Characterizations of the Interactions between Escherichia coli

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Characterizations of the Interactions between Escherichia coli Periplasmic Chaperone HdeA and its Native Substrates during Acid Stress Xing-Chi Yu, Chengfeng Yang, Jienv Ding, Xiaogang Niu, Yunfei Hu, and Changwen Jin Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00724 • Publication Date (Web): 10 Oct 2017 Downloaded from http://pubs.acs.org on October 11, 2017

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Biochemistry

Characterizations of the Interactions between Escherichia coli Periplasmic Chaperone HdeA and its Native Substrates during Acid Stress

Xing-Chi Yu1,2,§, Chengfeng Yang1,2,§, Jienv Ding2,3, Xiaogang Niu1,2, Yunfei Hu1,2,*, Changwen Jin1,2,3,4,*

1

College of Chemistry and Molecular Engineering, 2 Beijing Nuclear Magnetic Resonance Center, 3 College of Life Sciences, 4 Beijing National Laboratory for Molecular Sciences, Peking University, Beijing 100871, China Running Title: Substrate-regulated activation of HdeA

§ These authors contributed equally to this work. * To whom correspondence should be addressed: Yunfei Hu, Beijing Nuclear Magnetic Resonance Center, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China, Tel.: (86)-10-62767043, E-mail: [email protected]; Changwen Jin, Beijing Nuclear Magnetic Resonance Center, College of Chemistry and Molecular Engineering, College of Life Sciences, Peking University, Beijing 100871, China, Tel.: (86)-10-62756004, E-mail: [email protected].

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ABSTRACT The bacterial acid-resistant chaperone HdeA is a “conditionally disordered” protein that functions at low pH when it transits from a well-folded dimer to an unfolded monomer. The dimer dissociation and unfolding processes result in exposure of hydrophobic surfaces that allows binding to a broad range of client proteins. To fully elucidate the chaperone mechanism of HdeA, it is crucial to understand how the activated HdeA interacts with its native substrates during acid stress. Herein, we present an NMR study of the pH-dependent HdeA-substrate interactions. Our results show that the activation of HdeA is not only induced by acidification, but also regulated by the presence of unfolded substrates. The variable extent of unfolding of substrates differentially regulates the HdeA-substrate interaction, and the binding further affects the HdeA conformation. Finally, we show that HdeA binds its substrates heterogeneously, and the “amphiphilic” model for HdeA-substrate interaction is discussed.

TOC

Keywords: chaperone, acid resistant, protein interaction, HdeA, NMR.

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Introduction Pathogenic enteric bacteria have developed an intricate acid-resistant system to survive the acidic environment when passing through the mammdsalian stomach before colonizing the intestine (1-4). In the bacterial periplasmic space, two important chaperones HdeA and HdeB have been identified to play essential roles in preventing acid-induced aggregation of substrate proteins and helping them refold upon pH neutralization (5-7). Unlike the ATP-dependent chaperones such as DnaK and GroEL that rely on ATP-driven conformational changes to mediate the refolding or unfolding of their substrates, HdeA/B chaperones function in an energy-independent manner (3,8,9). In addition, HdeA has been classified as a “conditionally disordered” protein, as it is functionally activated only at low pH via an acid-induced dimer-to-monomer transition and partial unfolding (10-11). Although the importance of protein disorder has been widely suggested, direct evidences demonstrating the correlation between protein disorder and function are yet scarce due to the difficulty in studying disordered proteins (12). Therefore, investigations on HdeA may provide valuable insights in further understanding the relationships between protein disorder and function. Based on previous investigations, a general mechanism for HdeA activation has been established: HdeA exists as a well-folded dimer at neutral pH and is functionally inactive; the decrease of pH causes the protonation of several acidic residues and destabilizes the structure, leading to partial unfolding and a dimer-to-monomer transition; the unfolded HdeA exposes a large hydrophobic surface, enabling its interactions with a variety of unfolded substrate proteins (5,10,11,13-21). However, since the “activated state” of HdeA is largely disordered and its interactions with substrates relatively promiscuous (15), experimental data on the structural details of HdeA-substrate complexes are difficult to obtain. The lack of information on HdeA “in action” limits our understanding of its functional mechanism, as well as of how the conditional disordered structural feature is correlated with chaperone function. Although many native substrates of HdeA have been identified (18), previous studies mostly used model substrates such as malate dehydrogenase (MDH) and alcohol dehydrogenase (ADH) (13-16, 22). Since it has been observed that HdeA shows certain selectivity in binding substrate proteins (18, 21), we believe that using native protein substrates for studying HdeA-substrate interactions would better mimic the actual acid stress 3


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response in the bacterial periplasmic space. Herein we report an NMR study on the pH-dependent interactions between HdeA and three of its native substrates, SurA, MalE and OppA. We show that the acid-induced structural transition of HdeA occurs at approximately pH 3 in the absence of substrates, whereas the interaction between HdeA and its native substrates could actually start at pH higher than 3. We further observe that the pH conditions for HdeA to start interacting with different substrates depend on the intrinsic pH responses of the substrates themselves, and the exposed hydrophobic surface area of substrates may contribute to regulating the HdeA-substrate interactions. Finally, we demonstrate that HdeA functions as an amphiphilic molecule and binds its native substrates at variable stoichiometry, strongly supporting the “amphiphilic” model for HdeA chaperone function.

