Role of metal cations and oxyanions in the regulation of protein arginine phosphatase activity of YwlE from Bacillus subtilis

DOI: 10.1016/j.bbagen.2020.129698

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Biochimica et Biophysica Acta - General Subjects (2020)

Update date:2022-08-11

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Authors:

Huang, Biling

Huang, Chenyang

Huang, Shaohua

Liao, Xinli

Liu, Yan

Zhang, Yumeng

Zhao, Mingxiao

Zhao, Yufen

Zhao, Zhixing

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Article abstract of DOI:10.1016/j.bbagen.2020.129698

Protein arginine phosphorylation (pArg) is a relatively novel posttranslational modification. Protein arginine phosphatase YwlE negatively regulates arginine phosphorylation and consequently induces the expression of stress-response genes that are crucial for bacterial stress tolerance and pathogenic homolog Staphylococcus aureus virulence. However, little is known about the factors that affect the enzymatic activity of YwlE with the exception of the effect of oxidative stress. Herein, based on the hydrolysis of the chromogenic substrate p-nitrophenyl phosphate (pNPP) by YwlE, we investigate the role of metal cations and oxyanions in the regulation of YwlE activity. Interestingly, among the various cations that we tested, Ca²⁺ activates YwlE, while other cations, including Ag⁺, Co²⁺, Cd²⁺, and Zn²⁺, are inhibitory. Furthermore, as chemical analogues of phosphate, oxyanions play multiple roles in phosphatase activity. The regulatory switch Cys within the catalytic site regulates YwlE activity. Specifically, the thiol of this Cys could be alkylated by IAM (iodoacetamide) or oxidized by H₂O₂, resulting in enzymatic inhibition. Conversely, reducing reagents, such as DTT (dithiothreitol), β-me (β-mercaptoethanol), and TCEP (tris(2-carboxyethyl)phosphine) enhance YwlE activity. Additionally, as a stable analogue to pArg, pAIE binds to YwlE with a K_d of 149.1 nM and a binding stoichiometry n of 1.2 and inhibits YwlE with an IC₅₀ of 316.3 ± 12.73 μM. The inhibition and activation of YwlE may have broad implications for the physiology, pharmacology and toxicology of metal cations and oxyanions.

Full text of DOI:10.1016/j.bbagen.2020.129698

BB A -G e ne r a l S ub j e c t s 1 86 4 (2 020 )1 29698

Contents lists available at ScienceDirect

BBA - General Subjects

journal homepage: www.elsevier.com/locate/bbagen

Role of metal cations and oxyanions in the regulation of protein arginine

phosphatase activity of YwlE from Bacillus subtilis

Biling Huang^a, Zhixing Zhao^d, Chenyang Huang^c, Mingxiao Zhao^c, Yumeng Zhang^c, Yan Liu^c,

⁎

Xinli Liao^c, Shaohua Huang^a,b,, Yufen Zhao^a,b,c,e,

^aInstitute of Drug Discovery Technology, Ningbo University, Ningbo 315211, PR China

^bQian Xuesen Collaborative Research Center of Astrochemistry and Space Life Sciences, Ningbo University, Ningbo 315211, PR China

^cDepartment of Chemical Biology, College of Chemistry and Chemical Engineering, the Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen

361005, PR China

^dDepartment of Chemistry, College of Chemistry and Chemical Engineering, The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Xiamen University,

Xiamen 361005, PR China

^eKey Lab of Bioorganic Phosphorus Chemistry & Chemical Biology, Department of Chemistry, Tsinghua University, Beijing 100084, PR China

A R T I C L E I N F O

A B S T R A C T

Keywords:

Protein arginine phosphorylation

YwlE

Metal cations

Oxyanions

pAIE

Protein arginine phosphorylation (pArg) is a relatively novel posttranslational modiﬁcation. Protein arginine

phosphatase YwlE negatively regulates arginine phosphorylation and consequently induces the expression of

stress-response genes that are crucial for bacterial stress tolerance and pathogenic homolog Staphylococcus aureus

virulence. However, little is known about the factors that aﬀect the enzymatic activity of YwlE with the ex-

ception of the eﬀect of oxidative stress. Herein, based on the hydrolysis of the chromogenic substrate p-ni-

trophenyl phosphate (pNPP) by YwlE, we investigate the role of metal cations and oxyanions in the regulation of

YwlE activity. Interestingly, among the various cations that we tested, Ca²⁺activates YwlE, while other cations,

including Ag⁺, Co²⁺, Cd²⁺, and Zn²⁺, are inhibitory. Furthermore, as chemical analogues of phosphate, oxy-

anions play multiple roles in phosphatase activity. The regulatory switch Cys within the catalytic site regulates

YwlE activity. Speciﬁcally, the thiol of this Cys could be alkylated by IAM (iodoacetamide) or oxidized by H₂O₂,

resulting in enzymatic inhibition. Conversely, reducing reagents, such as DTT (dithiothreitol), β-me (β-mer-

captoethanol), and TCEP (tris(2-carboxyethyl)phosphine) enhance YwlE activity. Additionally, as a stable

analogue to pArg, pAIE binds to YwlE with a K_dof 149.1 nM and a binding stoichiometry n of 1.2 and inhibits

YwlE with an IC₅₀of 316.3

the physiology, pharmacology and toxicology of metal cations and oxyanions.

12.73 μM. The inhibition and activation of YwlE may have broad implications for

1. Introduction

such, pArg may be implicated in human diseases. The biological func-

tions of pArg protein in bacteria and eukaryotes are not completely

characterized to date.

