tris-HCl (pH 7.5), 1 mM KCl, 1 mM MgCl2, and 250 μM ATP at 25 °C. Every 5 min, aliquots were removed and the released phosphate was detected using a Malachite Green Assay Kit (Cayman, UK) by measuring the absorbance at 620 nm. All the experiments were independently performed twice (n = 2), and the results are the average of both. Error bars were used to show the range or variability of the data.

3. Results and Discussion 3.1. Hsp60 Reduces Protein Thermal Aggregation Given the sequence homology of H. pylori Hsp60 to the E. coli Hsp60 protein or GroEL [7,21], Hsp60 from H. pylori would be expected to function as a molecular chaperone. Here, we tested the ability of Hsp60 from H. pylori to prevent the heat-induced aggregation of several heat-sensitive enzymes. Thus, to test the ability of Hsp60 to prevent the aggregation of these enzymes, each enzyme was incubated at 48 °C in the absence or presence of Hsp60, and the samples light scattering was measured after 60 min. As shown in Figure 1, Hsp60 was able to reduce aggregation of the tested enzyme, resulting in decreased scattered light intensities in the case of rhodanese (96.2%), MDH (91.9%), CS (94.2%), and LDH (99.9%). The potential aggregation of Hsp60 alone was monitored by incubating the protein at the same temperature and measuring the sample light scattering BioChem 2021, 1, FOR PEER REVIEW 3 of 7 with time. No significant changes in light scattering were detected (not shown) that would BioChem 2021, 1 indicate either Hsp60 aggregation or disassembly by an increase or decrease in the light 21 scattering, respectively.

100 100 80 80 60 60 40 40 20 20

% Suppression by Hsp60 of Light Scattered Light of Hsp60 by Suppression % 0

% SuppressionScattered byHsp60Light of 0 Rhod1234 MDH CS LDH Rhod1 MDH2 CS3 LDH4

Figure 1. Hsp60 reduced the aggregation of proteins as measured by the reduction in light scattered intensity.Figure 1. Hsp60 Hsp60 (2 reducedµM) was the incubated aggregation in 50 of mM proteins Tris-HCl as measured pH 7.5 at 48by◦ theC for reduction 10 min and in light then scat- 1 µM oftered rhodanese intensity. or malate Hsp60 dehydrogenase(2 μM) was incubated (MDH) in or 50 citrate mM Tris-HCl synthase pH (CS) 7.5 orat lactate48 °C for dehydrogenase 10 min and then 1 μM of rhodanese or malate dehydrogenase (MDH) or citrate synthase (CS) or lactate dehy- (LDH) was added and the light scattering was recorded after 60 min. n = 2 biologically independent drogenase (LDH) was added and the light scattering was recorded after 60 min. n = 2 biologically experiments, and the result is the average of both. independent experiments, and the result is the average of both. Figure2 shows the time course of the light scattering of MDH (panel A) and CS (panel Figure 2 shows the time course of the light scattering of MDH (panel A) and CS (panel B) when these enzymes were incubated at 48 ◦C in the absence or presence of Hsp60. Light scatteringB) when wasthese detected enzymes to were increase incubated within at a 48 few °C minutes in the absence after the or addition presence of of MDH Hsp60. or Light CS toscattering the buffer was without detected Hsp60. to increase within a few minutes after the addition of MDH or CS to the buffer without Hsp60.

3 x 105

5 2.5 x 10 1 x 107

5 MDH alone 2 x 10 CS alone

5 1.5 x 10 5 x 106

1 x 105 MDH + Hsp60 CS + Hsp60 5 x 104 0 LightScattering (A.U.) Light Scattering (A.U.) Scattering Light 0 024681012 0 5 10 15 20 25 30 35 40 Time (min) Time (min)

(A) (B)