Materials and Methods Protein expression and purification The E. coli hdeA, surA, malE and oppA genes as well as the hdeA truncation mutant genes were cloned into pET-28a(+) plasmid (Novagen) and transformed into E. coli BL21(DE3)-T1R or BL21 Star(DE3)-T1R strains (Sigma-Aldrich) for protein expression. The cell cultures were first grown in 1L Luria-Bertani medium at 35 °C with 50 mg/L kanamycin. When the OD600 reached 0.8, the cells were collected by centrifugation at 4,000 g and resuspended in 500 mL M9 minimal medium with NH4Cl and glucose as the nitrogen and carbon sources, and with 50 mg/L kanamycin. After shaking for 1 hour at 35 °C, protein expression was induced by adding isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.4 mM. After 8 hr induction, the cells were centrifuged at 7,000 g, resuspended in 30 mM Tris-HCl buffer (pH 8.5) and frozen at -80 °C. For preparations of 13

C/15N-labeled or

15

N-labeled proteins,

15

NH4Cl and [13C6]-glucose or

15

NH4Cl only were

used in the M9 media. For purification of all proteins, the cells were freeze-thawed and subsequently sonicated. After centrifugation at 20,000 g, the supernatants were collected and loaded onto the anion exchange chromatography (Q-Sepharose, GE Healthcare). A sodium chloride gradient between 0 and 500 mM was used for protein elution using a buffer containing 30 mM Tris-HCl (pH 8.5). For HdeA protein, this initial purification procedure could be replaced by 4


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acid precipitation at pH 3.0 and neutralization back to pH 7.0, during which process most of the other endogenous proteins from the E. coli cells become precipitated. A subsequent gel-filtration chromatography (Superdex-75, GE Healthcare) was performed for all proteins and samples with purity > 90% could be obtained as judged by SDS-PAGE. The NMR samples were prepared in buffers containing 50 mM sodium phosphate and 45 mM citric acid at pH conditions ranging from 7.0 to 2.0. D2O was added to 10% for field lock and 2,2-dimethyl-2-silapentanesulfonic acid was used as the internal chemical shift reference.

Sample preparations for HdeA-substrate complexes at different pH conditions For pH-dependent interaction experiments monitored by 2D NMR or other methods, the HdeA-substrate complex samples were prepared using the following procedure: the HdeA and substrate protein samples were first mixed at neutral pH (pH = 7) at the desired molar ratios, and subsequently changed to the target pH conditions via extensive buffer exchange by using the Millipore centrifugation tubes with 3-kDa molecular weight cutoff. Since the substrates alone are prone to aggregation/precipitation in acidic environments, mixing HdeA with the substrates at neutral pH prior to acidification is not only an essential step for preventing the substrate aggregation/precipitation and ensuring an accurate control of protein concentrations, but also a better mimic of the acid-stress response process occurring in the bacterial periplasmic space. In the NMR monitored interaction experiments, since we use relatively high protein concentrations and small step sizes of pH variations, accurate calibrations of the sample pH values are essential for ensuring data consistency throughout all experiments. To this end, we prepared a stock of buffers containing 50 mM sodium phosphate and 45 mM citric acid at different pH conditions and calibrated the pH values using a pH electrode. The pH conditions of the stock of buffers were checked from time to time to ensure that no pH changes occurred, and all samples were prepared via extensive buffer exchanges against these same buffer stocks. Since HdeA itself is highly sensitive to pH changes, the chemical shifts of quite a few of its NMR resonances can be used to directly monitor the sample pH condition. In the experiments that we are observing the

15

N-HdeA signals, in particular the HdeA-substrate

interaction experiments in the pH range of 3.0-4.0, signals from well-folded free HdeA are 5


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visible in nearly all of the spectra, and those peaks that are highly sensitive to pH were carefully compared to verify that the pH values were consistent. In addition, a control sample containing

15

N-labeled HdeA was simultaneously prepared and underwent the same buffer

exchange processes for the cases that we could not directly observe the desired 15N-labeled HdeA signals in the experimental samples. Furthermore, additional measures were taken to directly confirm the pH consistency among various samples during the experiments. These include: 1) direct pH measurements of the NMR samples using a pH-meter with a micro-electrode, and 2) measurements of the 19F spectra of a chemical shift-based pH sensor 4-fluoroanilines (23) added in the NMR samples. Detailed results (as summarized in Supporting Table S1 and Supporting Fig. S1) demonstrated that the sample pH values were not perturbed by the presence of excess amount of substrate proteins.

Chemical shift assignments For assigning the HdeA signals in the HdeA-SurA complex, a sample containing 0.5 mM 13

C/15N-labeled HdeA and 1.0 mM unlabeled SurA was used. NMR spectra were acquired at

25 °C on Bruker Avance 600-, 700- and 800-MHz spectrometers, all equipped with four RF channels and a triple-resonance cryo-probe with pulsed field gradients. Two-dimensional (2D) 15

N-edited heteronuclear single quantum coherence (HSQC) spectroscopy, three-dimensional

(3D) HNCA, HNCACB, CBCA(CO)NH, HNCO, HBHA(CO)NH, (H)CC(CO)NH, (H)CCH-COSY, and (H)CCH-TOCSY experiments were collected for chemical shift assignments (24). For chemical shift assignments of HdeA alone at pH 3.0, a 13C/15N-labeled HdeA sample was used to collect the 2D

15

CBCA(CO)NH,

HBHA(CO)NH,

HNCO,

N-edited HSQC and conventional 3D HNCA, HNCACB,

(H)CCH-TOCSY experiments (23). 3D

15

N- and

(H)CC(CO)NH, 13

(H)CCH-COSY,

and

C-edited NOESY-HSQC spectra (mixing

times 100 ms) were performed to confirm the assignments and generate inter-proton distance restraints. 3D

13

C/15N-filtered

was recorded using a

13

C-edited NOESY-HSQC experiment (mixing time 100 ms)

13

C/15N-labeled and unlabeled mixed HdeA sample to obtain

inter-subunit distance restraints (25). All spectra were processed using the software package NMRPipe (26) and analyzed by the program NMRView (27). 6


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NMR structure calculation The solution structure of HdeA homodimer at pH 3.0 was calculated using the program CYANA (28) and refined by AMBER (29). Distance restraints were derived from inter-proton nuclear Overhauser effect (NOE). Inter-subunit distance restraints were extracted from the 15

N- and 13C-edited NOESY-HSQC spectra using the X-ray crystal structure of HdeA (protein

data bank accession number 1DJ8) as a reference, and further confirmed by the 13

C/15N-filtered

13

C-edited NOESY-HSQC experiment. Dihedral angles (φ and ψ) were

predicted from chemical shifts using TALOS (30). The initial structures were calculated using the CANDID module of the CYANA program (31). The 20 lowest-energy structures were selected as models for SANE to extend the NOE assignments (32). Two hundred structures were calculated by CYANA, and the 100 lowest-energy structures were used as initial structures and refined using AMBER. Finally, the 20 lowest-energy conformers were selected as representative structures.