Protein arginine phosphorylation (pArg), that is, the addition of a

phosphate group to the guanidinium group of arginine residues by

protein arginine kinase McsB, plays a role in protein-DNA interaction,

protein turnover, stress-response and bacterial pathogenicity [1 –3]. As

a cognate protein arginine phosphatase, YwlE negatively regulates

protein arginine phosphorylation and accordingly induces the expres-

sion of stress-response genes that are critical for bacterial stress toler-

ance and virulence. Recently, hundreds of pArg proteins were identiﬁed

in ywlE-deﬁcient mutant Bacillus subtilis under heat-shock and oxida-

tive-stress conditions [4,5]. Moreover, pArg is gradually emerging in

eukaryotic organisms. In a recent study, our laboratory ﬁrst identiﬁed

118 proteins containing 152 pArg sites in human cancer cells [6]. As

YwlE shares the active catalytic C^a(X)₄C^bR(S/T) motif (P-loop) with

low molecular weight protein tyrosine phosphatase (LMW-PTP), and

the Cys^aresidue is critical for its phosphatase activity [7]. The thiolate

of Cys^aﬁrst attacks the phosphorus atom of the phosphosubstrate and is

subsequently displaced by an active-site water molecule, which conse-

quently triggers the release of the phosphate from the substrate.

Therefore, YwlE activity is negatively regulated by oxidative stress due

to the oxidation of thiol of Cys [8]. The irreversible inhibitor cyc-SeCN-

amidine inactivates YwlE by the catalytic formation of a disulﬁde bond

in the catalytic site [9], an SO₃-amidine-based YwlE inhibitors that are

similar in size and charge distribution to a phosphoguanidinium moiety

^⁎Corresponding authors at: Institute of Drug Discovery Technology, Ningbo University, Ningbo 315211, PR China.

E-mail addresses: huangshaohua@nbu.edu.cn (S. Huang), zhaoyufen@nbu.edu.cn (Y. Zhao).

https://doi.org/10.1016/j.bbagen.2020.129698

Received 23 December 2019; Received in revised form 24 July 2020; Accepted 24 July 2020

A v a i l a b l e o n l i n e 2 8 J u l y 2 020

B. Huang, et al.

A-GeneralSubjects1864(2020)129698

competitively inhibit YwlE [8]. Nevertheless, in addition to oxidative

stress, it remains unknown whether other factors, such as metal cations

and oxyanions, regulate YwlE activity. Although YwlE is not considered

a metalloenzyme, metal cations play critical roles in its phosphatase

activity [10]. There are known and putative metal homeostasis systems

for Ca²⁺, Mg²⁺, Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺and Mn²⁺in Bacillus subtilis

[11]. Therefore, it is important to understand how the metal ions aﬀect

YwlE activity. Additionally, inorganic phosphate, a competitive in-

hibitor of phosphatase, is the hydrolytic product of the phosphatase

reaction. Oxyanions that are structurally analogous to phosphate con-

sequently inhibit phosphatase activity. Moreover, the stress tolerance

and virulence of the human pathogen Staphylococcus aureus is closely

related to protein arginine phosphorylation regulation mediated by

YwlE [3]. This ﬁnding taken together with our discovery of hundreds of

pArg proteins in human cancer cells suggests that understanding how

oxyanions regulate YwlE activity may facilitate the development of

anti-bacterial or anticancer drugs.

Here, we investigate the eﬀect of metal ions, oxyanions, phospha-

tase inhibitors, reducing reagents, and non-hydrolysable analogs of

pArg on the phosphatase activity of YwlE from Bacillus subtilis using the

chromogenic substrate p-nitrophenyl phosphate (pNPP), which is a

nonspeciﬁc phosphatase substrate favoring continuous assays of phos-

phatases at alkaline pH values. We also discuss the general problems

encountered with the inhibition and activation eﬀects in the literature.

37 °C overnight and then diluted with 500 mL fresh Luria-Bertani media

with the same antibiotics. Cells were allowed to grow at 37 °C under

220 rpm orbital shaking until an OD₆₀₀nm of 0.7 was reached. Protein

expression was induced with the addition of IPTG (isopropyl β-D-

thiogalactoside) to a ﬁnal concentration of 0.5 mM for 3 h at 37 °C

under 200 rpm orbital shaking. Cells were harvested by centrifugation

and lysed in lysis buﬀer [pH 8.0, 20 mM Tris-HCl, 300 mM NaCl, 1 mM

PMSF (phenylmethanesulfonyl ﬂuoride)] with cell disruptor. After

centrifugation at 11,000 rpm for 30 min to remove cell debris, the su-

pernatant was loaded on Ni²⁺-NTA column (BBI Life Sciences) for af-

ﬁnity capture of the His-tag target protein. The column was then wa-

shed with 5 mM imidazole and eluted with 50 mM imidazole. The

eluted protein was further puriﬁed using size-exclusion chromato-

graphy with a Superdex G75 column. The puriﬁed YwlE protein was

dialyzed into storage buﬀer [pH 7.6, 20 mM Tris/HCl, 100 mM NaCl],

and the concentration was determined using the Bradford assay.

The gene encoding YwlE was derived from Bacillus subtilis. The

mutant construct YwlE (C7S) was generated with a site-directed mu-

tagenesis kit (TAKARA) using the primers 5′-GGATATTATTTTTGTCA

GCACTGGAAATAC-3′ and 5′-GCGGCACGTATTTCCAGTGCTGACAAAA

ATAT-3′. The mutant construct YwlE (C7A) was generated similarly

using primers 5′-GGATATTATTTTTGTCGCTACTGGAAATAC-3′ and

5′-CACGTATTTCCAGTAGCGACAAAAATAATAT-3′. All constructs were

veriﬁed by DNA sequencing. The mutant plasmids were transformed

into E. coli BL21 (DE3) cells (Novagen) for mutant protein expression.

The expression and puriﬁcation of the mutant protein were carried out

using the same protocols used for the wild-type YwlE.