FigureFigure 2. 2.Time Time course course of of the the light light scattering scattering of of MDH MDH (A ()A and) and CS CS (B ()B without) without (closed (closed circles) circles) or or with with Hsp60 Hsp60 (open (open circles). circles). ForFor samples samples containing containing Hsp60, Hsp60, this this was was incubated incubated at at 2 µ2M μM in in 50 50 mM mM Tris-HCl Tris-HCl pH pH 7.5 7.5 at at 48 48◦C °C for for 10 10 min min and and then then MDH MDH (1 (1µM) μM) or CSor CS (1 µ(1M) μM) was was added added at t =at 0.t = n 0. = 2n biologically= 2 biologically independent independen experiments,t experiments, and the and result the resu is thelt averageis the average of the two.of the two. As shown in Figure2 no increase in the intensity of scattered light was observed when MDH orAs CS shown were incubatedin Figure 2 with no Hsp60.increase Thus, in the these intensity results of demonstrate scattered light that was Hsp60 observed from H.when pylori MDHcan function or CS were as incubated a molecular with chaperone Hsp60. Thus, at elevated these results temperature demonstrate by preventing that Hsp60 thefrom heat-induced H. pylori can aggregation function as of a thesemolecular model chaperon proteins.e at Our elevated ﬁndings temperature are consistent by prevent- with aning expected the heat-induced chaperone roleaggregation for Hsp60 of given theseits mo highdel degreeproteins. of homologyOur findings to GroELare consistent [7,21]. Givenwith thatan expected Hsp60 fromchaperoneH. pylori rolehas for been Hsp60 reported given its to high exist degree as a mixture of homology of dimers to GroEL and tetramers[7,21]. Given [22], andthat theHsp60 tested from enzymes H. pylori were has monomeric been reported (rhodanese), to exist as dimeric a mixture (MDH of dimers and CS),and and tetramers tetrameric [22], (LDH), and the in thesetested aggregation enzymes were assays, monomeric Hsp60 and (rhodanese), enzymes were dimeric mixed (MDH in a 1:1and tetrameric CS), and totetrameric monomeric (LDH), molar in ratio.these aggregation assays, Hsp60 and enzymes were mixedFigure in 3a shows1:1 tetrameric that sub-stoichiometric to monomeric molar amounts ratio. of Hsp60 did not suppress completely the observedFigure 3 light shows scattered that sub-stoichiometric by rhodanese. The amounts suppression of Hsp60 of 42% did lightnot suppress scattered com- by rhodanesepletely the was observed observed light with scattered a 1:0.5 by tetrameric rhodanese. Hsp60 The to suppression rhodanese stoichiometricof 42% light scattered ratio. Asby shown rhodanese in the was ﬁgure, observed complete with a suppression 1:0.5 tetrameric of lightHsp60 scattered to rhodanese by rhodanese stoichiometric in the ra- presencetio. As shown of Hsp60 in the occurred figure, only complete with asuppression 1:1 tetrameric of light Hsp60 scattered to monomeric by rhodanese rhodanese in the stoichiometricpresence of Hsp60 ratio. Ouroccurred light only scattering with a measurements 1:1 tetrameric forHsp60 Hsp60 to couldmonomeric not detect rhodanese any structuralstoichiometric changes ratio. at the Our tested light temperatures scattering measurements suggesting that for Hsp60 Hsp60 retained could itsnot quaternary detect any structurestructural during changes these at experiments.the tested temperatures Functional suggesting lower oligomers that Hsp60 have beenretained reported its quater- for thenary Hsp60 structure protein during from M.these tuberculosis experiments.[23]. Functional The unusual lower quaternary oligomers structure have been of H. reported pylori Hsp60 raises the question if, under in vivo or certain in vitro conditions, Hsp60 could

BioChem 2021, 1 22

undergo oligomerization like the Hsp60 from M. tuberculosis into a double ring structure

BioChem 2021, 1, FOR PEER REVIEW like that of GroEL [24]. Whereas, in the experiments reported here, Hsp60 by itself5 of 8 was an effective chaperone, it has been shown that the folding process of newly synthesized proteins in E. coli involves several molecular chaperones [8]. Thus, it remains to be seen BioChem 2021, 1, FOR PEER REVIEW whether Hsp60 from H. pylori can support the folding of other proteins and interact5 of with8

other chaperones. Moreover, the nature of the Hsp60-protein complexes detected here and the100 mechanismRho alone of release of the bound proteins remains to be elucidated.