ANS binding assays – The ANS binding assays for the SurA, MalE and OppA proteins were measured by the increase of fluorescence intensity for ANS using a Hitachi F7000 fluorescence spectrophotometer at 25 °C (33). All protein concentrations were 0.25 mg/mL (25 µM for HdeA and its truncated mutants, 5 µM for SurA, 4 µM for OppA and 6 µM for MalE), and the buffers were the same as used in the NMR titration experiments. Each protein sample was mixed with 100 µM ANS at specific pH values for 30 mins and all spectroscopy were collected three times for average. The samples were excited at 395 nm, and the emissions were recorded from 410 to 600 nm.

Size-exclusion chromatography Size-exclusion chromatography were performed using 16 mm × 70 cm or 16 × 100 cm columns (Superdex-200, GE Healthcare). For preparation of the HdeA-MalE and HdeA-OppA complexes, the proteins were mixed in a buffer containing 45 mM critic acid and 50 mM sodium phosphate at pH 7.0 and subsequently adjusted to pH 2.0 through extensive buffer exchange against a buffer containing 45 mM critic acid and 50 mM sodium 7


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phosphate at pH 2.0 using the Millipore centrifugation tubes with 3-kDa molecular weight cutoff. The mixtures at pH 2.0 were incubated at 37 °C for 1 hour and centrifuged at 10,000 g for 10 min before loading onto the columns. The control samples of OppA, MalE and HdeA alone were treated in the same way. The proteins were eluted at a flow rate of 2.0 ml/min using the same buffer at pH 2.0. The retention volumes for standard protein markers were calibrated at pH 7.0 using the same buffer containing 45 mM critic acid and 50 mM sodium phosphate.

Results Acid-induced structural transition of HdeA occurs around pH 3 It has been demonstrated that Escherichia coli HdeA adopts a well-folded dimer structure at neutral pH which is functionally inactive, and becomes active when it dissociates into partially unfolded monomers at low pH conditions (pH < 3) (5,13). To investigate the interaction between HdeA and its substrate proteins, we first studied the pH-induced conformational changes of HdeA alone. We performed a pH titration experiment covering the pH range from 7.0 to 2.0 monitored by 2D NMR spectroscopy (Supporting Fig. S2), which is similar to but extends the pH range investigated compared to a previous NMR study that covered the pH range from 6.0 to 3.0 (20). The titration results demonstrated that the acid-induced structural changes of HdeA could be divided into two stages with a transition point at approximately pH 3.0. In the pH range of 7.0-3.0, the 1H-15N heteronuclear single quantum coherence (HSQC) spectra show well-dispersed resonances and the chemical shift of each peak changes moderately with the varied pH, which is consistent with the previous report (20). When pH goes below 3.0, new signals are observed and mainly cluster in the center of the spectra, suggesting the appearance of an unfolded state or multiple unfolded states, whereas the well-dispersed peaks of the folded conformation gradually decrease in intensity. At pH 2.0, HdeA becomes significantly unfolded with strong signals clustering in the center of the spectrum, while the peaks of the folded conformation mostly disappear. The above results demonstrate that critical pH value for HdeA to start unfold is around pH 3.

pH-induced unfolding of three native substrates of HdeA 8


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Since many substrates of HdeA have been identified in vivo (18), we first probed the pH-induced unfolding processes of three native substrates SurA, MalE and OppA before investigating their interactions with HdeA. We expressed 15N-labeled SurA, MalE and OppA proteins and performed pH titration experiments in the absence of HdeA monitored by 1H-15N HSQC spectra (Supporting Fig. S3). The results show that all three proteins adopt well-folded three-dimensional structures at pH above 4 and become unfolded at pH below 3. At pH 3.5, both SurA and MalE are partially unfolded as the spectra show increased signals clustering in the central region, while the signal intensities of the well-dispersed peaks slightly decreased. Upon further acidification, SurA becomes fully unfolded at pH 3 while MalE becomes fully unfolded at pH 2.5 and lower. In contrast to the above two substrates, OppA appears much more sensitive to acid stress and is already fully unfolded at pH 3.5. In addition, we conducted the ANS binding assays for the three substrates to evaluate their exposure of hydrophobic surface area at different pH conditions. Consistent with the NMR observations, OppA exhibits a drastic structural change from pH 4.0 to 3.5 and exposes significantly larger hydrophobic surface area at pH 3.5 compared to the other two proteins (Supporting Fig. S4).

pH-dependent interactions between HdeA and its native substrate SurA In order to characterize the HdeA-substrate interactions during acid stress, we chose SurA as one representative substrate to repeat its pH titration experiment in the presence of HdeA.

15

N-labeled SurA was mixed with unlabeled HdeA at pH 7.0, and the mixture was

subsequently buffer exchanged to various pH conditions (more details of the sample preparation and pH calibration are described in Materials and Methods). The HSQC spectra of

15

N-labeled SurA in the presence of HdeA recorded at different pH conditions are

summarized in Supporting Fig. S5, which suggest that HdeA does not change the overall unfolding process of SurA: SurA remains folded at pH higher than 4 and becomes unfolded at pH lower than 3. We further compared the HSQC spectra of 15N-labeled SurA in the presence or absence of HdeA at different pH conditions. We observed that the HSQC spectra of 15N-labeled SurA are highly similar with or without HdeA at pH higher than 3 (Supporting Fig. S6), 9


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suggesting that the two proteins have no interaction or the interaction is not strong under neutral and near neutral conditions. On the other hand, the HSQC spectra of 15N-labeled SurA show significant differences with or without HdeA at pH lower than 3 (Supporting Fig. S6), indicating that the two proteins obviously interact with each other under acidic conditions. Similarly, we performed a series of pH titration experiments using

15

N-labeled HdeA

mixed with unlabeled SurA so that we can characterize the structural changes of HdeA during interaction by observing the NMR signals from HdeA.

15

N-labeled HdeA was mixed with

unlabeled SurA at pH 7.0, and the mixture was subsequently exchanged to buffers with various pH conditions prior to NMR experiments. The results shown in Fig. 1 again demonstrate obvious interaction between HdeA and SurA under acidic pH conditions.