2. Materials and methods

2.1. Reagents and materials

Brieﬂy, p-nitrophenyl phosphate (pNPP) was purchased from J&K

Scientiﬁc (Shanghai, China). Tris, HEPES, PIPES (piperazine-1,4-bi-

sethanesulfonic acid), DTT (dithiothreitol), β-me (β-mercaptoethanol)

and TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) were

purchased from Sangong Biotech. NaCl (sodium chloride), CaCl₂(cal-

cium chloride), ZnCl₂(zinc chloride), CuCl₂(cupric chloride), MgCl₂

(magnesium chloride), MnCl₂(manganese chloride), NiCl₂(nickel

chloride), LiCl (lithium chloride), NaNO₃(sodium nitrate), Na₂CO₃

(sodium carbonate), Na₂SO₄(sodium sulfate), Na₂B₄O₇(sodium bo-

rate), Cd(NO₃)₂(cadmium nitrate), K₂C₂O₄(potassium oxalate mono-

2.3. McsB phosphorylated Arg-peptide

The synthetic Arg-peptide (40 μM) (S1·F) was enzymatically phos-

phorylated using protein arginine kinase McsB (15 μM) in reaction

buﬀer-A [pH 8.0, 20 mM Tris-HCl] supplemented with 1 mM adenosine

triphosphate (ATP) and 5 mM MgCl₂at 30 °C for 1 h. Phosphorylated

Arg-peptide (pArg-peptide, molecular weight: 1509.8 Da) was analyzed

by RP-HPLC and mass spectrometry. RP-HPLC was performed on an

Agilent 1260 instrument equipped with an Agilent ZORBAX 300Extend-

C18 (3.5 μm, 2.1 × 100 mm) column at a ﬂow rate of 0.8 mL/min. The

elution was performed employing gradients of solvent A (0.1% formic

acid in water) and solvent B (95% acetonitrile in water with 0.1%

formic acid). A Bruker micrOTOF-Q-II mass spectrometry was con-

nected using split ﬂow for mass detection. The MS parameters were as

follows: nebulizer 8 bar, dry gas 4.5 L/min, temperature 200 °C, posi-

tive mode, target mass, and scan range m/z 110–2000. Data processing

and analysis were performed using Data Analysis 4.1 (Bruker).

hydrate),

Na₂MoO₄•2H₂O

(sodium

molybdate

dihydrate),

Na₂WO₄•2H₂O (sodium tungstate dihydrate), H₃BO₃(boric acid),

AgNO₃(silver nitrate), CoCl₂(cobaltous chloride), H₂O₂(hydrogen

peroxide), NaF (sodium ﬂuoride), Na₃VO₄(sodium vanadate), and

Na₄P₂O₇(sodium pyrophosphate) were obtained from Sinopharm

Chemical Reagent, and β-glycerophosphate was obtained from Tokyo

Chemical Industry. IAM (iodoacetamide), pTyr (phosphotyrosine), pSer

(phosphoserine) and phosphocreatine were obtained from Sigma-

Aldrich. The protease/phosphatase inhibitor cocktail was obtained

from Thermo Fisher (No. 88668). The synthesis of pAIE (2-((2-ammo-

nioethyl)amino)-2-iminoethyl phosphonic acid) was performed ac-

cording to our previous work [12].

2.4. YwlE hydrolyzed pArg-peptide, pNPP, phosphocreatine and pArg in

vitro

N-terminally acetylated Arg-peptide derived from CtsR

(MW = 1429.8 Da, sequence see Fig. S1·F) was synthesized and HPLC

puriﬁed to 95% purity. The peptide was then lyophilized and dissolved

in distilled water as stock solution (5 mg/mL) for further assays.

Dephosphorylation of pArg-peptide was performed with YwlE

(1 μM) in buﬀer-A [pH 8.0, 20 mM Tris-HCl] at 30 °C for 1 h. Analysis of

dephosphorylation of pArg-peptide was performed using the same

process mentioned above via RP-HPLC and Bruker micrOTOF-Q-II.

Dephosphorylation of pNPP, phosphocreatine and pArg by YwlE

were performed at 30 °C for 1 h. For control assays, pNPP, phospho-

creatine and pArg were dissolved in buﬀer-A, separately. D₂O was

added to a ﬁnal concentration of 10% (v/v) to prepare NMR samples.

The reactions were characterized by ³¹P spectra acquired at 298 K using

a Bruker Avance III 600 MHz spectrometer equipped with triple re-

sonance cryogenic probes. Spectral width of ³¹P spectra were set to

400.0 ppm with a transmitter frequency oﬀset of −50.0 ppm. All the

spectra with the same parameters were processed and analyzed using

Topspin 3.2 (Bruker).

2.2. Expression and puriﬁcation of McsB, YwlE, YwlE (C7A) and YwlE

(C7S)

Expression and puriﬁcation of McsB (Geobacillus stearothermophilus)

were performed according to our previous work [12]. The plasmid

encoding the Bacillus subtilis ywlE gene inserted into vector pET-21a(+)

containing a C-terminal 6 × His-tag was a generous gift from Prof.

Changwen Jin at Peking University. The plasmid was transformed into

E. coli BL21 (DE3) cells (Novagen). The cells were ﬁrst cultured in 5 mL

fresh Luria-Bertani media supplemented with 50 μg/mL ampicillin at

B. Huang, et al.

A-GeneralSubjects1864(2020)129698

Fig. 1. YwlE from Bacillus subtilis hydrolyzed the substrates in vitro. (A) 12% SDS-PAGE analysis of puriﬁed YwlE, YwlE (C7A) and YwlE (C7S). (BeC) YwlE

dephosphorylated the pArg-peptide that was phosphorylated by protein arginine kinase McsB (EIC spectra were extracted from RP-HPLC with ESI-MS). The three

peaks (retention time: 17.71 min, 17.25 min and 17.23 min) represented the Arg-peptide, and the emerging peak (retention time: 18.25 min) represented a

phosphorylated pArg-peptide. YwlE catalyzed the hydrolysis of (D) pNPP, (E) phosphocreatine and (F) small molecule pArg, which were not hydrolyzed by YwlE

(C7A) or YwlE(C7S). The peaks labeled black triangle were resonance signals of phosphate (PO₄³⁻) released from the substrates.