10060 Rho alone + 0.5 Hsp60 4080

2060 + 0.5 Hsp60+ 1.0 Hsp60 Relative Light Scattering (A.U.) 400 1 2 3 Hsp604 alone 20 + 1.0 Hsp60 Relative Light Scattering (A.U.) 0 1 2 3 Hsp604 alone Figure 3. Effect of Hsp60 on the light scattered by rhodanese. Rhodanese (1 μM) was incubated µ withoutFigure Hsp60 3. Effect or with of Hsp60 (0.5 on theμM) light or with scattered Hsp60 by (1 rhodanese.μM) in Tris- RhodaneseHCl, pH 7.5 (1 at 4M)8 °C was and incubated then the lightwithout scattering Hsp60 was or with recorded Hsp60 after (0.5 60µM) min. or n with = 2 Hsp60biological (1 µlyM) independent in Tris-HCl, experiments, pH 7.5 at 48 and◦C and the then Figure 3. Effect of Hsp60 on the light scattered by rhodanese. Rhodanese (1 μM) was incubated resultthe is lightthe average scattering of the was two. recorded after 60 min. n = 2 biologically independent experiments, and the without Hsp60 or with Hsp60 (0.5 μM) or with Hsp60 (1 μM) in Tris-HCl, pH 7.5 at 48 °C and then result is the average of the two. 3.2.the lightATPase scattering Activity was ofrecorded Hsp60 after 60 min. n = 2 biologically independent experiments, and the result3.2. is the ATPase average Activity of the of two. Hsp60 Given the ability of H. pylori Hsp60 to function as a molecular chaperone, it would Given the ability of H. pylori Hsp60 to function as a molecular chaperone, it would be3.2. expected ATPase Atoctivity have an of ATPaseHsp60 activity like its homologue the E. coli Hsp60 protein or GroELbe expected[25]. It has to been have reported an ATPase that activityboth potassium like its homologueand magnesium the E. are coli requiredHsp60 for protein the or Given the ability of H. pylori Hsp60 to function as a molecular chaperone, it would ATPaseGroEL activity [25]. of It hasthe E. been coli reported Hsp60 or that GroEL both protein potassium [26]. and Potassium magnesium appeared are required to increase for the be expected to have an ATPase activity like its homologue the E. coli Hsp60 protein or the affinityATPase of activity GroEL of for the ATP,E. coli whileHsp60 magnesium or GroEL proteinis required [26]. since Potassium the magnesium appeared- tobound increase GroEL [25]. It has been reported that both potassium and magnesium are required for the formthe of afﬁnitythe nucleotide of GroEL binds for ATP, GroEL. while Thus, magnesium the ability is requiredof Hsp60 since to hydrolyze the magnesium-bound ATP was ATPase activity of the E. coli Hsp60 or GroEL protein [26]. Potassium appeared to increase testedform in the of theabsence nucleotide or presence binds of GroEL. potassium Thus, and/or the ability magnesium. of Hsp60 The to results hydrolyze in Figure ATP 4 was the affinity of GroEL for ATP, while magnesium is required since the magnesium-bound showtested that inpotassium the absence and or magnesium presence of are potassium required and/or by Hsp60 magnesium. to hydrolyze The resultsATP. W ine Figurealso 4 form of the nucleotide binds GroEL. Thus, the ability of Hsp60 to hydrolyze ATP was examinedshow thatthe potassiumpotential GTPase and magnesium activity of are Hsp60. required However, by Hsp60 as shown to hydrolyze in Figure ATP. 4, Wethe also tested in the absence or presence of potassium and/or magnesium. The results in Figure 4 hydrolysisexamined of GTP the by potential Hsp60 from GTPase H. pylori activity was of not Hsp60. observed However, in the aspresence shown of in potassium Figure4, the show that potassium and magnesiumH. are pylori required by Hsp60 to hydrolyze ATP. We also and/orhydrolysis magnesium. of GTP by Hsp60 from was not observed in the presence of potassium examinedand/or the magnesium. potentialB GTPase activity of Hsp60. However, as shown in Figure 4, the hydrolysis of GTP by Hsp60 from H. pylori was not observed in the presence of potassium and/or magnesium. 100 B