Figure 1. NMR monitored interactions between HdeA and SurA. Overlay of the 1H-15N HSQC spectra of

15

N-labeled HdeA in the presence (black) or absence (red) of unlabeled SurA at pH

conditions of 4.0, 3.0 and 2.5. The concentration of

15

N-HdeA was 0.2 mM, and the molar ratio of

HdeA and SurA is 1:2.

Interaction with substrates affects HdeA conformation As mentioned above, the HSQC spectrum of HdeA alone at pH 3.0 shows a set of well-dispersed peaks corresponding to the folded conformation, while it shows multiple conformations with more than one set of peaks at pH 2.5 (Supporting Fig. S2). As shown in Fig. 1, interaction with substrate SurA at pH 3.0 causes the intensities of the well-dispersed peaks of HdeA to decrease, accompanied by the appearance of new signals in the central region of the spectra. At pH 2.5, interaction with SurA also results in intensity reduction for the well-dispersed peaks, as well as the intensity increase for signals in the central region of

10


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the spectra. The peaks clustering in the center of the spectra reflect unfolded conformation, suggesting that there is a larger portion of disordered conformation in the HdeA-SurA complex compared to free HdeA. To investigate whether HdeA shows similar behavior when binding to different substrates, we also recorded the pH titration experiments using

15

N-HdeA mixed with the

other two substrates OppA and MalE. Comparisons of 15N-HdeA spectra with or without the substrates (SurA, OppA and MalE) at different pH values are summarized in Supporting Fig. S7. Similar to SurA, interactions with both OppA and MalE at pH 3.0 also cause the intensity decrease and/or signal disappearance of the major set of well-dispersed peaks (representing well-structured conformation) in the

15

N-labeled HdeA spectra, accompanied by the

appearance of new peaks clustering in the central region (representing disordered conformation). Considering the fact that the level of intensity decrease is even for all the well-dispersed peaks, that is we do not observe more significant intensity decrease in a certain region compared to other regions of the protein, the results are most readily explained by a population shift of HdeA conformational equilibrium in the presence of the substrate. In addition, the number of newly appeared peaks is much fewer compared to the total number of residues in HdeA, suggesting that we are only observing part of the protein sequence. Therefore, the newly appeared peaks most probably originate from some flexible regions of HdeA in complex with the substrates (vide infra).

HdeA starts interact with different substrates at different pH A closer inspection into the spectra reveals that although the HdeA-substrate binding event is obvious at pH lower than 3, the interactions actually start at higher pH conditions. For example, in the pH titration experiments using 15N-HdeA mixed with unlabeled SurA, we could already observe the appearances of a number of minor peaks in the central region of the spectra in the pH range of 3.5-3.0 (Fig. 2A). Although these newly appeared minor peaks are relatively weak, they are repeatable from experiment to experiment. Moreover, as will be described in more details later, these new peaks correspond to the N-terminal amides of residues Lys5, Ala6 and Ala7 of HdeA. They start to become observable at pH around 3.5 and are quite apparent at pH 3.0. Similarly, in the pH titration experiments using 15N-HdeA mixed 11


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with unlabeled MalE or OppA, we also observed the appearances of the minor peaks of the N-terminal Lys5-Ala7 residues at pH higher than 3.0 (Fig. 2A). These observations suggest that the interactions between HdeA and its native substrate proteins start at pH conditions slightly higher than 3.0, and the signals of the N-terminal segment are sensitive to substrate binding.

Figure 2. Interactions between HdeA and different substrates monitored by NMR. (A) Enlarged view of local spectral region showing the appearances of the minor peaks for residues Lys5-Ala7 of 15

N-HdeA in the presence of different substrates. The spectra of

15

N-HdeA in the presence or absence

of substrate proteins are shown in black and red, respectively. The concentrations of 15N-HdeA used in the experiments were 2 mg/mL (0.2 mM), whereas the substrate concentrations were kept at 18 mg/mL for all substrate proteins (0.40 mM for SurA, 0.44 mM for MalE, and 0.31 mM for OppA,) to account for their different molecular weights. (B) Overlay of the 1H-15N HSQC spectra of 15N-labeled HdeA in the presence (black) or absence (red) of unlabeled OppA at pH 3.0.

When we compare the HSQC spectra of 15N-HdeA in the presence of different substrate proteins, we are aware that the extent to which the substrates affect the HdeA conformation is substrate dependent. As shown in Fig. 1, the well-folded state still constitutes a large portion of the whole population of HdeA at pH 3 in the presence of SurA. The situation is mostly similar when MalE is used as the substrate (Supporting Fig. S7). However, in the case of OppA, the unfolded state of HdeA dominates the population at pH 3.0 and the folded state has nearly vanished (Supporting Fig. S7 and Fig. 2B). Therefore, it appears that different substrates have differential effects on HdeA. To better describe this phenomenon, we tried to estimate the critical pH value for HdeA to start interact with each of these substrates by 12


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observing the appearances of the N-terminal minor peaks of 15N-labeled HdeA. As shown in Fig. 2A, in the cases of SurA and MalE, we could observe the minor peaks of these residues at pH about 3.5, whereas in the case of OppA, the peaks were already observable at pH 3.8. Since HdeA undergoes dimer-to-monomer transition during acid stress, one concern is that whether slight fluctuations of the HdeA concentration among different samples might result in changes of the dimer-monomer equilibrium and thus cause the spectral changes. As Gajiwala & Burley have determined in their work (5), the dimer dissociation constant values Kd of HdeA at pH 7, 4 and 2 are about 0.25 µM, 1 µM and 45 µM, respectively. Here we are observing spectral changes in the pH range of 3.5-3.8, and the Kd values under which conditions are expected to be between 1 and 45 µM. The 0.2 mM HdeA concentration used in the experiments is much higher than the expected range, and slight concentration fluctuations are not likely to cause spectral changes observed in Fig. 2. To further rule out this possibility, we compared the HSQC spectra of HdeA at concentrations ranging from 2 mM to 0.02 mM at pH 3.5 and 3.0 (Supporting Fig. S8), and found them to be essentially identical. No peak shifts, peak intensity changes or the appearances of the N-terminal minor peaks were observed upon decrease of the HdeA concentrations, suggesting that the Kd values under these pH conditions should be smaller than 20 µM. In addition, to rule out the possibility that the spectra changes might be the result of protein crowding, we collected the HSQC spectrum of HdeA in the presence of an unrelated protein BSA instead of the native substrates (Supporting Fig. S9), which is also essentially identical to the spectrum of free HdeA. The results clearly demonstrate that the spectral changes observed in Fig. 2 are substrate-specific and not caused by protein crowding or HdeA concentration fluctuations. Taken together, the results indicate that HdeA can interact with its native substrates at pH higher than 3, and the starting pH for HdeA binding is substrate dependent.