2.5. Phosphatase assay and competition experiments for Ca²⁺against

Zn²⁺, Cu²⁺, Cd²⁺, Co²⁺, and Ag⁺

(for pNP) or A_{312 nm}(for pNPP) were used as parameters and converted

into the percentage of activity (% activity of YwlE for activator or %

residual activity of YwlE for inhibitor) relative to the control reaction

according to the equations below:

The phosphatase assay was performed at 30 °C using 20 mM Tris/

HCl, 100 mM NaCl (pH 7.6) buﬀer containing 20 μM YwlE and 50 mM

pNPP as the substrate. The eﬀects of ions on YwlE activity were tested

by serial dilution of the ions. The phosphatase reaction was monitored

by UV absorption at 405 nm (absorption maximum of the generated

pNP) using a SHIMADZU UV-VIS spectrophotometer UV-2700. The

progress curves were generated, and the IC₅₀or EC₅₀values were ob-

tained by plotting the percentage of YwlE activity versus ion con-

centrations using the Doseresp model in the OriginPro software.

For competition experiments, 100 μM metal cations were used.

After determining that YwlE activity was not inﬂuenced when

[Na⁺] < 10 mM (S4), 100 μM Na⁺was added to the control assays to

%activity = (I_A1/I_A0) ∗ 100%

(1)

(I_A0: I_{A405 nm}of control assay without additives, I_A1: I_{A405 nm}of assay

with additives)

%activity = (1 − I_A1/I_A0) ∗ 100%

(2)

(I_A0: I_{A312 nm}of control assay without additives, I_A1: I_{A312 nm}of assay

with additives)

2.7. Isothermal titration calorimetry assay

maintain ion strength. In the control assays, 100 μM Ca²⁺, Zn²⁺, Cu²⁺

Cd²⁺, Co²⁺, and Ag⁺were incubated with YwlE. For competition ex-

periments, 100 μM Ca²⁺was added together with the single inhibitory

cation (100 μM) during YwlE incubation. UV absorbances of the sub-

strate pNPP and the product pNP were used to characterize the com-

petition eﬀects.

Isothermal titration calorimetry experiments were conducted at

25 °C using TA NANO ITC. YwlE was prepared in storage buﬀer [pH 7.6,

20 mM Tris/HCl, 100 mM NaCl] at a ﬁnal concentration of 25 μM or

20 μM, and pAIE and metal cations were dissolved in water at 1.0 mM

and 10 mM, respectively. The sample was centrifuged before the assay

to remove any protein precipitation. pAIE (or metal cations) solution

(in the syringe) was titrated into YwlE solution (in the sample cell).

Consecutive injections of 2 μL of pAIE (or metal cations) were dispensed

into the protein sample at 50-s intervals. The initial injection was dis-

carded from the data analysis. Control experiments consisted of in-

jecting pAIE (or metal cations) into buﬀer and showed no signiﬁcant

eﬀect of dilution. The data were analyzed, and thermodynamic para-

meters were obtained by ﬁtting the data to a single-site-binding model

2.6. Calculation of YwlE activity

Triplicate data were collected for all experiments, and error bars for

each point were calculated from the triplicate replications. We nor-

malized the activity of YwlE as 100% in the absence of additives, which

was set as blank control. The absorbance peak intensity (I) of A_{405 nm}

B. Huang, et al.

A-GeneralSubjects1864(2020)129698

using OriginPro software.

dephosphorylated by YwlE in vitro (Fig. 1E-F). These ﬁndings demon-

strate that protein arginine phosphatase YwlE depicts broad substrate

speciﬁcity in vitro.

2.8. Michaelis-Menten equations for YwlE against metal cations

To measure the Michaelis-Menten equations for YwlE against metal

cations, we established optimal experiments containing 20 μM YwlE,

100 μM metal cations (Zn²⁺, Ca²⁺, Cd²⁺, Co²⁺, and Ag⁺) and pNPP

(50, 100, 200, 300, and 400 mM). The phosphatase assay was per-

formed at 30 °C using 20 mM Tris/HCl, 100 mM NaCl (pH 7.6) buﬀer.

The phosphatase reaction was monitored based on absorbance at

405 nm.

3.2. Ca²⁺enhances the phosphatase activity of YwlE, but Zn²⁺, Cu²⁺

Cd²⁺, Co²⁺, Ag⁺and Mn²⁺inhibit YwlE

Metals are sequesters to prevent undesirable consequences (reactive

species generation, and biomolecule damage) and to provide nutritional

immunity by preventing the growth of pathogens in mammals.

Moreover, metal-binding proteins evolved to maintain these extremely

low cytosolic metal ion concentrations and protect the cell against cy-

totoxic increases in intracellular metal ions [15]. Metal cations play

critical roles in phosphatase activity; however, some of these phos-

phatases are not considered metalloenzymes [10]. Metal ion interac-

tions with phosphatases extend the roles of metal ions in phosphatase

action and phosphorylation signaling. Zn²⁺and Mg²⁺inhibits and

activates protein tyrosine phosphatase PTP1B, respectively [16,17].

Zn²⁺, Cu²⁺, Ca²⁺and Mg²⁺inhibit protein histidine phosphatase

PHPT1 [18]. Co²⁺signiﬁcantly increases the activity of alkaline

phosphatase PfuAP, whereas Mg²⁺and Ca²⁺only have modest eﬀects

[19]. Ni²⁺provides signiﬁcant activation for bacteriophage λ protein

phosphatase λPP, while Mn²⁺is a physiologically relevant activation

metal ion in E. coli [20]. Mn²⁺, Co²⁺and Fe²⁺activate PP1 and PP2A

[21]. PP2B phosphatase is activated by Ca²⁺[22]. Therefore, metal

cations act as inhibitors or activators for phosphatases to regulate

phosphatase activity.