100 60

80 40

60 20

Relative Hydrolysis (%) Hydrolysis Relative 40 0 K+/ATP Mg2+/ATP K+/Mg2+ATP K+/GTP Mg2+/GTP K+/Mg2+/GTP 20

Relative Hydrolysis (%) Hydrolysis Relative µ Figure0 4. ATP or GTP hydrolysis by Hsp60. Hsp60 (1 M) was incubated with 50 mM Tris-HCL, ◦ pH 7.5 atK+/ATP 25 MgC2+ in/ATP the K+/Mg presence2+ATP K+/GTP of Mg 12+/GTP mM K+ KCl/Mg2+/GTP or 1 mM MgCl2 or both and 250 µM ATP, and in the Figurepresence 4. ATP of or 1 GTP mM potassiumhydrolysis orby 1 Hsp60. mM magnesium Hsp60 (1 μ orM) both was and incubated 250 µM with GTP. 50 Then, mM hydrolysis Tris-HCL, of ATP pH 7.5 at 25 °C in the presence of 1 mM KCl or 1 mM MgCl2 or both and 250 μM ATP, and in the or GTP was determined, as described in Materials and Methods. n = 2 biologically independent presence of 1 mM potassium or 1 mM magnesium or both and 250 μM GTP. Then, hydrolysis of experiments, and the result is the average of the two. ATPFigure or 4.GTP ATP was or determinedGTP hydrolysis, as described by Hsp60. in Hsp60 Materials (1 μ M)and was Methods. incubated n = 2with biological 50 mMly Tris -HCL, pH 7.5 at 25 °C in the presence of 1 mM KCl or 1 mM MgCl2 or both and 250 μM ATP, and in the independent3.3. Binding experiments, of GTP to and Hsp60 the result is the average of the two. presence of 1 mM potassium or 1 mM magnesium or both and 250 μM GTP. Then, hydrolysis of To determine if GTP was not hydrolyzed by Hsp60 because of the protein’s inability to 3.3.ATP Binding or GTP wasof GTP determined to Hsp60, as described in Materials and Methods. n = 2 biologically independentbind the experiments, nucleotide, weand used the result the ﬂuorescent is the average nucleotide of the two. analog TNP-GTP [27]. An increase inTo the determine ﬂuorescence if GTP of was TNP-GTP, not hydrolyzed in the presence by Hsp60 of a because protein, of is t consideredhe protein’s a inability reliable test to3.3. bind forBinding the the assessment nucleotide,of GTP to ofHsp60 we the used nucleotide-binding the fluorescent capacity nucleotide of the analog protein TNP [28-GTP]. Figure [27]5. shows An increase in the fluorescence of TNP-GTP, in the presence of a protein, is considered a To determine if GTP was not hydrolyzed by Hsp60 because of the protein’s inability reliable test for the assessment of the nucleotide-binding capacity of the protein [28]. to bind the nucleotide, we used the fluorescent nucleotide analog TNP-GTP [27]. An Figure 5 shows that Hsp60 had a significant effect on the fluorescence spectrum of TNP- increase in the fluorescence of TNP-GTP, in the presence of a protein, is considered a GTP, suggesting that Hsp60 has a GTP-binding site. The significance of the binding of reliable test for the assessment of the nucleotide-binding capacity of the protein [28]. GTP to Hsp60 in the function and structure of the chaperone remains to be determined. Figure 5 shows that Hsp60 had a significant effect on the fluorescence spectrum of TNP- GTP, suggesting that Hsp60 has a GTP-binding site. The significance of the binding of GTP to Hsp60 in the function and structure of the chaperone remains to be determined.