The N- and C-termini of HdeA are flexible in the HdeA-substrate complexes Although the critical pH condition for HdeA to start interaction is substrate-dependent, the HSQC spectra of

15

N-HdeA upon complex formation at low pH are essentially similar

when using different substrates (Fig. 3A). A main feature of the spectra of HdeA in complex with its substrates is that the observable signals are strong and narrowly dispersed, reflecting 13


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an intrinsically disordered and highly flexible conformation. In addition, the number of signals is much fewer than the number of residues of the protein, suggesting that only the flexible segments are observable, whereas the other regions, which are most probably involved in interaction with the substrates, cannot be detected. To illustrate which segments of HdeA remain flexible in the HdeA-substrate complexes, we prepared an HdeA-SurA complex using

13

C/15N double-labeled HdeA and unlabeled SurA for assignments of the observable

HdeA signals in the complex. The triple resonance experiments were collected at pH 3.0 to ensure optimal sample stability, and the assignments can represent all acidic conditions since the chemical shifts of these signals remain mostly the same through the pH range of 3.0 to 2.0. As shown in Fig. 3B, the observed signals for

15

N-HdeA in the HdeA-SurA complex

correspond to its N- and C-termini, covering the Asp2-Val13 and Asn73-Met89 segments, among which only Pro12, Lys87 and the starting residue Met1 could not be assigned/observed. These residues map onto the highly-charged regions of the protein primary sequence, and have been identified to play essential roles in HdeA chaperone function (14). Although crystal structures of HdeA dimer (PDB accession code 1DJ8 and 1BG8) have been determined at pH 4.0 and 3.6 (5, 34), about 8-9 residues in the N-terminus and 2-4 residues in the C-terminus are missing in their electron density maps. We therefore determined the solution NMR structure of HdeA at pH 3.0, under which condition the main conformation of HdeA maintains a well-folded dimer (Supporting Fig. S10). The atomic coordinates have been deposited in the Protein Data Bank under the accession code 5WYO, and the related NMR data have been deposited in the Biological Magnetic Resonance Bank under the accession code 36045. The structural statistics are summarized in Table S2 and the representative lowest-energy conformer is shown in Fig. 3C. While the overall fold of HdeA in solution at pH 3.0 is generally similar to the crystal structures, we can observe near complete signals including those from the flexible regions. In particular, the residues that remains observable in the HdeA-substrate complexes map onto the three-dimensional structure in two regions: the Asp2-Val13 segment corresponds to the N-terminal loop that interacts with the 50s loop from the other subunit at the dimeric surface, whereas the Asn73-Met89 segment corresponds to the α4 helix that locates on the periphery of the dimer structure (Fig. 3C). The chemical shifts of all these signals in the HdeA-substrate complexes 14


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are close to the random coil values, indicating that in complex with substrate proteins under acidic conditions, the α4 helix unwinds and together with the N-terminal loop adopts disordered conformation.

Figure 3. The N- and C-termini of HdeA are flexible in its complex with substrates. (A) Comparison of the spectra of

15

N-labeled HdeA in complex with its substrate proteins SurA (green),

MalE (red) and OppA (black) at pH 2.5. The three spectra are arbitrarily displayed with offset for clarity reason. (B) Overlay of the spectra of 15N-labeled HdeA in the absence (red) and presence (black) of unlabeled SurA at pH 3.0. The backbone assignments of signals corresponding to the complex state of HdeA are labeled. The strong peaks in the dashed square in panel (B) correspond to lysine sidechain signals. (C) Mapping of the flexible residues of HdeA observable in the complexed state (colored in green) onto the dimeric structure of HdeA at pH 3. The secondary structural elements, the N- and C-termini, as well as the 50s loop are labeled.

Substrate binding further influences the C-terminus conformation As demonstrated by the pH titration experiments (Supporting Fig. S2), when the pH decreases to lower than 3, HdeA undergoes an order-to-disorder structural transition. At pH 2.5, the spectrum shows a mixture of folded and unfolded conformations. We compared the 2D HSQC spectra of

15

N-HdeA alone and in complex with substrates at pH 2.5 (Fig. 4) to

investigate whether the conformations of HdeA N- and C-termini are different in the free and complexed states. The peaks corresponding to the N-terminal residues in the HdeA-substrate complexes mostly overlay well with signals in the free HdeA spectrum, suggesting the chemical environments for this segment in the free and complexed states are essentially similar. On the other hand, the peaks for many residues in the C-terminal region, for example 15


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residues K77, K79, G80, K84, I85, K86 and D88, do not show signals at the corresponding positions in the free state as in the complexed state. This suggests that the local conformation of the C-terminal segment of HdeA is further affected by substrate binding. Comparison of the 2D HSQC spectra of 15N-HdeA alone and in complex with substrates at pH 2.0 provides similar results (Supporting Fig. S11).

Figure 4. Spectra comparison of 15N-HdeA in the free and complexed states. The 2D 1H-15N HSQC spectra of free

15

N-HdeA (red) and

15

N-HdeA-SurA complex (black) at pH 2.5 are overlaid. The full

spectra and enlarged view of the central region are shown in panel (A) and (B), respectively. For clarity reason, the spectra are displayed at relatively high contour level so that for the

15

N-HdeA-SurA

complex sample only the signals corresponding to the complexed state are visible. The assignments for the HdeA N- and C-terminal residues in the complexed state are labeled. (C) Enlarged view of the local spectral regions showing the signals from K79, K84, I85 and K86 in the complexed state (black). In the spectrum of the free HdeA (red), no signals are observed in the corresponding positions.