3. Results and discussion

3.1. YwlE depicts broad substrate speciﬁcity in vitro

To ensure that the phosphatase activity is derived from YwlE itself

but not from contaminants, we veriﬁed the activity of YwlE and its

mutants. The C^a(X)₄C^bR(S/T) redox sensing cysteine nanoswitch is

crucial for YwlE function as depicted by the YwlE homolog from

Geobacillus stearothermophilus. It has been demonstrated that neither the

YwlE(C9S) nor the YwlE(C9A) mutant protein from Geobacillus stear-

othermophilus exhibits any signiﬁcant phosphatase activity [9,13]. Re-

sidue C7 in YwlE from Bacillus subtilis is homologous to C9 in YwlE from

Geobacillus stearothermophilus based on sequence alignment analysis.

Thus, we prepared YwlE and mutants (YwlE(C7A) and YwlE(C7S))

proteins that we puriﬁed through Ni²⁺-NTA aﬃnity and Superdex G75

size-exclusion chromatography (Fig. 1A). The molecular weights of

proteins were identiﬁed using ESI-MS (S1).

The Arg-peptide was speciﬁcally phosphorylated on the guanidi-

nium group of the Arg residue by protein arginine kinase McsB with a

phosphorylation eﬃciency of ~33% that was calculated from the in-

tegral area of individual peaks (Fig. 1B-C). The three peaks (retention

time: 17.71 min, 17.25 min and 17.23 min) represented the Arg-peptide

(1429.8 Da). The emerging peak (retention time: 18.25 min) re-

presented a phosphorylated pArg-peptide with a mass of 1509.8 Da,

which is 79.0 Da (the mass of PO₃²⁻) greater than the unmodiﬁed

substrate (1429.8 Da). The unambiguous identiﬁcation of Arg as the

site of modiﬁcation on the Arg-peptide was conﬁrmed by ECD-MS/MS.

[1] The pArg-peptide species was no longer observed after YwlE

treatment, suggesting that YwlE is capable of dephosphorylating pArg-

peptide in vitro. Additionally, as shown in Fig. 1B, YwlE hydrolyzed

pArg-peptide without metal cations except for the Na⁺from the YwlE

storage buﬀer, conﬁrming that YwlE is not a metalloenzyme in vitro.

Consistently, YwlE contains a P-loop signature and a global fold

common to all low molecular weight protein tyrosine phosphatases

(LMW-PTPs) that are generally not recognized as metalloenzymes.

pNPP is analogous to the side-chain of pTyr and is a nonspeciﬁc

substrate for the measurement of the enzymatic activity of protein

tyrosine phosphatases [14]. After determining that small molecule

phosphotyrosine (pTyr) was not hydrolyzed by YwlE and its mutants

(data not shown), we tested whether pNPP could be hydrolyzed by

YwlE by monitoring the ³¹P spectra using NMR spectroscopy. As con-

trols, we found that pNPP was quite stable in buﬀer-A in the absence of

YwlE at various temperatures or for longer incubation times. However,

a considerable amount of pNPP was hydrolyzed in the presence of YwlE.

Prokaryotes evolved in a reductive environment when certain

transition metal ions became available [23]. Metalloregulatory proteins

control metal ion uptake, storage and eﬄux systems to regulate metal

ion homeostasis. There are known and putative metal homeostasis

systems in Bacillus subtilis for Ca²⁺, Mg²⁺, Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺and

Mn²⁺[11]. In Staphylococcus aureus, the clpC operon encodes ctsR, clpC,

mcsA and mcsB genes. The expression of the clpC operon in S. aureus is

induced in response to various types of heavy metals, such as copper

and cadmium [24]. There are no reports on the eﬀects of metal cations

on YwlE.

Intriguingly,

K_0.5= 33.1

Ca²⁺

0.92 μM (Fig. 2A). Ca²⁺binds to YwlE with a dis-

enhances

YwlE

activity

with

sociation constant K_d= 3.0 × 10⁻⁵M at the cooperative-binding mode

(Hill coeﬃcient n = 5.02) (Table 1, S4), which does not cause the

conformational change of YwlE based on the CD analysis (S5).

In contrast, Zn²⁺and Cu²⁺almost completely inhibit YwlE with

97.2% and 100% reduction in activity at the concentration of 50 μM

with distinct IC₅₀values, respectively (Table 1, S6). Zn²⁺is a powerful

inhibitor of most phosphatases; however, alkaline phosphatase requires

Zn²⁺and Mg²⁺in its active site for its proper function [25]. Zn²⁺and

Cu²⁺prefer to coordinate sulfur ligands [26]; however, Cu²⁺can also

catalyze the oxidation of thiol groups by O₂to form the disulﬁde bond

(SeS) (S7A) [27]. We used ESI-MS to assess whether the addition of

Cu²⁺could oxidize the thiol groups of YwlE protein. According to the

results (S7B), Cu²⁺caused a 2-Da loss in YwlE's molecular weight. This

ﬁnding demonstrates that Cu²⁺catalyzes the formation of the disulﬁde

bond between Cys7 and Cys12 within the catalytic site rather than

binding with thiols, which induces the conformational change of YwlE

(S8). Cys oxidation closes catalytic pocket to inhibit YwlE. In contrast,

the activity of YwlE is reversed by the reducing reagent DTT, which

reduces the disulﬁde bond to thiol groups and drives the catalytic

pocket to an open conformation.

As shown in Fig. 1D, the intensity of δ_31P= −3.1 ppm (representation

3−

of pNPP) decreased and δ_31P= 1.5 ppm (representation of PO₄

re-

lease from pNPP) emerged when pNPP was incubated with YwlE but not

with its two mutants, which apparently are not active against pNPP. In

addition, UV absorbance at 405 nm, which represents p-nitrophenol

(pNP), was observed when pNPP was treated with YwlE (S2). This

ﬁnding signiﬁes that YwlE eﬃciently catalyzes the hydrolysis of pNPP

in vitro. Hence, pNPP can act as a chromogenic substrate for char-

acterization of the enzymatic activity of YwlE.