BioChem 2021, 1, FOR PEER REVIEW 5 of 7

ATP or GTP was determined, as described in Materials and Methods. n = 2 biologically independent experiments, and the result is the average of the two. ATP or GTP was determined, as described in Materials and Methods. n = 2 biologically independ- ent3.3. experiments, Binding of and GTP the to result Hsp60 is the average of the two. To determine if GTP was not hydrolyzed by Hsp60 because of the protein’s inability 3.3.to Binding bind the of nucleotide,GTP to Hsp60 we used the fluorescent nucleotide analog TNP-GTP [27]. An in- creaseTo determine in the fluorescence if GTP was of TNP-GTP,not hydrolyzed in the by presence Hsp60 ofbecause a protein, of the is considered protein’s inability a reliable BioChem 2021, 1 23 to testbind for the the nucleotide, assessment we of used the thenucleotide-bin fluorescentding nucleotide capacity analog of the TNP-GTP protein [28].[27]. FigureAn in- 5 creaseshows in thatthe fluorescence Hsp60 had a of significant TNP-GTP, effect in the on presence the fluorescence of a protein, spectrum is considered of TNP-GTP, a reliable sug- testgesting for the that assessment Hsp60 has of athe GTP-binding nucleotide-bin site.ding The capacity significance of the of proteinthe binding [28]. Figureof GTP 5 to showsHsp60that that in Hsp60 Hsp60the function had had a a significant andsignificant structure effect effect of on the on the thechaperone fluorescence fluorescence remains spectrum spectrum to be determined. of of TNP-GTP, TNP-GTP, suggesting sug- gestingthat that Hsp60 Hsp60 has has a GTP-binding a GTP-binding site. Thesite. significanceThe significance of the of binding the binding of GTP of to Hsp60GTP to in the Hsp60function in the function and structure and structure of the chaperone of the chaperone remains remains to be determined. to be determined.

20 TNP-GTP + Hsp60

20 15 TNP-GTP + Hsp60 TNP-GTP

15 10 TNP-GTP

10 5

5 0 Fluorescence Intensity (A.U.) Intensity Fluorescence 500 520 540 560 580 600 Wavelength (nm) 0 Fluorescence Intensity (A.U.) Intensity Fluorescence 500 520 540 560 580 600 Wavelength (nm) Figure 5. Binding of GTP to Hsp60 analyzed by fluorescence spectroscopy. The fluorescence of TNP-GTPFigure at 5. 50Binding μM in 600 of GTP μL of to 50 Hsp60 mM tris analyzed buffer, bypH fluorescence7.8, with and spectroscopy. without Hsp60 The was fluorescence measured of FigureusingTNP-GTP 5. an Binding excitation at of 50 GTP µwavelengthM to in Hsp60 600 µ Lof analyzed of 409 50 nm mM andby tris fluorescence its buffer, emission pH 7.8,spectroscopy.recorded with and from without The 500 fluorescenceto 600 Hsp60 nm. was n of= measured2 bio- TNP-GTPlogicallyusing at independent an50 excitationμM in 600 experiments, wavelengthμL of 50 mM and of tris 409 th buffer,e nmresult and pH is itsthe7.8,emission averagewith and of recorded without the two. Hsp60 from 500 was to measured 600 nm. n = 2 using biologicallyan excitation independent wavelength experiments,of 409 nm and and its theemission result recorded is the average from of500 the to two. 600 nm. n = 2 bio- logically3.4. Temperature independent Dependence experiments, of theand ATPase the result Activity is the average of Hsp60 of the two. 3.4. Temperature Dependence of the ATPase Activity of Hsp60 Given that the ATPase of GroEL was previously shown to be stimulated at elevated 3.4.temperatures TemperatureGiven [29,30],Dependence that the we ATPase investigated of the ofATPase GroEL the Activity wasability previously of of Hsp60 Hsp60 shownfrom H. to pylori be stimulated to hydrolyze at elevated ATP temperatures [29,30], we investigated the ability of Hsp60 from H. pylori to hydrolyze ATP at elevatedGiven that temperatures. the ATPase ofFigure GroEL 6 shows was previous the ATPasely shown activities to be of stimulated Hsp60 in atthe elevated 22–67 °C ◦ temperaturesrangeat elevatedin the [29,30], presence temperatures. we ofinvestigated potassium Figure the6and shows ability magnesium. the of Hsp60 ATPase As from activitiesshown H. pylori in of the Hsp60to figure,hydrolyze in maximum the 22–67ATP C range in the presence of potassium and magnesium. As shown in the figure, maximum at ATPaseelevated activity temperatures. was observed Figure 6at shows 62 °C. the Thus, ATPase it would activities be expected of Hsp60 that in the 22–67ATPase °C of ATPase activity was observed at 62 ◦C. Thus, it would be expected that the ATPase of rangeHsp60 in fromthe presence H. pylori of observed potassium here and in that ma gnesium.temperature As range,shown would in the befigure, needed maximum to trigger Hsp60 from H. pylori observed here in that temperature range, would be needed to trigger ATPasethe release activity of boundwas observed proteins at from 62 °C. Hsp60, Thus, li itke would in GroEL-mediated be expected that protein the ATPase folding of[8]. the release of bound proteins from Hsp60, like in GroEL-mediated protein folding [8]. Hsp60However, from H.this pylori question observed awai herets further in that experimentation. temperature range, would be needed to trigger However, this question awaits further experimentation. the release of bound proteins from Hsp60, like in GroEL-mediated protein folding [8]. However, this question awaits further experimentation. 12

