Mevalonate 5-diphosphate mediates ATP binding to the mevalonate diphosphate decarboxylase from the bacterial pathogen Enterococcus faecalis

Abstract

The mevalonate pathway produces isopentenyl diphosphate (IPP), a building block for polyisoprenoid synthesis, and is a crucial pathway for growth of the human bacterial pathogen Enterococcus faecalis. The final enzyme in this pathway, mevalonate diphosphate decarboxylase (MDD), acts on mevalonate diphosphate (MVAPP) to produce IPP while consuming ATP. This essential enzyme has been suggested as a therapeutic target for the treatment of drug-resistant bacterial infections. Here, we report functional and structural studies on the mevalonate diphosphate decarboxylase from E. faecalis (MDD_EF). The MDD_EF crystal structure in complex with ATP (MDD_EF–ATP) revealed that the phosphate-binding loop (amino acids 97–105) is not involved in ATP binding and that the phosphate tail of ATP in this structure is in an outward-facing position pointing away from the active site. This suggested that binding of MDD_EF to MVAPP is necessary to guide ATP into a catalytically favorable position. Enzymology experiments show that the MDD_EF performs a sequential ordered bi-substrate reaction with MVAPP as the first substrate, consistent with the isothermal titration calorimetry (ITC) experiments. On the basis of ITC results, we propose that this initial prerequisite binding of MVAPP enhances ATP binding. In summary, our findings reveal a substrate-induced substrate-binding event that occurs during the MDD_EF-catalyzed reaction. The disengagement of the phosphate-binding loop concomitant with the alternative ATP-binding configuration may provide the structural basis for antimicrobial design against these pathogenic enterococci.

Introduction

Multidrug-resistant microorganisms have been found in humans and food animals due to the overuse of antibiotics (1). In the United States, a surveillance of antibacterial resistance from the Centers for Disease Control and Prevention reports the deaths and the cost resulting from infectious diseases and emphasizes the urgency to control drug-resistant bacteria (2). This critical list includes vancomycin-resistant enterococci (VRE),² which cause a range of enterococcal infections such as bacteremia, urinary tract infections, intra-abdominal and pelvic infections, central nervous system infections, skin and skin structure infections, and infective enterocarditis (2–4).

Enterococci are Gram-positive and facultative anaerobes colonized in the gastrointestinal tract. The first clinical isolates of vancomycin-resistant enterococci were reported in Europe in 1988 (5, 6). The VRE bacteria are intrinsically insensitive to detergents and antibiotics (clindamycin, cephalosporins, and aminoglycosides) and keep gaining drug resistance to other newly invented antibiotics, such as linezolid and daptomycin (7–11). The emergence of drug-resistant enterococci results in difficulties of treatment of VRE infections (12–14). For the protection of patients in health care settings, new approaches and new antimicrobial agents against enterococcal infections are urgently needed.

In 2000, Wilding et al. (15) reported that the mevalonate pathway is pivotal in the Gram-positive bacterial pathogens, enterococci, staphylococci, and streptococci. This pathway includes six enzymes, acetyl-CoA acetyltransferase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase (EC 4.1.1.33), that ultimately produce IPP. IPP then serves as a building block for polyisoprenoid synthesis in living organisms ranging from bacteria to humans (16–18). In bacteria, isoprenoid production is also involved in the biosynthesis of bacterial cell wall and electron carriers in the respiratory chain (16, 19–21).

The five enzymes in the mevalonate pathway (except acetyl-CoA acetyltransferase) have been identified to be critical for bacterial growth (15). Experiments with the mammalian enzyme have shown that the ATP-dependent decarboxylation of MVAPP catalyzed by MDD is a rate-limiting step in IPP synthesis (22), implicating MDD as a suitable drug target for the disease treatment. Interestingly, in Streptococcus pneumoniae, an in vitro experiment has indicated that a higher level of MVAPP can also inhibit mevalonate kinase (23). This implies that the mevalonate pathway is sophisticatedly regulated by its downstream products and strengthens the case for MDD as a promising drug target for treatments of enterococcal infections.