Co²⁺, Cd²⁺and Ag⁺inhibit YwlE with various IC₅₀values that are

greater than that of Zn²⁺(Table 1, S6). In addition, Mn²⁺inhibits YwlE

activity in a certain concentration range (S9), and the greatest level of

inhibition of YwlE activity (i.e., 37.8%) was noted with 25 μM Mn²⁺

We also investigated the eﬀect of other metal cations on YwlE activity

and found that Li⁺, Ni²⁺and Mg²⁺show only modest eﬀects (S10).

The counterions (Cl⁻and NO₃⁻) have no eﬀect on phosphatase activity

Additionally, small molecules phosphocreatine and pArg were also

B. Huang, et al.

A-GeneralSubjects1864(2020)129698

Fig. 2. The eﬀects of metal cations on YwlE. (A) Ca²⁺enhances YwlE activity with K_0.5= 33.1

0.92 μM. (B) Histogram representation of competition assays for

Ca²⁺against Ag⁺, Cd²⁺, Co²⁺, Zn²⁺, and Cu²⁺. The error bars indicate the standard deviation of triplicate experiments. (C) Lineweaver-Burk plot depicting the

mode for Ag⁺, Cd²⁺, Co²⁺, Zn²⁺, and Ca²⁺binding with YwlE. The blue dash line represents the Lineweaver-Burk plot of YwlE using diﬀerent pNPP concentrations

without metal cations. The error bars indicate the standard deviation in triplicate experiments.

at the corresponding concentration ranges.

YwlE performs nucleophilic substitution reaction to depho-

binding to YwlE is an important biological process that should be as-

sessed using comprehensive studies via metadynamics, molecular dy-

namics simulations and quantum chemical calculations in combination

with experimental investigations, ideally at the molecular level.

sphorylate the phosphosubstrates. Metal cations have diﬀerent eﬀect on

the arrangements and activities of water molecules that are necessary

for the hydrolysis of the phosphocysteine intermediate in the second

catalytic step of the reaction [7]. Competition experiments involving

3.3. The eﬀect of oxyanions on YwlE activity

the Ca²⁺activator and an inhibitory cation, such as Zn²⁺, Cu²⁺, Cd²⁺

Co²⁺, or Ag⁺, were performed to explore the binding location within

the enzyme and metal cation preference. Ca²⁺interferes with the ef-

fects of Ag⁺, Cd²⁺, Co²⁺and Zn²⁺on the phosphatase activity of YwlE

to various degrees, while the inhibition of YwlE activity by Cu²⁺cannot

be reversed by the activator Ca²⁺(Fig. 2B). Cd²⁺and Zn²⁺are most

aﬀected by Ca²⁺, and the phosphatase activity of YwlE exhibits 64.91%

and 17.52% enhancement by Cd²⁺or Zn², respectively, in the presence

of Ca²⁺compared with alone. This eﬀect is likely due to the weaker

binding aﬃnity of Cd²⁺and Zn²⁺with YwlE than that of Ca²⁺based

on ITC analysis (Table 1). Consistently, as Ag⁺and Co²⁺exhibit

stronger binding aﬃnity to YwlE, adding activator Ca²⁺into the assay

only increases the YwlE activity by 5.77% and 9.22%, respectively.

Activator Ca²⁺and inhibitory metal cations comprehensively inﬂuence

YwlE activity. To further deﬁne the mode of inhibition of inhibitory

cations, we determined the YwlE inhibition pattern (Fig. 2C). These

results indicate that Zn²⁺, Cd²⁺, Co²⁺and Ag⁺act as classic compe-

titive inhibitors. In contrast, activator Ca²⁺results in a decrease in K_m

(K_m= 180.375 μM) but the same υ_max(1.25 mM/min) and K_cat

(3.57 min⁻¹) for YwlE without metals (K_m= 206.125 μM) (Fig. 2C,

Table 1). This ﬁnding demonstrates that inhibitory ions are actually

competitive with activator Ca²⁺. Therefore, we hypothesize that they

synergistically bind to YwlE, suggesting that YwlE exhibits similar

substrate preference for these metal cations and the same binding lo-

cation.

Inorganic phosphate, a competitive inhibitor of phosphatase, is the

hydrolytic product of a phosphatase reaction. At present, commercial

broad-spectrum phosphatase inhibitors target protein serine/threonine

phosphatases and protein tyrosine phosphatases. NaF, Na₂P₂O₇and β-

glycerophosphate target protein serine and threonine phosphatases,

while Na₃VO₄inhibits the activity of protein tyrosine phosphatases.

Here, we investigate the eﬀect of commercial phosphatase inhibitors on

the protein arginine phosphatase YwlE (Table 2). Na₃VO₄, NaF,

Na₄P₂O₇and β-glycerophosphate reduce YwlE activity with distinct

IC₅₀values. The protein tyrosine phosphatase inhibitor Na₃VO₄inhibits

YwlE more prominently than protein serine/threonine phosphatases

inhibitors probably because YwlE shares the active catalytic motif

C(X)₄CR(S/T) with LMW-PTPs. Vanadate exhibits structural similarities

with phosphate and is considered to be a transition state analog of

phosphate within phosphatase [28]. Moreover, vanadium compounds

have received extensive interest as antidiabetic agents and potential

anticancer drugs [29,30]. We evaluated the eﬀect of commercial pro-

tease/phosphatase inhibitor (named PPI) tablets (protease inhibitors:

aprotinin, bestain, E-64 and leupeptin; phosphatase inhibitors: Na₃VO₄,

NaF, Na₄P₂O₇, and β-glycerophosphate) on YwlE (S11). PPI exhibits an

excellent ability to inhibit YwlE at a higher ﬁnal concentration (3[×] or

higher). Hence, samples for phosphoproteome assessment require

higher concentrations of inhibitors to adequately inhibit phosphatase

activity and preserve the phosphorylation states.