Truncations of the N- and C-termini result in elevated unfolding and interaction pH To further assess the impact of the N- and C-terminal segments on HdeA-substrate interactions, we constructed two HdeA truncation mutants HdeA-∆3 and HdeA-∆9, each having 3 or 9 residues in the N- and C-termini deleted. Inspection of the 1H-15N HSQC spectra indicates that the mutated proteins adopt similar structures as the wild-type HdeA (wt-HdeA) at neutral pH, whereas pH titration experiments by NMR show that both mutants start to unfold at higher pH values than wt-HdeA (Supporting Fig. S12A). In particular, HdeA-∆3 shows a more significant population of the disordered state at pH 3.0 compared to wt-HdeA, and HdeA-∆9 already shows a substantial population of the disordered state at pH 16


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7.0. The ANS binding assay also indicates that the exposure of hydrophobic surfaces of wt-HdeA, HdeA-∆3 and HdeA-∆9 gradually increase at pH 3.5 (data not shown), which reflects the different extent of unfolding among the three constructs. Furthermore, we performed the pH titration experiments for the two mutants in the presence of unlabeled SurA. The results reveal that the truncated mutants start interacting with the substrate at higher pH values compared to wt-HdeA, as the newly appeared minor peaks are of similar intensity level for HdeA-∆3 at pH 3.4 as for wt-HdeA at pH 3.2, whereas for HdeA-∆9 the interaction is already obvious at pH 4.0 (Supporting Fig. S12B). The observations suggest that truncation of the charged N- and C-terminal segments not only results in an elevated unfolding pH for HdeA, but also enables it to interact with the substrate protein at higher pH range.

HdeA-substrate complexes are heterogeneous Since the NMR signals corresponding to the hydrophobic region of HdeA become completely unobservable upon complex formation, it is hard to directly characterize the mode of interaction between HdeA and its substrates. The disappearance of the signals may originate from high molecular weight, intermediate exchange on the NMR timescale, or the intrinsic heterogeneous property of the complexes. Previously reported ultracentrifugation data on the complex between HdeA and a model substrate MDH at acidic pH (16) strongly suggest the heterogeneous nature of HdeA-MDH complex. Herein we tried to investigate the binding stoichiometry between HdeA and its native substrates by size-exclusion chromatography. The size-exclusion chromatography profiles of the three substrates alone or mixed with HdeA at ~1:3 molar ratio at pH 2.0 are shown in Fig. 5A. The data show that SurA does not interact strongly with HdeA and shows a large free fraction, whereas OppA and MalE show stronger interaction with HdeA at this pH condition. We further used OppA and MalE to collect a series of size-exclusion chromatography data with varying HdeA:substrate molar ratio. As shown in Fig. 5B-C, both HdeA-OppA and HdeA-MalE complexes are apparently heterogeneous at low HdeA:OppA molar ratio, showing multiple elution peaks. As more HdeA is added into the sample, the elution peaks of the complexes shift towards higher molecular weight. When the HdeA:OppA (or HdeA:MalE) 17


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molar ratio reaches 5:1, the complexes are still heterogeneous showing asymmetric elution peaks, while the elution peak corresponding to free HdeA start to appear. When the HdeA:OppA (or HdeA:MalE) molar ratio further increase to 10:1 or 20:1, the elution peaks corresponding to the complexes appear to become more symmetric but is relatively broad, while at the same time the elution peaks corresponding to free HdeA become significantly stronger. The above results demonstrate that the binding stoichiometry between HdeA and its substrates varies with the amount of HdeA available, and the binding of HdeA seems to reach a plateau when the HdeA:substrate molar ratio reaches beyond 10:1.

Figure

5. Size-exclusion

chromatography

profiles

of

HdeA-substrate complexes. (A)

Size-exclusion chromatography profiles of the three substrates alone (black) or mixed with HdeA at ~1:3 molar ratio (red) at pH 2.0. All samples were mixed at pH 7.0, buffer exchanged to pH 2.0 and incubated at 37 °C for 1 hour before loading onto a 16 mm × 70 cm Superdex 200 size-exclusion column. The protein concentrations used were ~0.3 mM for the substrate proteins and ~1.0 mM for HdeA prior to loading. The retention volumes for standard protein markers were calibrated at pH 7 and the corresponding molecular mass are indicated. (B-C) Size-exclusion chromatography profiles of OppA (B) and MalE (C) proteins at pH 2.0 with varying HdeA concentrations. All samples contain 0.3 mM OppA or MalE, and the substrate:HdeA molar ratios are indicated by different colors. All samples were mixed at pH 7.0, buffer exchanged to pH 2.0 and incubated at 37 °C for 1 hour before loading onto a 16 mm × 100 cm Superdex 200 size-exclusion column. The retention volumes for standard protein markers were calibrated at pH 7 and the corresponding molecular weights are indicated. For comparison, the control profiles for OppA, MalE and HdeA at pH 2.0 alone are shown underneath. 18


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Estimating the binding stoichiometry between HdeA and its substrates We further tried to estimate the binding stoichiometry between HdeA and OppA/MalE by analyzing the integrated peak volumes in the size-exclusion chromatography profiles. Results suggest that when HdeA and substrates were mixed at 10:1 molar ratio, the actual binding stoichiometry in the complexes is about 6:1 to 7:1 for OppA, and about 5:1 to 6:1 for MalE. When HdeA and substrates were mixed at 20:1 molar ratio, the actual binding stoichiometry in the complexes is about 10:1 to 12:1 for both proteins. Alternatively, we also collected the flow-through of the HdeA-substrate complex peak, changed the buffer back to neutral condition for protein refolding, loaded onto the size-exclusion column for a second time at pH 7 to separate the two proteins and calculated their amount. For the HdeA-OppA complex, we obtained a binding stoichiometry of about 10:1 when the two were mixed at 20:1 molar ratio, which is highly consistent with the results obtained from peak volume analysis. For the HdeA-MalE complex, however, since MalE does not refold well and mostly precipitated during the buffer exchange process, we could only retrieve and quantify the amount of the HdeA component. Comparison of the amount of HdeA purified from the complex peak with the amount originally added into the mixture suggested that the binding stoichiometry is also about 10:1 when HdeA and MalE were mixed at 20:1 molar ratio. The two methods are consistent with each other and together suggest an average binding stoichiometry of about 10:1 when the substrates become saturated. This is also consistent with the estimated molecular weight of near 200 kDa from the average retention volume for the complexes. However, we should keep in mind that estimation of the complex molecular weight is not accurate since it is based on the assumptions that the complexes adopt a globular shape and that the molecular weight calibrated from standard proteins at pH 7 can be adapted to the condition of pH 2.