In this research, we have studied MDD from Enterococcus faecalis (MDD_EF) from functional, biophysical, and structural points of view to decipher the substrate-binding mechanism of MDD_EF. MDD proteins trigger the irreversible ATP-dependent decarboxylation of MVAPP to produce IPP in the last step of the mevalonate pathway (Fig. 1) (24). Several residues (aspartate, lysine, serine and arginine) in the active site of MDD proteins have been identified as being involved in substrate binding and enzyme catalysis (25–27). In 2012, Barta et al. (27) reported several crystal structures of the wild-type and mutant forms of MDD from Staphylococcus epidermidis (MDD_SE) with or without substrates/analogues binding to the active-site cleft. From their structural models, they proposed that MVAPP is the first substrate binding to the deeper pocket in the active site of MDD_SE, where Ser-106, Arg-144, Ser-192, and Arg-193 hold MVAPP in position. Next, ATP sits in the ATP-binding pocket interacting with Ser-94 and Asn-96, and the phosphate tail is clamped by Ser-106, Ser-107, and the phosphate-binding loop (Ala-101, Gly-103, and Leu-104), followed by enzyme catalysis (27). The substrate-binding order of MDD isolated from chicken liver was examined with results that suggest avian MDD performs a sequential ordered bi-substrate mechanism with MVAPP as the first substrate (28, 29). However, no kinetic evidence was presented that prokaryotic MDDs act on substrates in the same manner,. A case in point is the enzyme mechanism of proteins in the GHMP kinase family (galactokinases, homoserine kinases, mevalonate kinases, and phosphomevalonate kinases), which vary due to different biological sources (27). In this work, we aimed to obtain a crystal structure of MDD_EF bound with ATP alone to investigate structural differences between the MDD_EF–ATP structure and those complex structures of MDD_SE (supplemental Table S1). With that, we could better understand how the two substrates initially bind to the active site, which may aid in our rational drug design in vitro and in silico.

Figure 1.

Decarboxylation reaction of MVAPP to IPP by MDD. MVAPP and MgATP are the two substrates of MDD enzymes. MgADP, CO₂, phosphate, and IPP are the products.

Results

MDD_EF protein expression and purification

Protein expression and purification conditions are detailed under “Experimental procedures.” Overexpressed His-tagged MDD_EF in Escherichia coli (BL21) was purified via nickel-affinity chromatography (Fig. 2A). Eluted protein fractions were examined by SDS-PAGE (Fig. 2B). Fractions containing MDD_EF were collected for TEV treatment to remove the fusion N-terminal His tag from MDD_EF (Fig. 2C) (“Experimental procedures”). Finally, we obtained a His tag-removed MDD_EF (less than 5% impurities, Fig. 2C, flow-through) with a protein yield of 80–120 mg/liter of culture and adjusted the protein concentration to 8–10 mg/ml.

Figure 2.

Protein purification and structure determination of MDD_EF. A, purification profile of MDD_EF from a nickel affinity column. The detailed procedures of protein purification are discussed under “Experimental procedures.” mAU, milli-absorbance unit. B, SDS-polyacrylamide gel for the MDD_EF protein purification. M, protein marker; Pellet, sample in the pellet after French press; Flow through, an aliquot of flow-through containing proteins that cannot be trapped on the Ni-NTA column; fractions from 12 to 18 were combined. C, SDS-polyacrylamide gel for the TEV-protease treatment profile of MDD_EF. The lane 0 hr shows the His-tagged MDD_EF without TEV treatment. The TEV treatment process is done in 2 days (Day1 and Day2; 4 and *16 hrs indicate the TEV treatment at different time courses; *, dialysis against buffer without DTT and EDTA; RT, treatment at room temperature; Flow through, an aliquot containing MDD_EF proteins without a His tag which cannot be trapped on a Ni-NTA column; Strip, an aliquot after 1 column volume of strip buffer (containing 50 mm EDTA) flowing through the column. D, superposition of MDD_EF and the MDD_EF–ATP complex. Two structures of MDD_EF (gray) and MDD_EF–ATP (pink) were Cα-aligned (r.m.s.d. = 0.174 Å). The position of the phosphate-binding loop (97–104) can be determined in MDD_EF–ATP but not MDD_EF. The missing residues (dashed line) in MDD_EF range from 183 to 192; in MDD_EF–ATP, residues from 184 to 189 are missing.

Crystal structure of MDD_EF-ATP and the apo form of MDD_EF

The detailed procedures for obtaining co-crystals of MDD_EF bound with ATP (MDD_EF–ATP, PDB accession number 5V2L) and apo-MDD_EF (MDD_EF, PDB accession number 5V2M), data collection, and structure refinement are summarized under “Experimental procedures.” The data collection and refinement statistics of both structures are listed in Table 1. According to the CATH protein structure classification database analysis, the overall structure of the MDD family of proteins contains α and β secondary structure elements (α and β class) (Fig. 2D), and the folding architecture is characterized as a two-layer sandwich (30). The protein family annotation (Pfam) (31) indicates MDD proteins belong to the GHMP kinase (galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase) superfamily, which catalyzes ATP-dependent reactions. The overall structures of MDD_EF and MDD_EF–ATP superimpose well with an r.m.s.d of 0.174 Å. In MDD_EF, the phosphate-binding loop (97–104) and residues from 183 to 192 are missing. Similarly, in MDD_EF–ATP, residues from 184 to 189 cannot be determined. However, the phosphate-binding loop can be observed (Fig. 2D). In the literature, the phosphate-binding loop in MDD is known for ATP binding (27). In our crystal structure of MDD_EF–ATP, the phosphate-binding loop does not interact with the ATP molecule (Fig. 3A), although ATP binds to the ATP-binding pocket via the interactions between ATP and the conserved residues (Ser-93, Asn-95, and Ser-105 (Fig. 3B).

