Structural Insights into the Regulation of Staphylococcus aureus

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Structural insights into the regulation of Staphylococcus aureus phosphofructokinase by tetramer-dimer conversion Tian Tian, Chengliang Wang, Minhao Wu, Xuan Zhang, and Jianye Zang Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00028 • Publication Date (Web): 25 Jun 2018 Downloaded from http://pubs.acs.org on June 26, 2018

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Biochemistry

Structural insights into the regulation of Staphylococcus aureus phosphofructokinase by tetramer-dimer conversion Tian Tian1,2, Chengliang Wang1,2, Minhao Wu1,2, Xuan Zhang1,2* and Jianye Zang1,2*

1

Hefei National Laboratory for Physical Sciences at Microscale CAS Center for Excellence in

Biomacromolecules, Collaborative Innovation Center of Chemistry for Life Sciences, and School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, China. 2

Key Laboratory of Structural Biology, Chinese Academy of Sciences, Hefei, Anhui 230026,

China

*To whom correspondence may be addressed. Address correspondence to Jianye Zang: School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, China. Phone: 86-551-63603433, E-mail: [email protected] Address correspondence to Xuan Zhang: School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, China. Phone: 86-551-63603435, E-mail: [email protected] Accession number: The coordinates for SaPfk, SaPfk/ADP, SaPfk/ATP, SaPfk/F6P, SaPfk/AMP-PNP, and SaPfk/F6P/AMP-PNP-Mg have been deposited in the Protein Data Bank under accession codes 5XOE, 5XZA, 5XZ9, 5XZ7, 5XZ6 and 5XZ8, respectively.

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ABSTRACT Most reported bacterial Phosphofructokinases (Pfks) are tetramers that exhibit activity allosterically regulated via conformational changes between the R- and T-states. We report that the Pfk from Staphylococcus aureus NCTC 8325 (SaPfk) exists as both an active tetramer and inactive dimer in solution. Multiple effectors, including pH, ADP, ATP, and adenylyl-imidodiphosphate (AMP-PNP), cause equilibrium shifts from the tetramer to dimer, whereas the substrate F6P stabilizes SaPfk tetrameric assembly. Crystal structures of SaPfk in complex with different ligands and biochemical analysis reveal that the flexibility of the Gly150-Leu151 motif in helix α7 plays a role in tetramer-dimer conversion. Thus, we propose a molecular mechanism for allosteric regulation of bacterial Pfk via conversion between tetramer and dimer in addition to the well-characterized R-/T-state mechanism.

Short title: Regulation of SaPfk activity by tetramer-dimer conversion

Key words: phosphofructokinase, crystallography, enzyme catalysis, allosteric effectors, equilibrium, molecular mechanism.


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Biochemistry

ABBREVIATIONS ADP, Adenosine 5'-diphosphates; AMP, Adenosine 5'-monophosphates; AMP-PNP, Adenylyl imidodiphosphate; ATP, Adenosine 5'-triphosphates; cAMP, cyclic AMP; DTT, Dithiothreitol; F6P, D-fructose 6-phosphate; GDP, Guanosine 5'-diphosphates; IPTG, isopropyl 1-β-D-galactopyranoside; ITC, Isothermal Titration Calorimetry; kDa, kilodalton; LB, Luria-Bertani; IPTG, isopropyl 1-β-D-galactopyranoside; SDS: Sodium dodecyl sulfate; MES,

2-Morpholinoethanesulfonic

acid;

ADP-Mg,

ADP-magnesium;

NADH,

nicotinamide-adenine dinucleotide reduced form; Ni-NTA, nickel-nitriloacetic acid; PCR, polymerase chain reaction; PEG, polyethylene glycol; PEP, phosphoenolpyruvate; Pfk, Phosphofructokinase; PGA, phosphoglycolate; RMSD, root mean square deviation; SEC, Size-exclusion chromatography; Tris, tris(hydroxymethyl)aminomethane; Vmax, maximum velocity; WT, wild type; β-ME, β-Mercaptoethanol.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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INTRODUCTION Phosphofructokinase (Pfk, EC 2.7.1.11) is an ATP-dependent 6-phosphofructokinase that catalyzes the key control step of glycolysis by converting D-fructose 6-phosphate (F6P) and ATP to fructose 1,6-bisphosphate and ADP. Bacterial Pfks are allosteric enzymes that form a “dimer of dimers” tetrameric assembly. Subunits A and B contact each other via Interface I, while subunits C and D form the same interaction due to the 222 symmetry of the tetramer (Figure 1A). Dimer AB and CD form a tetramer via dimer-dimer interface (Interface II, Figure 1B). Interface II is responsible for the substrates binding, while Interface I contains the binding site for allosteric effectors (Figure 1A and 1B). Two allosteric effectors for bacterial Pfks have been reported, including the allosteric activator ADP-Mg and the inhibitor phosphoenolpyruvate (PEP), both of which are K-type allosteric effectors (change the affinity of the enzyme for the substrate so that the Km value is altered) of the enzyme for F6P without changing its maximal activity1, 2. By comparing the crystal structures of Pfk from Bacillus stearophilus (BsPfk) in complex with phosphoglycolate (PGA; a PEP analog; PDB: 6PFK) 3 and BsPfk bound to F6P and ADP-Mg (PDB ID: 4PFK) 4, an R-/T-state model was proposed for the allosteric behavior of bacterial Pfks (Figure 1C)

1, 3, 5

. In this model, the

quaternary structure of the substrate- and activator-bound R-state of Pfk undergoes a 7° relative rotation of dimer CD with respect to dimer AB as compared with the inhibitor-bound T-state of Pfk, thereby repacking the dimers across Interface II, which increases the binding affinity of the enzyme for the F6P substrate. 3