As prokaryote evolved [23], metals, such as Fe²⁺(Fe³⁺), Mn²⁺

Oxyanions are structurally analogous to phosphate and inhibit

Cu²⁺(Cu⁺), Ni²⁺, Co²⁺and Zn²⁺, are required for the growth of many

microorganisms. Arginine phosphorylation of proteins plays a critical

role in the response to diﬀerent stress conditions in Bacillus subtilis,

including oxidative stress and heat shock. Therefore, metal cation

phosphatases. Therefore, we investigated the role of other oxyanions in

2−

the regulation of YwlE activity. We found that oxyanions B₄O₇

CO₃²⁻, SO₄²⁻, and NO₃minimally aﬀect the hydrolysis of pNPP by

−

3−

2−

YwlE (Table 2, S12). Interestingly, as activators, BO₃

and MoO₄

Table 1

The eﬀects of metal cations on the phosphatase activity of YwlE.

Metal cations

I: inhibition A: activation

K_0.5or IC₅₀

Dissociation constant K_d(M)

K_m(μM)

Ca²⁺

Zn²⁺

Ag⁺

33.1

5.5

55.5

23.2

17.3

0.92 μM (K_0.5

)

3.0 × 10⁻⁵

6.8 × 10⁻⁴

2.3 × 10⁻⁸

9.8 × 10⁻⁴

5.0 × 10⁻⁸

180.375

206.125

518.625

405.000

251.375

0.003 μM (IC₅₀

)

0.32 μM (IC₅₀

0.11 μM (IC₅₀

0.71 μM (IC₅₀

Cd²⁺

Co²⁺

B. Huang, et al.

A-GeneralSubjects1864(2020)129698

Table 2

(Fig. 3E), indicating that H₂O₂catalyzed the formation of a disulﬁde

bond between two Cys within the catalytic site. In addition, two mutant

proteins (YwlE (C7S) and YwlE (C7A)) do not show signiﬁcant phos-

phatase activity for phosphoguanidinium, suggesting that Cys within

the catalytic motif is crucial for YwlE activity.

We found that reducing reagents DTT, β-Me, and TCEP reduce

disulﬁde bonds to improve YwlE activity (Fig. 3D). TCEP improves

YwlE activity by 27.3% at 25 μM and by 35.8% at 200 μM. In the assay,

detection of pNP by absorbance at 405 nm gradually decreases as TCEP

increases. This ﬁnding is attributed to the fact that TCEP lowers the

solution pH from 8.0 to an acidic range, but the detection of pNP by

absorbance at 405 nm depends on its intense yellow color at a basic pH.

The pH also aﬀects the nucleophile eS anion state of Cys with pKa

(-SH) = 8.4. We also monitored the substrate pNPP by absorbance at

312 nm to characterize YwlE activity. YwlE activity was gradually en-

hanced by 41.5% and 13.1% as DTT and β-Me concentrations increased

to 200 μM, respectively.

Previous studies revealed that the transcriptional factor HrcA ex-

hibits increased phosphorylation levels under oxidative stress. [5]

Phosphorylation of R64, which is located within the winged helix-turn-

helix DNA binding domain of HrcA, can potentially serve as a universal

mechanism for regulating protein-DNA interactions. Dissociation of

phosphorylated protein from DNA drives the expression of stress-re-

sponse genes, which is vital for bacterial virulence. Thus, as a sensor of

cell stress, YwlE is inactivated by oxidative stress [8]. The Cys within

the catalytic site regulates YwlE activity by acting as a regulatory

switch.

The eﬀects of anions on YwlE phosphatase activity.

Anions

I: inhibition A: activation

K_0.5or IC₅₀

3−

VO₄

35.0

41.0

57.5

43.9

N/A

–

0.78 μM (IC₅₀

)

F⁻

1.08 μM (IC₅₀

4.34 μM (IC₅₀

1.39 μM (IC₅₀

β-glycerophosphate

4−

P₂O₇

WO₄

2−

C₂O₄

2−

BO₃

2−

MoO₄

B₄O₇

2−

CO₃

SO₄

NO₃

–

2−

−

NS: no signiﬁcant eﬀect; N/A: not applicable.

enhance pNPP hydrolysis by YwlE (Table 2, S13A-B). MoO₄²⁻enhances

phosphocreatine hydrolysis. Nevertheless, small molecule pArg forms a

stable complex with MoO₄²⁻, which decreases the hydrolysis of pArg

3−

[31,32]. Activator (BO₃

promotes a conformational change that locks the substrate in a caged

Michaelis complex and activates the hydrolysis process [33]. YwlE is

or MoO₄²⁻) interacts with enzyme and

2−

weakly inhibited by C₂O₄

and WO₄

(Table 2, S13C-D), which are

generally weak inhibitors. WO₄²⁻, which often mimics the substrate

phosphate group in complex with phosphatase, is located at the center

of the active catalysis site [34]. The inorganic salt Na₂WO₄, a protein

tyrosine phosphatase PTP1B inhibitor, is a potential antiplatelet agent

[35]. Moreover, hundreds of pArg proteins have been identiﬁed in

human cells [6], and pArg may be implicated in human diseases. Pro-

tein arginine kinase and cognate protein arginine phosphatase are po-

tential drug targets. Therefore, such an inhibition has broad implica-

tions for the physiology, pharmacology and toxicology of oxyanions.