DISCUSSION As a representative of the “conditionally disordered” protein, HdeA is functionally activated when it becomes unfolded under acidic conditions. Previous studies have 19


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demonstrated that acidification causes the disruption of a number of electrostatic interactions and results in unfolding and thus activation of HdeA at pH lower than 3 (10, 11, 20). Our current study suggests that although the unfolding of HdeA starts at pH lower than 3 when it is alone, its interactions with substrates can start at pH higher than 3. Interactions with substrates result in the appearance of signals with disordered conformational characteristics. These results suggest that the unfolding and activation of HdeA is not only modulated by pH changes, but can also be regulated and facilitated by the presence of acid-denatured substrates. In addition, we observed that the substrate OppA, which begins to unfold at higher pH and exposes larger hydrophobic surface area (as demonstrated by NMR and ANS binding assays), also starts interacting with HdeA at higher pH conditions compared to SurA or MalE. The observations imply that the intrinsic structural properties, namely the acid sensitivities and acid-induced hydrophobic surface exposure of the substrates themselves, may play a role in regulating the activation of HdeA chaperone function. On the other hand, experimental data from the HdeA truncation mutants suggests that the increased hydrophobic exposure of HdeA itself also results in elevated pH values for it to interact with the substrates. Furthermore, Foit and co-workers have shown that an HdeA D20A D51A double mutant which displays partially unfolded conformations at near-neutral pH, also shows constitutive chaperone activity at pH 5 and is even partially active at pH 7 (10). Taken together, it appears that the extent of hydrophobic surface exposure of both parties of the interaction, either the chaperone or the client, contributes to the modulation of their binding event. This notion can be further supported by a recently published work by Zhang et al in which they compared the in vivo client specificities between two acid chaperones HdeA and HdeB (21). They discovered that the hydrophobic surfaces of HdeB-preferred clients are significantly exposed at HdeB activation pH (pH ~ 4.5), whereas HdeA-preferred clients become sufficiently exposed only at low pH range. These results together highlight that the pH-dependent unfolding events of both substrates and chaperone itself modulate the interaction, which may act to ensure that the interactions occur at the appropriate time and with the correct partner. Up to date, the detailed information concerning HdeA interaction with its native substrates has been scarce, largely due to the fact that both HdeA and the substrate protein are 20


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unfolded during interaction. The interaction between two disordered proteins is highly promiscuous as suggested by previous FRET results (15), rendering it difficult for high-resolution structural investigations. Our results demonstrate that the N-terminal loop (Asp2-Val13) and C-terminal helix (Asn73-Met89) of HdeA become fully disordered and are highly flexible in the HdeA-substrate complexes, showing strong peaks in the center of the HSQC spectra. Apart from these regions, we were not able to observe the NMR signals from other regions of the HdeA sequence, suggesting that they are most probably involved in HdeA-substrate interactions which are highly heterogeneous. As Hong and Wu et al. described in their earlier studies, the sequence of HdeA displays a conserved “amphiphilic” feature with highly charged residues at the two terminal regions and highly hydrophobic residues in the internal region, which is essential for the chaperone function at low pH (14). In their study, they constructed HdeA-∆N and HdeA-∆C mutants in which the first 11 and last 15 residues were separately deleted, and showed that both mutants were highly soluble on their own under acidic condition, but tend to co-precipitate with denatured substrates during acid treatment (14). The observation indicated that deletions of the N- and C-termini do not disrupt the HdeA-substrate interactions but strongly affect the solubility of the HdeA-substrate complexes. In our current study, we used the HdeA-∆3 and HdeA-∆9 truncation mutants to investigate the effects of HdeA structural instability on its interactions with substrates at pH higher than 3. Since the N-terminal segment contributes to dimer packing and the C-terminal segment forms a helix, truncations from both termini affect the HdeA stability and result in both HdeA partial unfolding and HdeA-substrate interactions at higher pH values. Therefore, it appears that the integrity of the N- and C-termini of HdeA may play dual roles in its chaperone function. On one hand, the structural integrity of the termini ensures the chaperone inactivity of HdeA under neutral and near-neutral pH conditions, and thus prevents unwanted interactions of HdeA with periplasmic proteins. On the other hand, the highly-charged termini become exposed and adopt flexible disordered conformation under acidic pH conditions, which may help promote the solubility of HdeA-substrate complexes. Moreover, we observed that although the binding stoichiometry between HdeA and its substrates can vary depending on the available amount of HdeA, a plateau can be reached 21


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when excess amount of HdeA is added into the sample. Despite the fact that OppA and MalE have different molecular weights (58 and 41 kDa, respectively), the maximum binding stoichiometry between them and HdeA is essentially similar (about 10 molecules of HdeA to 1 molecule of substrate protein). This observation suggests that the condition in which the substrates become saturated by HdeA does not depend on the substrate mass (or sequence length), but may be limited by the intrinsic property of the chaperone HdeA itself. We should also keep in mind that ten times excess amount of HdeA may not reflect the actual physiological condition, since under acid stress a large amount of periplasmic proteins would undergo acid-induced denaturation and require chaperone protection. Based on our current study and results from previous reports, a hypothesis of the HdeA functional mechanism is summarized as follows: At neutral and near-neutral pH, HdeA adopts a well-folded dimer structure with hydrophobic surface buried in the structure core. When pH decreases, the protonation of acidic residues located in N- and C-termini as well as the 50s loop causes the weakening or breaking of several electrostatic interactions, thus destabilizing the structure and resulting in partial unfolding. At the same time, the decrease of pH also causes the substrate proteins to become unfolded. The exposed hydrophobic surface of HdeA could bind the hydrophobic surface of client protein to form an energetically favorable complex, and the binding stoichiometry is variable. The binding drives the population equilibrium of HdeA to shift towards unfolding, and under acid stress conditions when unfolded substrate proteins are abundant, would shift the equilibrium fully towards the formation of the HdeA-substrate complexes. Upon complex formation, both the N- and C-termini of HdeA adopt flexible disordered conformation, which helps promote the solubility of the complexes. On the other hand, residues in the central hydrophobic region of HdeA protein sequence become unobservable upon complex formation, which is most probably due to the heterogeneous nature of the HdeA-substrate interactions. It is highly possible that the dynamic exchanges of HdeA binding to different sites of the substrates, or the dynamic conformational exchanges of HdeA itself when interacting with the substrates, may lead to the signal disappearances of the hydrophobic region.