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Biochemistry

Figure 1 Tetrameric R- and T- state structures of Pfk from Bacillus stearophilus (BsPfk). (A) Effector and (B) substrate binding sites along Interface I and II of BsPfk, respectively. Subunit A, B, C and D are shown as surface and colored blue, pink, cyan and orange respectively. The effectors binding in Interface I and the substrates binding in Interface II are shown as dots and colored purple for ADP, green for PGA, and yellow for F6P. (C) The R-/T-state model for the allosteric behavior of BsPfk. Dimer AB of R-state structure (4PFK) and T-state structure (6PFK) are overlaid and shown as surface, while dimer CD in both structures are shown as cartoon and colored wheat and cyan, respectively. Bacterial Pfks were reported as tetramers, except that of the Thermus thermophilus Pfk (TtPfk) tetramer, which is disassociated to dimers in the presence of PEP 6. The addition of either F6P or ADP-Mg or the removal of PEP from the solution results in reassembly of the catalytically active tetramer 6. Despite this, the allosteric characteristics of TtPfk and BsPfk are quite similar, with the hyperbolic plot of enzyme activity versus F6P concentration


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

changing to a sigmoidal shape only in the presence of PEP

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7, 8

. By introducing a

tryptophan-shifted mutant W179Y/Y164W, BsPfk shares similar behavior with TtPfk that dissociates into inactive dimers with PEP and reversely returns to tetramer with the addition of F6P9. These findings suggested that quaternary structural changes probably play an important role in allosteric communication of TtPfk and BsPfk. However, no dimeric structure of bacterial Pfk has been reported and the molecular mechanism of tetramer-dimer conversion remains unclear. Here, we observed that Pfk from Staphylococcus aureus NCTC 8325 (SaPfk) exists as both tetramer and dimer in solution and the tetramer-dimer conversion is regulated by multiple effectors. To elucidate the molecular basis of tetramer-dimer conversion, we determined both the dimeric and tetrameric structures of SaPfk. Consistently, SaPfk only crystallized as a tetramer upon F6P binding, while mutation of key residues involved in F6P binding results in the formation of inactive SaPfk dimers only. Sequence alignment and structural analysis indicated a unique Gly150-Leu151 (G-L) motif on the SaPfk dimer-dimer interface. Mutation of G-L to G150D-L151A (D-A) stabilized the SaPfk tetramer, with the mutant showing higher affinity for F6P and catalytic activity, but less sigmoidal kinetics than the wild-type (WT) variant. These findings demonstrated that SaPfk activity is regulated by undergoing tetramer-dimer conversion modulated by substrates and allosteric effectors.


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Biochemistry

EXPERIMENTAL PROCEDURES Cloning, expression and purification S.aureus Pfk (GeneID: 3919277) gene was amplified using PCR (polymerase chain reaction) method with S.aureus genome as the template. The amplified product was purified using Normal DNA Purification Kit (TIANGEN Cat: DP204) and digested with BamHI and XhoI (TAKARA, Japan), and inserted into a similarly digested pET-22b-C6H vector. Mutagenesis was performed following a PCR-mediated site directed protocol using wild type plasmid as the template and primers with the desired mutation. All plasmids were verified by sequence analysis. The SaPfk plasmids were transformed into E.coli Rosetta2 (DE3) (Novagen) cells and grown with shaking overnight at 37°C in a 100 mL Luria–Bertani (LB) medium containing 100 µg/mL ampicillin. Fifteen milliliter overnight starter culture was then transferred into 1 L LB medium and incubated at 37°C until reaching an OD600 of 0.6-0.8. The culture was then induced with 0.4 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and incubated for 20 h at 16°C. Cells were harvested by centrifugation at 6,760 g for 8 min at 4°C and resuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 5 mM Imidazole, 5% Glycerol, 2 mM β-ME). The cells were then homogenized by sonication and the lysate was centrifuged at 23,800 g for 30 min at 4°C. The resulting supernatants containing target protein fused with C-terminal hexahistidine-tag was subjected to a nickel-nitriloacetic acid (Ni-NTA, QIAGEN) affinity chromatography column pre-equilibrated with lysis buffer. The column was washed with 50 column volumes of washing buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 50 mM Imidazole, 5% Glycerol, 2 mM β-ME) to remove contaminants and the bound protein was harvested with 3 column volumes of elution buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 250 mM Imidazole, 5% Glycerol, 2 mM β-ME). The eluted protein was concentrated by centrifugal ultrafiltration (Millipore, 10 kDa cutoff) at 4,000 g and further purified using a HiLoad 16/60 Superdex 200 pg size exclusion column (GE healthcare) equilibrated with SEC


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buffer 1 (20 mM Tris-HCl pH 7.5, 200 mM NaCl). The mutant proteins were expressed and purified as described above.

Size-exclusion chromatography for oligomeric states analysis The oligomeric states of SaPfk (wild type and mutants) at different concentrations, at various pH levels, or with the addition of different substrates and effectors were verified using Superdex 200 10/300 GL or Superdex 200 5/150 GL size-exclusion chromatography (GE healthcare) at 15 °C. All the experiments were performed with 20 mM Tris-HCl pH 7.5, 200 mM NaCl except for experiments with different pH levels. To exclude the effect of pH level on SaPfk, the pH values of gel filtration buffer and the stock solutions of ADP, PEP, ATP and AMP-PNP were adjusted to 7.5. The buffers for different pH levels were listed in Table S1.

Crystallization The purified SaPfk protein was concentrated to 114 µM in 20 mM Tris-HCl pH 7.5, 200 mM NaCl for crystallization. Initial crystal screening was carried out at 10°C using sitting-drop vapour-diffusion method with commercial kits (Hampton Research). Equal volumes of protein (1 µl) and reservoir solutions were mixed and equilibrated against 100 µl well solution. The optimized condition for native SaPfk crystals was 0.1 M MES pH 6.0, 0.15 M (NH4)2SO4, 15% PEG 4,000 (w/v). The

Crystals

of

SaPfk/ADP,

SaPfk/ATP,

SaPfk/F6P,

SaPfk/AMP-PNP

and

SaPfk/F6P/AMP-PNP-Mg were obtained by co-crystallization of native SaPfk with ligands at a molar ratio of protein to ligands of 1:10. The final crystals of SaPfk/ADP and SaPfk/AMP-PNP for data collection were obtained from the same condition, which contains 0.1 M Sodium citrate pH 5.5, 15% PEG 6,000 (w/v). SaPfk/ATP crystals were grown from 0.5 M (NH4)2SO4, 0.1 M HEPES pH7.5, 30% MPD (v/v). The optimized condition for SaPfk/F6P consists of 0.2 M sodium acetate, 0.1 M Sodium citrate pH 5.5, 5% PEG 4,000 (w/v). The condition for SaPfk/F6P/AMP-PNP-Mg crystals was the same as that for native SaPfk crystals.