3.5. pAIE, a stable analog of pArg, inhibits YwlE

pAIE (2-((2-ammonioethyl)amino)-2-iminoethyl phosphonic acid),

a stable analog of the side chain of pArg, replaces the hydrolytically

labile PeN bond in pArg with a stable PeC bond according to “Grimm's

hydride displacement law” (Fig. 4A) [39]. The newly introduced -CH₂-

could serve as a potential hydrogen bond donor in protein-protein in-

teractions. pAIE binds to YwlE with a K_d= 149.1 nM and a binding

stoichiometry n of 1.2 (roughly one pAIE for one YwlE protein) based

on ITC analysis (Fig. 4B). To test the eﬀect of pAIE on YwlE activity, we

3.4. Two cysteines within active site are crucial for YwlE activity

Oxidation/reduction is a key regulatory event in protein tyrosine

phosphatase function. YwlE shares the active catalytic C(X)₄CR(S/T)

motif (P-loop) with LMW-PTPs. Residue C7 (YwlE(Bacillus subtilis),

which is equal to Cys9 in YwlE (Geobacillus stearothermophilus)) acts as a

strong nucleophile, attacking the phosphorus center of the substrate,

and as a phosphate acceptor, producing a covalent phosphocysteine

intermediate in the second reaction step. Therefore, the redox state

(-SH) of Cys7 inﬂuences YwlE activity. Fuhrmann and colleagues have

developed an inhibitor cyc-SeCN-amidine that catalyzes the formation

of a disulﬁde bond between Cys9 and Cys14 within the active site of

YwlE (Geobacillus stearothermophilus) [9].

determined its IC₅₀value. The IC₅₀for pAIE is 316.3

12.73 μM

(Fig. 4C), indicating that pAIE is able to inhibit the phosphatase activity

of YwlE. pAIE mimics the size, the charge distribution and electrostatic

map of a phosphoguanidinium moiety. We used autodock software to

dock pAIE onto YwlE to better understand their interactions. As illu-

strated in Fig. 4D, pAIE forms multiple hydrogen bonds with the

backbone NH of Phe40, the hydroxyl group of Thr11, and the carboxyl

group of Asp118. Thus, pAIE potentially blocks the catalytic pocket of

YwlE to inhibit its phosphatase activity. This ﬁnding demonstrates that

pAIE, as a non-hydrolysable pArg mimic, is a potential phosphatase

inhibitor.

IAM (iodoacetamide) is an irreversible alkylation agent that has an

acetamide (AM) group that could covalently react with thiols (Fig. 3A).

We found that IAM decreases YwlE activity by 99.7% with an IC₅₀of

7.8

0.33 μM (Fig. 3B). Upon incubation with IAM, YwlE monoalk-

Protein arginine kinase McsB is a crucial component of the stress

and pathogenicity mechanisms in Staphylococcus aureus [3], suggesting

that modulating protein arginine phosphorylation may represent an

attractive strategy to treat bacterial infections. Therefore, under-

standing the interaction of inhibitors with YwlE is important for im-

proving the design of potential antibacterial agents.

ylation (+ 1 AM) and dialkylation (+ 2 AM) were observed (Fig. 3E)

due to incomplete reaction between YwlE and IAM. It's our under-

standing that both mono- and di-alkylation are capable of fully in-

hibiting YwlE as the addition of an AM group on either C7 or C12 alone

would have blocked the catalytic pocket.

H₂O₂, an oxidant, is a signaling agent that mediates diverse phy-

siological responses [36]. Protein tyrosine phosphatase is an important

target of H₂O₂produced during signal transduction processes. The Cys

residue in the active site exists as a thiolate anion (eS) at biological pH

[37] and is sensitive to oxidation by various oxidants, including H₂O₂.

H₂O₂induces the formation of a cyclic sulfenamide or a disulﬁde

(Fig. 3A). Such oxidations of Cys abolish phosphatase activity and can

be reversed by cellular thiols. [38] H₂O₂reduces YwlE activity by

Diﬀer from the inhibition of YwlE by pAIE, which does not inhibit

kinase activity of McsB (data not shown). pAIE mimics pArg that is the

phosphorylated product mediated by McsB. McsB prefers to release the

product in an open conformation. Kinase inhibitor are the largest class

of new cancer drugs [40], it's necessary to explore the relationship

between pArg modiﬁcation and human diseases with the discovery of

pArg proteins in human cells, and to develop protein arginine kinase

inhibitors.

95.4% with an IC₅₀of 260.4

that H₂O₂caused a 2-Da reduction in the YwlE molecular weight

9.26 μM (Fig. 3C). Mass-spec revealed

B. Huang, et al.

A-GeneralSubjects1864(2020)129698

Fig. 3. The two Cys residues within the catalytic motif regulate the phosphatase activity of YwlE. (A) Iodoacetamide (IAM) and (B) H₂O₂inhibit YwlE with diﬀerent

IC₅₀values. (C) (E) IAM and H₂O₂alkylates and oxidizes Cys, respectively. The ESI-MS spectra, including the mass (black) and charge status (red), are shown

(theoretical mass of mono-alkylation YwlE = 16,855.23 Da, di-alkylation YwlE = 16,927.27 Da and oxidized YwlE = 16,783.19 Da). (D) DTT, β-Me and TCEP

enhance YwlE activity.

B. Huang, et al.

A-GeneralSubjects1864(2020)129698

Fig. 4. Stable analog pAIE inhibits YwlE. (A) The structures of pAIE (up) and pArg (down). (B) ITC measurements of the binding between pAIE and YwlE. (C) pAIE

inhibits YwlE with IC₅₀of =316.3

12.73 μM. (D) A 2D map of the key residues that interact with pAIE.

4. Conclusions

toxicology and pharmacology of metal cations and oxyanions.

The signiﬁcance of protein arginine phosphorylation in organisms

has been a subject of increasing interest. Protein arginine phosphor-

ylation is negatively regulated by YwlE. Therefore, YwlE activity is of

importance for bacterial stress tolerance and virulence. Moreover, the

discovery of pArg in eukaryotes also opened the possibility that pArg

may be related to human diseases. Thus, protein arginine kinases and

phosphatases are important targets for potential drug development.

In this report, for the ﬁrst time, we explore the roles of metal ca-