Supporting Information 22


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Addition information including sample pH calibration, pH titration, NMR structure and substrate interactions of HdeA.

Funding Information C. J. received funding from the National Key R&D Program of China, Grant 2016YFA0501201. Y. H received funding from the National Natural Science Foundation of China, Grant 31370718.

Acknowledgements All NMR experiments were performed at the Beijing NMR Center and the NMR facility of National Center for Protein Sciences at Peking University. This research was supported by Grant 2016YFA0501201 from the National Key R&D Program of China to C. J., and Grant 31370718 from the National Natural Science Foundation of China to Y. H.

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Biochem. J. 412, 389-397. 15. Tapley, T. L., Korner, J. L., Barge, M. T., Hupfeld, J., Schauerte, J. A., Gafni, A., Jakob, U., Bardwell, J. C. (2009) Structural plasticity of an acid-activated chaperone allows promiscuous substrate binding. Proc. Nat. Acad. Sci. U. S. A. 106, 5557-5562. 16. Tapley, T. L., Franzmann, T. M., Chakraborty, S., Jakob, U., Bardwell, J. C. (2010) Protein refolding by pH-triggered chaperone binding and release. Proc. Nat. Acad. Sci. U. S.

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Escherichia coli HdeA. Nat. Struct. Biol. 5, 763-764.

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Figure 1. NMR monitored interactions between HdeA and SurA. Overlay of the 1H-15N HSQC spectra of 15Nlabeled HdeA in the presence (black) or absence (red) of unlabeled SurA at pH conditions of 4.0, 3.0 and 2.5. The concentration of 15N-HdeA was 0.2 mM (2 mg/mL), and the molar ratio of HdeA and SurA is 1:2. 177x60mm (300 x 300 DPI)


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Figure 2. Interactions between HdeA and different substrates monitored by NMR. (A) Enlarged view of local spectral region showing the appearances of the minor peaks for residues Lys5-Ala7 of 15N-HdeA in the presence of different substrates. The spectra of 15N-HdeA in the presence or absence of substrate proteins are shown in black and red, respectively. The concentrations of 15N-HdeA used in the experiments were 2 mg/mL (0.2 mM), whereas the substrate concentrations were kept at 18 mg/mL for all substrate proteins (0.40 mM for SurA, 0.44 mM for MalE, and 0.31 mM for OppA,) to account for their different molecular weights. (B) Overlay of the 1H-15N HSQC spectra of 15N-labeled HdeA in the presence (black) or absence (red) of unlabeled OppA at pH 3.0. 114x47mm (300 x 300 DPI)


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Biochemistry

Figure 3. The N- and C-termini of HdeA are flexible in its complex with substrates. (A) Comparison of the spectra of 15N-labeled HdeA in complex with its substrate proteins SurA (green), MalE (red) and OppA (black) at pH 2.5. The three spectra are arbitrarily displayed with offset for clarity reason. (B) Overlay of the spectra of 15N-labeled HdeA in the absence (red) and presence (black) of unlabeled SurA at pH 3.0. The backbone assignments of signals corresponding to the complex state of HdeA are labeled. The strong peaks in the dashed square in panel (B) correspond to lysine sidechain signals. (C) Mapping of the flexible residues of HdeA observable in the complexed state (colored in green) onto the dimeric structure of HdeA at pH 3. The secondary structural elements, the N- and C-termini, as well as the 50s loop are labeled. 170x54mm (300 x 300 DPI)


Biochemistry

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Figure 4. Spectra comparison of 15N-HdeA in the free and complexed states. The 2D 1H-15N HSQC spectra of free 15N-HdeA (red) and 15N-HdeA-SurA complex (black) at pH 2.5 are overlaid. The full spectra and enlarged view of the central region are shown in panel (A) and (B), respectively. For clarity reason, the spectra are displayed at relatively high contour level so that for the 15N-HdeA-SurA complex sample only the signals corresponding to the complexed state are visible. The assignments for the HdeA N- and Cterminal residues in the complexed state are labeled. (C) Enlarged view of the local spectral regions showing the signals from K79, K84, I85 and K86 in the complexed state (black). In the spectrum of the free HdeA (red), no signals are observed in the corresponding positions. 155x52mm (300 x 300 DPI)


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Biochemistry

Figure 5. Size-exclusion chromatography profiles of HdeA-substrate complexes. (A) Size-exclusion chromatography profiles of the three substrates alone (black) or mixed with HdeA at ~1:3 molar ratio (red) at pH 2.0. All sample was incubated at pH 2.0 for 1 hour before loading onto a 16 mm × 70 cm Superdex 200 size-exclusion column. The protein concentrations used were ~0.3 mM for the substrate proteins and ~1.0 mM for HdeA prior to loading. The retention volumes for standard protein markers were calibrated at pH 7 and the corresponding molecular mass are indicated. (B-C) Size-exclusion chromatography profiles of OppA (B) and MalE (C) proteins at pH 2.0 with varying HdeA concentrations. All samples contain 0.3 mM OppA or MalE, and the substrate:HdeA molar ratios are indicated by different colors. All sample was incubated at pH 2.0 for 1 hour before loading onto a 16 mm × 100 cm Superdex 200 size-exclusion column. The retention volumes for standard protein markers were calibrated at pH 7 and the corresponding molecular mass are indicated. For comparison, the control profiles for OppA, MalE and HdeA alone are shown underneath. 167x73mm (300 x 300 DPI)


Characterizations of the Interactions between Escherichia coli

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