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Biochemistry

TABLE 1: Data collection and structure refinement statistics. SaPfkA/F6P/AMP-PNPSaPfkA(apo)

SaPfkA/AMP-PNP

SaPfkA/F6P

SaPfkA/ATP

SaPfkA/ADP

Mg PDB entry

5XOE

5XZ6

5XZ7

5XZ8

5XZ9

5XZA

C2

I2

I222

I222

C2

C2

116.230,36.949,87.8

89.410,37.010,101.1

73.560,80.200,121.3

118.75,36.79,89.8

119.09,

0

36.81,89.86

90.00,123.82,90.0

90.00,

0

90.00

Data Collection Space group Unit cell parameters

a, b, c (Å)

α, β, γ (°)

75.08, 79.99, 120.55 10

35

00

90.00,123.31,90.00

90.00,103.36,90.00

90.00,90.00,90.00

124.01,

90.00,90.00,90.00

Resolution (Å)a

50.0-3.0(3.05-3.0)

50.0-2.6(2.64-2.6)

50.0-1.6(1.69-1.60)

50-1.95(2.06-1.95)

50-2.0(2.05-2.0)

50.0-1.9(1.93-1.9)

Rmerge (%)b

10.5(47.4)

10.6(45.1)

10.7(41.7)

13.3(37.7)

9.6(50.9)

7.4(37.8)

Mean I / σI

13.2(2.4)

10.7(2.6)

11.6(4.3)

9.6(5.3)

12.5(3.1)

20.8(4.0)

Wilson plot B factors(Å2)

39.1

19.6

12.6

16.7

38.5

11.9

Completeness (%)

99.5(98.6)

98.1(97.8)

100.0(100.0)

99.4(100.0)

99.9(100)

99.6(98.8)


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Redundancy

4.6(4.5)

3.5(3.4)

6.2(5.3)

5.5(5.5)

4.9(4.9)

3.8(3.8)

Number of unique reflections

6551(340)

9970(486)

47521(6845)

26662(3860)

22161(1627)

25669(1269)

Resolution (Å)

50.0-3.0

50.0-2.7

50.0-1.6

50.0-1.95

50.0-2.0

50.0-1.9

Rworkc/Rfreed(%)

22.00/28.40

21.95/28.80

17.06/19.28

20.68/26.07

20.64/24.26

18.92/22.92

No. of atoms

2411

2459

2842

2645

2561

2643

Bond lengths (Å)

0.0135

0.0106

0.0082

0.0120

0.0112

0.0104

Bond angles (°)

1.708

1.668

1.431

1.6835

1.664

1.6858

50.40

25.82

17.08

27.95

28.34

27.68

91.40

94.90

97.37

97.43

92.12

95.33

8.60

5.10

2.63

2.57

7.88

4.67

0

0

0

0

0

0

Refinement

R.m.s. deviations

B-factors(Å2) Ramachandran plot Most favored regions (%) Additionally

allowed

regions (%) Outliers (%) a

The values in parentheses refer to statistics in the highest shell.

b

Rsym =|Ii－|/|Ii| where Ii is the intensity of the ith measurement, and is the mean intensity for that reflection.


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Biochemistry

c

Rwork =|FP－FP(calc)|/FP.

d

Rfree was calculated with 5.1% of the reflections in the test set.

e

Statistics for the Ramachandran plot from an analysis using MolProbity.


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Data collection and structure determination The crystals were gradually transferred into cryoprotectant solution supplemented with 20% (v/v) glycerol and flash-cooled in liquid nitrogen. X-ray diffraction data were collected at 100 K at the Shanghai Synchrotron Radiation Facility (SSRF) beamline BL17U1 with a crystal to detector distance of 475 mm for SaPfk/ADP, 260 mm for SaPfk/ATP, 260 mm for SaPfk/AMP-PNP, 260 mm for SaPfk/F6P, and 320 mm for SaPfk/F6P/AMP-PNP-Mg, respectively. The wavelength for data collection was 0.9791 Å. Individual frames were collected using 1 s for each 1.0° oscillation over a range of 180° for SaPfk/ADP, 180° for SaPfk/ATP,

180°

for

SaPfk/AMP-PNP,

180°

for

SaPfk/F6P,

and

180°

for

SaPfk/F6P/AMP-PNP-Mg, respectively. Diffraction data were indexed, integrated and scaled using iMosflm

10

, POINTLESS and SCALA

11

from the CCP4 suite

12

. Data collection and

processing statistics are shown in Table 1. The structure of native SaPfk was determined by molecular replacement using Phaser program from CCP4 suit 13 with the structure of Pfk from Bacillus Stearothermophilus (BsPfk, PDB ID: 3U39) as the search model. Structures of SaPfk in complex with various ligands were determined using molecular replacement with native SaPfk structure as the model. The models were refined using REFMAC5

14

and manually adjusted in Coot 15. Geometry of all

the final models was validated using MolProbity

16

. The final refinement statistics are

summarized in Table 1. All the figures were prepared using PyMOL (http://www.pymol.org). The coordinates for SaPfk, SaPfk/ADP, SaPfk/ATP, SaPfk/F6P, SaPfk/AMP-PNP, and SaPfk/F6P/AMP-PNP-Mg have been deposited in the Protein Data Bank under accession codes5XOE, 5XZA, 5XZ9, 5XZ7, 5XZ6 and 5XZ8, respectively.