10 12 8 10 6 8 4 6 2 Phosphate Released 4 (micromol/min/micromol Hsp60) (micromol/min/micromol 0 2 20 30 40 50 60 70 Phosphate Released o (micromol/min/micromol Hsp60) (micromol/min/micromol Temperature ( C) 0 20 30 40 50 60 70 Figure 6. TemperatureTemperature dependence ( ofoC) the ATPase activity of Hsp60 in the presence of potassium and ◦ Figuremagnesium 6. Temperature in the 22–67dependenceC range. of the Hsp60 ATPase (1 µ M)activi wasty incubatedof Hsp60 in at the the presence indicated of temperature potassium with and50 magnesium mM Tris-HCl, in the pH 22–67 7.5, 1°C mM range. KCl, Hsp60 1 mM (1 MgCl μM) 2was, and incubated 250 µM ATP,at the and indicated the ATPase temperature activity was Figuredetermined 6. Temperature as described dependence in Materials of the ATPase and Methods. activity n of = 2Hsp60 biologically in the presence independent of potassium experiments, and and magnesiumthe result is in the the average 22–67 °C of range. the two. Hsp60 (1 μM) was incubated at the indicated temperature

4. Conclusions Our results demonstrate that Hsp60 from H. pylori can function as a molecular chaperone at heat-shock temperatures. Given that that Hsp60 from H. pylori contains a signiﬁcant exposure of hydrophobic surfaces [15] and that these are also present in proteins subjected to elevated temperatures, our results suggest that the interaction between Hsp60 and other proteins undergoing heat-induced denaturation may be mediated by hydrophobic interactions. The ability of Hsp60 to protect a protein exposed to elevated temperatures BioChem 2021, 1 24

is signiﬁcant in light of the fact that under in vivo conditions of heat shock, like those in which the synthesis of the Hsp60 proteins is stimulated, partially thermally-denatured proteins may actually exist.

Author Contributions: J.A.M. designed the study, directed the project, and drafted the manuscript. J.L.I. and C.M.B. carried out the experiments and made the graphs and figures. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by a development grant to J.A.M from the California State University Program for Education and Research in Biotechnology (CSUPERB). Institutional Review Board Statement: The study did not involve humans or animals. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest.

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