Isothermal Titration Calorimetry (ITC) The binding affinity of wild type SaPfk and its mutants with the substrate F6P was analyzed by ITC using a MicroCal iTC200 instrument (GE healthcare) at 16 ºC in 20 mM Tris-HCL pH 7.5, 200 mM NaCl. Experimental data was fitted to a single binding site model


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Biochemistry

and analyzed using ITC data analysis module of OriginPro 2017 (MicroCal Inc.) provided by the manufacturer.

Enzyme activity assay All enzyme activity assays were measured spectrophotometrically on the CLARIOstar (BMG LABTECH) at 340 nm that relies on coupling the particular enzymatic step to the appropriate NADH-linked reaction, with the use of commercially available enzymes (Sigma) as described in previous studies

17, 18

. Kinetic studies were carried out in 200 µl reaction

system at 37 °C contained 100 mM Tris-HCl pH 7.5, 5 mM MgCl2, 0.1 mM NADH, 1 mM ATP, 1 U aldolase, 2.52 U triosephosphate isomerase and 1.376 U glycerolphosphate dehydrogenase. The concentration of SaPfk used in activity assays was 150 nM for wild type, R245A and R164A, and 50 nM for G150D-L151A, respectively. Kinetic parameters were generated by non-linear fitting analysis of the Hill equation using OriginPro 2017 (MicroCal Inc.) to fit the parameters of the equations:

ν=

∙ [F6P]

. + [F6P]

K =

[E]

where ν is the PFK activity in the presence of a given concentration of F6P ([F6P]), is the maximal reaction rate calculated at saturating concentrations of F6P, is the turnover number, .is the affinity constant for F6P, which is equal to the concentration of F6P responsible for half-activation of the enzyme by F6P, is the hill coefficient and provides a measure of the cooperativity of F6P binding to PFK. An value greater than 1 ( > 1) or less than 1 ( < 1) indicates that two or more binding sites exist in the protein and there is positive or negative cooperativity with respect to substrate binding, respectively; if the n value equal to 1 ( = 1), the Hill equation is reduced to Michaelis-Menten equation. In our study, the value is greater than 1, therefore the Hill equation was used for data


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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analysis. The calculated kinetic parameters were listed in Table 2. /. indicates the catalytic efficiency of the enzyme, efficient enzymes have higher values. Table 2 Kinetics of SaPfk and its mutants. Vmax

K0.5

kcat

kcat / K0.5

n

(µM ·min-1)

(µM)

(s-1)

(M-1·s-1)

(hill coefficient)

Wild Type

57.7±1.6

115.5±7.7

6.4

5.6×104

2.6±0.4

G150D-L151A

75.6±1.5

115.2±6.0

25.2

2.2×105

1.9±0.2

Protein

Kinetic parameters were generated by non-linear fitting analysis of the Hill equation using OriginPro 2017 (MicroCal Inc.) to fit the parameters of the equations: ∙ [F6P]

. + [F6P] K = [E]

ν=


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Biochemistry

RESULTS SaPfk exists as both tetramer and dimer Bacterial Pfks are homotetramers with molecular mass of ~35 kDa per subunit and allosterically regulated by ADP-Mg (activator) and PEP (inhibitor), which stabilize the R- and T-states of Pfk 1, 3, 5, respectively. Until now, the dimeric structure of bacterial Pfk in apo form has not been reported. When SaPfk was purified by size-exclusion chromatography (SEC), two peaks were observed with apparent molecular mass of 136 kDa and 63 kDa, corresponding to the tetrameric and dimeric forms, respectively (Figure 2A). We further examined the tetramer-dimer equilibrium of SaPfk at various protein concentrations. As shown in Figure 2B, SaPfk tends to form more tetramers at higher concentrations. When the protein concentration was reduced to 57 µM, about one third of SaPfk are dimers.

Figure 2 SaPfk exists as both tetramers and dimers. (A) SEC (Size-exclusion chromatography) for oligomeric states analysis of SaPfk at 57 µM. The apparent molecular weights of each peak calculated from the elution volume based on the standard curve are labeled in black. The molecular weights of protein standards are labeled in gray. (B) SEC results for SaPfk at 57 (black), 114 (red) and 570 (blue) µM. The peaks corresponding to the tetrameric and dimeric SaPfk are labeled.

SaPfk oligomeric state is regulated by ATP, ADP, AMP-PNP, F6P, and pH


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All the reported bacterial Pfks are tetramers except that TtPfk tetramers has been reported to disassociate into dimers in the presence of PEP6. Because SaPfk exists as both dimer and tetramer in solution, we evaluated the effects of substrates and various effectors on the equilibrium of tetramer-dimer of SaPfk.

Figure 3 The tetramer-dimer conversion of SaPfk is regulated by multiple effectors. (A) Effect of pH level at 5.5 (green), 6.0 (blue), 6.5 (red) and 7.5 (black) on the tetramer-dimer conversion of SaPfk (114 µM). (B) The tetramer-dimer conversion of SaPfk (114 µM) is regulated by ADP (pink), ATP (purple), AMP-PNP (green) and F6P (red). PEP (cyan) has no effect on the tetramer-dimer equilibrium of SaPfk. F6P regenerate tetramers against the addition of AMP-PNP (blue). Same total amount of WT proteins (114 µM) were incubated without (apo) or with 1 mM ligand and loaded for SEC analysis. As shown in Figure 3A, the SaPfk favors dimeric state at low-pH conditions. Nearly all SaPfk proteins exist as dimers when pH is lower than 6.5. Additionally, the presence of 1 mM ADP, ATP, or AMP-PNP (an ATP analog) shifts the equilibrium towards dimer, with AMP-PNP having the most significant effect (Figure 3B, S1). The more ADP (Figure S1A), ATP (Figure S1B) or AMP-PNP (Figure S1C) was added in the solution, the more SaPfk dimer was formed. These results demonstrated that the tetramer-dimer equilibrium of SaPfk can be shifted to dimer by multiple effectors (lower pH levels, ADP, ATP and AMP-PNP). By contrast, SaPfk proteins exist as tetramers in the presence of 1 mM F6P, which indicates


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that the formation of SaPfk tetramers is stabilized by F6P (Figure 3B). However, PEP has no effect on the tetramer-dimer conversion of SaPfA, although it has been reported to be an inhibitor of TtPfk by dissociating tetramers to dimers (Figure 3B).

Dimeric structure of SaPfk To investigate the molecular mechanism underlying the equilibrium between SaPfk tetramer and dimer, we solved the crystal structure of SaPfk and the complex structures of SaPfk/ADP, SaPfk/ATP, and SaPfk/AMP-PNP at 3.0 Å, 1.9 Å, 2.0 Å, and 2.6 Å resolution, respectively. Consistent with the biochemical results, both SaPfk and SaPfk in complex with ADP, ATP, and AMP-PNP crystallized as homodimers (dimer AB) (Figure 4, S2). The overall structure of SaPfk subunit is similar to that of BsPfk subunit (PDB: 4PFK), with a root mean square deviation (RMSD) value of 0.557 Å for 240 Cα atoms. Two SaPfk subunits (subunit A and B) form a head-to-tail homodimer via interactions between the N-terminal domain of one subunit with the C-terminal domain of the other, and vice versa (Figure S2A). The interface between subunit A and B (Interface I) is primarily mediated by helices α1, α3, α7, α8, α12, and α14 from each subunit (designated as α1′, α3′, α7′, α8′, α12′, and α14′ from the adjacent subunit) (Figure S2A). The dimeric structures of ATP-, ADP-, and AMP-PNP-bound SaPfk are similar to that of SaPfk (Figure S2B, 4A and S2C), with a RMSD value of 0.261 Å for 578 Cα atoms, 0.259 Å for 558 Cα atoms, and 0.246 Å for 594 Cα atoms, respectively (Figure S3A-S3C). The less Cα atoms superposed in ATP- and ADP- bound structures are due to the different conformations of loopα7-β6, loopα9-β8, and loopα10-β9. Both ATP and ADP molecules are located in the N-terminal domain and stabilized by Cys73 from α4, Gly104 and Ser105 from α6, and Gly102 from loopβ4–α6 (Figure S2B and 4B). The ATP analog AMP-PNP occupies the ATP-binding site in the SaPfk/AMP-PNP structure. In addition to the four residues that contribute to ATP binding in the SaPfk/ATP structure, two more residues, Thr127 and Arg173, are involved in AMP-PNP binding by forming hydrogen bonds with the third phosphate group of AMP-PNP (Figure S2C).


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Figure 4 Crystal structures of SaPfk dimer. (A) The ribbon diagram of the overall structure of ADP-bound SaPfk dimer with subunit B colored pink and the secondary structural elements of subunit A colored blue for α-helices, green for β-strands and yellow for loops. The α-helices and β-strands are labeled in black. The interface formed by the two subunits is indicated as Interface I. Subunit A and B are labeled in blue and pink, respectively. (B-C) The details of the (B) ADP and (C) citrate binding sites in ADP-bound SaPfk structure. The residues involved in ligand binding and the ligands are depicted as sticks with the carbon atoms colored green and yellow, respectively. Hydrogen bonds are shown as black dash. Residues from the adjacent subunit are labeled in red and indicated with prime. (D) Structural comparison of the citrate binding site in SaPfk/ADP (pink) with the native BsPfk (orange, PDB ID:3U39), the PGA (yellow, PDB ID: 6PFK) and ADP-Mg (green, PDB ID: 4PFK)


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binding sites in BsPfk. Citrate, PGA, ADP and Mg are labeled in pink, yellow, green and black, respectively. Residues from SaPfk and BsPfk are labeled in pink and green. Prime indicates the residues from the adjacent subunit. Both the SaPfk/ADP and SaPfk/AMP-PNP structures have ligands bound to the effector site at Interface I between the two subunits of the dimer (Figure 4C, S2C). The side chains of Arg21 and Arg25 from one subunit and Arg156′ from the adjacent subunit form a basic pocket for citrate binding in SaPfk/ADP structure (Figure 4C), while the AMP-PNP-bound SaPfk structure shows a glycerol molecule bound within the same pocket (Figure S2C). As previously mentioned, either ADP-Mg or PEP binds in the BsPfks effector-binding site allosterically regulate BsPfks activity. By comparing the citrate binding site of SaPfk/ADP structure with that in BsPfk bound either PGA (PDB code: 6PFK) 3 or ADP-Mg (4PFK) 4, we found that one of the carboxylate groups of citrate can be overlaid with the terminal phosphate group of PGA and ADP (Figure 4D). The key residues of BsPfk involved in ADP-Mg and PGA binding are similar, with Arg21 and Arg25 from one subunit and Arg154′ from the adjacent subunit responsible for interactions with the β-phosphate group of ADP and the phosphate group of PGA. Arg25 and Arg211′ stabilize the α-phosphate group of ADP, as well as the carboxylate group of PGA. In the ADP-Mg-bound BsPfk structure, the side chain of Glu187′ coordinates the magnesium to form the complete ADP-Mg-binding site together with the arginine residues. These BsPfk residues involved in PEP and ADP-Mg binding are highly conserved in SaPfk; however, the side chains of Arg156′ and Arg213′ (corresponding to Arg154′ and Arg211′ of BsPfk) in the SaPfk/ADP structure (dimeric state) shift outward from the center of the effector-bind pocket relative to their counterparts in the BsPfk structures (tetrameric state). These conformational changes result in a more open effector site in the SaPfk dimer, which might impair the binding of PEP and ADP-Mg.

F6P stabilizes the SaPfk tetramer assembly To investigate the mechanism associated with F6P inducing and stabilizing the formation of the SaPfk tetramer and its antagonistic effect on dissociation of the SaPfk tetramer induced by AMP-PNP, we co-crystallized SaPfk with F6P in the presence or absence of AMP-PNP.


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Consistent with the results of biochemical analysis, we obtained the tetrameric SaPfk structure with the addition of F6P (Figure S4). Additionally, SaPfk/F6P/AMP-PNP-Mg also formed a tetramer, confirming the antagonistic effect of F6P against AMP-PNP-mediated dissociation (Figure 5A).

Figure 5 Crystal structures of SaPfk tetramer. (A) Ribbon diagram of the interface II in SaPfk/F6P/AMP-PNP-Mg structure. The details for the F6P/AMP-PNP-Mg binding site (the complete active site) is shown in zoom in window. AMP-PNP, F6P, residues from one subunit and the adjacent subunit are shown as sticks with the carbon atoms colored purple, yellow, green and orange, respectively. Hydrogen bonds are shown as black dash. Residues from the adjacent subunit are labeled in red and indicated with prime. Subunit A, B, C and D are labeled in blue, pink, cyan and orange, respectively. (B) SEC results for oligomeric states analysis of SaPfk mutants R164A (red) and R245A (blue) comparing to WT (black).


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In both the SaPfk/F6P and SaPfk/F6P/AMP-PNP-Mg structures, four SaPfk subunits formed a dimer of dimers (dimer AB and CD), with each subunit binding an F6P molecule at the dimer-dimer interface (Interface II) (Figure S4A, S4B and 5A). Both the SaPfk/F6P and SaPfk/F6P/AMP-PNP-Mg tetrameric structures can be overlaid to the R-state structure of BsPfk (Figure S4C). Interface II is mainly formed by the interaction of Loopβ1–α1, Loopα2– α3, and α11 from the N-terminal domain of one subunit with α7′ from the C-terminal domain of the adjacent one, and vice versa (Figure S4D). The N- and C-terminal regions of α7 are buried within Interfaces I and II, respectively, indicating the essential role of α7 in regulating SaPfk oligomerization (Figure S4D). The four binding pockets for F6P are located within interface II between two SaPfk dimers in the tetrameric structures of SaPfk/F6P/AMP-PNP-Mg and SaPfk/F6P. In one of the binding pocket, the fructose moiety of F6P is buried in one side of the pocket formed by residues Asp129, Met171, Gly172, Arg173, Gly224 and Arg254 from subunit A via hydrogen bonds and hydrophobic interactions (Figure 5A). The other side of the pocket is composed of positively charged residues including Arg72, Arg254, His251 from subunit A and Arg164′, Arg245′ from the adjacent subunit D, which accommodates the negatively charged phosphate group of F6P. The other three F6P binding sites are identical to the pocket described above. In SaPfk/F6P/AMP-PNP-Mg and SaPfk/F6P complex, the four F6P molecules buried in binding pockets to lock the interactions between two SaPfk dimers in the tetramer. To verify this, we mutated two key residues involved in F6P binding, Arg164 and Arg245, to alanine. As expected, both R164A and Rg245A mutants were unable to bind F6P (Figure S5) and existed only as dimers in solution (Figure 5B). These results demonstrated that F6P and its binding site play an essential role in the formation of the SaPfk tetramer.

The molecular mechanism associated with dimer-tetramer conversion Given that the overall structures of either dimeric SaPfk, SaPfk/ADP, SaPfk/ATP, and SaPfk/AMP-PNP (Figure S3) or tetrameric SaPfk/F6P and SaPfk/F6P/AMP-PNP-Mg (Figure S6) are almost identical, we only compared the two high-resolution structures: the dimeric SaPfk/ADP (1.9 Å) and the tetrameric SaPfk/F6P/AMP-PNP-Mg (1.9 Å) structures.


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Figure 6 Structural comparison of SaPfk dimer with tetrameric Pfks. (A) Comparison of the overall structure of SaPfk/ADP (green) with that of SaPfk/F6P/AMP-PNP-Mg (grey). The subunit from the other dimer in SaPfk/F6P/AMP-PNP-Mg is colored orange. The conformational changes of α7, β9 and α9 are highlighted in red dash square and the details are shown in (B) zoom in window. The Gly150, Leu151, Arg245 and Arg164 residues are shown as sticks and labeled in black. F6P is shown as sticks with the carbon atoms colored yellow. Loopα3-α4′ and α11′ from the adjacent subunit are labeled in blue and indicated by blue arrows. The conformational change of α7 is indicated by red arrow. (C) Structural comparison of the α7 in dimeric SaPfk (green) with those in 3PFK (BsPfk, yellow), 4PFK (BsPfk in complex with F6P and ADP-Mg, orange), 4I4I (BsPfk in complex with PEP, pink), 1PFK ( E. coli Pfk in complex with FBP and ADP-Mg, wheat), 2PFK (E. coli Pfk, grey) and 1ZXX ( Lactobacillus delbrueckii Pfk, blue). (D) Multiple sequence alignment of SaPfk with other bacterial Pfks. The unique G-L motif in SaPfk is highlighted with red arrows. The NCBI entries of the Pfks used for sequence alignment are listed in Table S2. (E) SEC results for oligomeric states analysis of SaPfk mutant G150D-L151A at variable concentrations. As shown in Figure 6A, the SaPfk N-terminal domains of both structures showed highly similar conformations, whereas helices α7, α9, and strand β9 in the C-terminal region exhibits


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Biochemistry

large conformational changes upon F6P binding. One major difference involves changes in the orientations of the two arginine residues essential for F6P binding (Figure 6B). In the SaPfk dimer, the electron density of the Arg164 and Arg245 side chains is missing due to the flexibility of Loopα7–β6 and Loopα10–β9, respectively (Figure S7A). However, in the SaPfk/F6P/AMP-PNP-Mg structure, the Arg164 side chain flips to interact with F6P. In addition, the Arg245 side chain is stabilized by F6P, resulting in the extension of strand β9 (Figure 6B, S7B). The reorientation of the Arg164 and Arg245 side chains upon F6P binding in SaPfk is similar to the conformational changes of Arg162 and Arg243 side chains from T-state to R-state BsPfk, indicating the highly conserved mechanism for F6P binding among bacterial Pfks (Figure S7C). Another major difference between the two structures is the conformational change in α7, which is longer in SaPfk/F6P/AMP-PNP-Mg structure relative to that of SaPfk/ADP, with four residues extended into the C-terminus (from Ala159 to His162). The α7 C-terminus bends inwards from Gly150 and Leu151 to interact with α11′ and Loopα2′–α3′ from the adjacent subunit to facilitate formation of the SaPfk tetramer (Figure 6B). Structural comparison of dimeric SaPfk with other tetrameric bacterial Pfks (both the T- and R-state) confirmed the uniqueness of the α7 conformation observed in the dimeric SaPfk structure (Figure 6C). Sequence alignment revealed that the G-L motif only exists in S. aureus, with Gly150 in other bacterial Pfks replaced by either a glutamate or an aspartate residue (Figure 6D). The unique property of Gly150 might confer more conformational flexibility to α7, which plays an important role in SaPfk dimer-tetramer conversion. Otherwise, the α7 C-terminus in dimeric SaPfk is oriented outward and clashes with α11′ and Loopα2′– α3′ from the adjacent dimer in tetrameric SaPfk (Figure 6A and 5B). This flexibility of α7 might mediate the equilibrium between tetrameric and dimeric SaPfk. Therefore, mutating the G-L motif to D-A (SaPfkG150D-L151A) might stabilizes the tetrameric form relative to that observed in WT SaPfk. As expected, SaPfkG150D-L151A is incapable of promoting complete dimer-to-tetramer conversion (Figure 6E). Although mutation of the G-L motif to D-A facilitates this shift, the tetramer remains unstable in the absence of F6P. Here, SaPfk only crystallized as a tetramer


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upon the addition of F6P, indicating that F6P stabilizes the SaPfk tetramer. Structural analysis revealed that the ordering of the Arg164 and Arg254 side chains upon F6P binding resulted in the extension of β9 and the conformational changes in Loopα7–β6 (Figure 6B). Additionally, F6P orients two SaPfk dimers close enough to facilitate interactions between α7 from one dimer with Loopβ1– α1, Loopα2–α3, and α11′ from the other dimer, thereby allowing formation of the dimer-dimer interface to stabilize the tetramer. Mutation of the F6P-binding site disrupts this activity, resulting in complete disassociation of the SaPfk tetramer (Figure 5B). These findings illustrated the molecular mechanism associated with SaPfk dimer-tetramer conversion, demonstrating that the flexibility of the G-L motif enables α7 to function as a switch at interface II upon F6P binding to promote assembly or disassembly of the SaPfk tetramer.

Kinetic studies of SaPfk Most reported bacterial Pfks are tetramers exhibiting catalytic activity that is mediated allosterically. Only the TtPfk tetramer is disassembled by the allosteric effector PEP, leading to loss of enzymatic activity6. Here, we found that SaPfk exists as both dimer and tetramer, with the tetramer form stabilized upon F6P binding. Therefore, we hypothesized that SaPfk activity is regulated by tetramer-dimer conversion. To confirm this, we compared the enzymatic activities of SaPfkWT and SaPfkG150D-L151A. As shown in Figure 7A, SaPfkWT exhibits an “S-shaped” sigmoidal saturation curve, with Vmax and kcat/ K0.5 values of 57.7±1.6 µM·min−1 and 5.6×104 M−1·s−1, respectively (Table 2). Because SaPfkG150D-L151A favors to form active tetramers, we observed a 3-fold higher binding affinity of this variant for F6P (~2.7 µM) relative to that of SaPfkWT (~6.5 µM) (Figures S5 and 7B) and V max and kcat/ K0.5 values of 75.6±1.5 µM·min−1 and 2.2×105 M−1·s−1, respectively (Table 2). The higher V max and kcat/ K0.5 values SaPfkG150D-L151A relative to those of SaPfkWT indicated higher catalytic activity on the part of the mutant. Additionally, the SaPfkG150D-L151A variant exhibited a lower Hill coefficient (1.9 vs. 2.6 for SaPfkWT) (Table 2).


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Figure 7 Kinetic studies of SaPfk. (A) Kinetic studies of 150 nM SaPfk (WT) and 50 nM SaPfkG150D-L151A (G150D-L151A) were carried out at 37 °C in 200 µl reaction system containing 100 mM Tris-HCl pH 7.5, 5 mM MgCl2, 0.1 mM NADH, 1 mM ATP, 1 U aldolase, 2.52 U triosephosphate isomerase and 1.376 U glycerolphosphate dehydrogenase. (B) ITC results for the binding affinity of F6P to SaPfk G150D-L151A mutant.


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DISCUSSION In this study, we observed both dimeric and tetrameric states of SaPfk, and the equilibrium between active tetramer and inactive dimer is regulated by several allosteric effectors, including protein concentration, pH, ADP, ATP, AMP-PNP and F6P. Although TtPfk tetramer-dimer conversion has been reported among bacterial Pfks, the dissociation of TtPfk tetramer to dimer only occurs in the presence of PEP6. The BsPfk mutant W179Y/Y164W also undergoes reversible dissociation of tetramer to dimer upon PEP binding, however, no dissociation of the wild type enzyme tetramer has been observed 9. Conversely, PEP has no effect on the tetramer-dimer conversion of SaPfk (Figure 3B). These observations indicate a distinct regulatory mechanism for SaPfk that differs from other reported bacterial Pfks. Firstly, SaPfk exists as both tetramer and dimer, with equilibrium between the two states dependent on enzyme concentration. Secondly, SaPfk tetramer-dimer conversion is more sensitive than that observed in TtPfk. Multiple effectors, including ADP, ATP, low pH, and AMP-PNP, shift the equilibrium toward dimer. Thirdly, stable SaPfk tetramers only form upon F6P binding, and mutations of the F6P-binding site results in formation of dimers alone. These results showed that F6P triggers and stabilizes formation of the SaPfk tetramer, and demonstrated a different allosteric regulatory mechanism for SaPfk among bacterial Pfks. Because all the reported bacterial Pfks except TtPfk are tetramers either in the presence or absence of ligands/effectors, it seems that there could be major structural changes at the dimer-dimer interface (Interface II) (Figure 5, S4) of SaPfk to destabilize its tetrameric structure. Unfortunately, crystallization of apo form SaPfk tetramer has been unsuccessful. We only obtained the tetrameric SaPfk structure with the addition of F6P. Therefore, we compared the interface II of SaPfk/F6P/AMP-PNP-Mg with that of BsPfk/F6P/ADP-Mg (PDB entry: 4PFK) using PDBsum online server (www.ebi.ac.uk/pdbsum/) (Figure S8). Multiple sequence alignment reveals that most residues at interface II of SaPfk and BsPfk are highly conserved (Figure S9A). Structural comparison shows no major conformational differences along interfaces (Figure S9B). Although residues Arg72, Thr147, Arg164 and Arg245 are involved in interface II formation in SaPfk but their counterparts are not involved


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Biochemistry

in BsPfk interface II (Figure S8, S9A), these residues are highly conserved among bacterial Pfks, and the side chains of each residue in both structures can be overlaid quite well (Figure S9C). Additionally, Arg72, Arg164 and Arg245 are active site residues required for substrate binding, the slight difference of Arg72 side chains is likely due to the binding of different ligands, with ADP-Mg and AMP-PNP-Mg in BsPfk and SaPfk, respectively. The residues that contribute to BsPfk interface but not SaPfk interface are also highly conserved, except Glu161 is replaced with an alanine in SaPfk (Figure S9A and S9D). However, the E161A mutation of BsPFK has been previously reported to have very similar behavior to that of wild type. Taken together, the interface II of SaPfk is quite similar to BsPfk and these interface residues are not likely the key residues for the dimer-tetramer conversion of SaPfk. By contrast, structural comparison of inactive dimeric SaPfk with active tetrameric SaPfk reveals that the α7 helix may play an important role for the equilibrium of tetramer and dimer, whereas F6P triggers and stabilizes dimer-to-tetramer conversion. Because the G-L motif in α7is flexible, that allows conformational change in α7, thereby mediating assembly or disassembly of the SaPfk tetramer. Mutation of G-L to D-A promotes a higher degree of tetramer formation than that observed in WT SaPfk, supporting our hypothesis. Based on these results, we proposed that SaPfk kinetics with respect to F6P are sigmoidal due to the existence of a dimeric state, and that the G-L mutant might exhibit higher affinity to F6P and decreased sigmoidal kinetics relative to the WT variant. Consistently, SaPfk displayed sigmoidal kinetics with respect to F6P (Figure 7A), and SaPfkG150D-L151A showed 3-fold higher binding affinity to F6P (Figures 6B and S5) and 5-fold higher enzymatic activity and lower sigmoidal kinetics relative to WT (Table 2). Because the crystallization attempts of tetrameric apo form SaPfk and SaPfk in complex with PEP or ADP-Mg binding at effector sites have been unsuccessful, the contribution of the R-T states model on the allosteric regulation of SaPfk cannot be excluded. However, our results clearly demonstrated that the tetramer-dimer conversion modulated by F6P and allosteric effectors plays a critical role in the regulation of SaPfk catalytic activity.


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S. aureus is a virulent pathogen that causes infections in animals and human addition to a glycolytic enzyme, SaPfk is also a member of the RNA degradosome

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19 20

. In that

regulates S. aureus RNA metabolism, formation of biofilm, and the expression of virulence factors, thereby playing an important role in S. aureus growth and infection 20-22. However, the function of SaPfk in the RNA degradosome remains unknown. The existence of dimeric SaPfk and its unique allosteric regulatory mechanism suggests that SaPfk might have multiple functions, such as be involved in RNA degradosome, through its different oligomeric states.


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ACKNOWLEDGEMENT We thank the staff at beamline BL17U1 of the Shanghai Synchrotron Radiation Facility for assistance with data collection. This work was supported by the National Key Research and Development Program of China (Nos. 2017YFA0503600, 2016YFA0400903), and the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (No. 31621002). This work was also supported by the National Natural Science Foundation of China (No. U1532109) to JZ. This work was also supported by the Anhui Provincial Natural Science Foundation (No. 1608085QC52), the National Natural Science Foundation of China (No. 31700671) to XZ.

CONFLICT OF INTEREST The authors declare that they have no conflict of interest.

AUTHOR CONTRIBUTION Jianye Zang, Xuan Zhang provided the scientific direction and the overall experimental design for the studies. Tian Tian designed and performed the biochemical experiments. Chengliang Wang was responsible for the crystal structure studies. Tian Tian, Xuan Zhang and Jianye Zang wrote the manuscript.

SUPPORTING INFORMATION Table S1: SEC buffer used for different pH levels. Table S2: NCBI accession ID for multiple sequence alignment. Figure S1: SEC results of SaPfk with the addition of ADP, ATP and AMP-PNP. Figure S2: Crystal structures of SaPfk dimer. Figure S3: Structural comparison of SaPfk dimeric structures. Figure S4: Crystal structures of SaPfk tetramer. Figure S5: ITC results for the binding affinity of F6P to the SaPfk wild type (WT), R245A


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and R164A mutant. Figure S6: Structural comparison of SaPfk tetrameric structures. Figure S7: Conformational change of the Arg245 and Arg164 side chains upon F6P binding. Figure S8: Analysis of the interface II of SaPfk/F6P/AMP-PNP-Mg with that of BsPfk/F6P/ADP-Mg using PDBsum online server. Figure S9: Structural comparison of SaPfk interface II with that of BsPfk.


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Structural Insights into the Regulation of Staphylococcus aureus

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