Table 1.

Data collection and refinement statistics of MDD_EF-ATP and MDD_EF

r.m.s.d. is root mean square deviation.

	MDDEF–ATP	MDDEF
Data collection
Space group	P21212	P21212
Cell dimensions:
a, b, c (Å)	82.0, 97.7, 46.3	81.9, 98.1, 46.1
α = β = γ (°)	90.0	90.0
Wavelength (Å)	1.0332	0.9794
Resolution range (Å)	40.0–2.1 (2.18–2.10)	20.0–2.0 (2.07–2.00)
Rmerge (%)	7.5 (40.9)	6.4 (12.9)
〈I〉/〈σI〉	24.1(4.3)	17.4 (10.6)
Completeness (%)	99.8 (99.8)	95.4 (86.7)
Redundancy	4.4	5.9
Refinement
Resolution range (Å)	31.4–2.1 (2.17–2.09)	19.7–2.0 (2.07–1.99)
No. reflections in working set	21,102	23,223
Rworka/Rfreeb	0.172/0.192	0.179/0.205
No. of atoms
Protein	2483	2624
Solvent	112	154
Ligand	36	10
Average B-factor (Å2)	28.1	25.5
Protein	27.5	25.2
Solvent	30.0	30.9
Ligand	58.8	24.7
r.m.s.d. from ideal geometry
Bond lengths (Å)	0.012	0.012
Bond angles (degrees)	1.07	1.10
Ramachandran plot
Most favored (%)	98.7	98.7
Allowed (%)	1.3	1.3
Outliers (%)	0.0	0.0
Occupancy of ligandc	ATP(0.90), PO43−(0.80)	SO42−(0.61, 0.39)

^a R_work = Σ‖F_o| − |F_c‖/Σ|F_o|.

^b R_free was calculated against 5–5.3% of the reflections removed at random.

^c Phosphate and sulfate ions are in the individual crystallization conditions.

Figure 3.

Superimposition of complex structures of MDD_EF–ATP and MDD from S. epidermidis bound with FMVAPP and ATPγS (MDD_S.E.–FMVAPP–ATPγS). A, ribbon model of the crystal structure of MDD_EF–ATP is shown in pink with the bound ATP molecule as a stick model. B, ATP molecule is surrounded by the SA-omit map (mF_o − DF_c at a contour of 3σ, cropped at 5 Å from ATP). Residues (Gln-68, Ser-93, Asn-95, and Ser-105) that form hydrogen bonds with ATP are shown as stick models. The hydrogen bonding partners (Gln-68–O and ATP–O2′, 3.3 Å; Ser-93–Oγ and ATP–N₆, 3.0 Å; Asn-95–Oδ and ATP–N₆, 2.9 Å; Asn-95–Nδ and ATP–N₇, 3.3 Å; Ser-105–N and ATP–Oα, 2.9 Å) are connected by dashed lines. C, overlay of the models of MDD_SE–FMVAPP–ATPγS (PDB code 4DPT, green) and MDD_EF–ATP (pink) (r.m.s.d. = 0.66 Å) is depicted with the phosphate-binding loops emphasized. D, ligands from two structures are shown as stick models (FMVAPP and ATPγS from MDD_SE–FMVAPP–ATPγS; ATP from MDD_EF–ATP). The arrows in black indicate the distinct orientations of the phosphate tails of ATPγS and ATP from these two structures.

Structural differences between MDD_EF-ATP and MDD_SE–FMVAPP–ATPγS infer the pivotal role of MVAPP in ATP binding

By comparing our crystal structure of MDD_EF–ATP with the tertiary crystal structure of MDD_SE (MDD_SE–FMVAPP–ATPγS, PDB code 4DPT) (27), three major differences were found in the phosphate-binding loop (Fig. 3C) and the ATP/ATPγS configurations (Fig. 3D). First, the phosphate-binding loop in MDD_EF–ATP remains in an open conformation and does not bend down to form contacts with ATP, if compared with the phosphate-binding loop in MDD_SE–FMVAPP–ATPγS (Fig. 3C). The largest distance moved by the backbone atoms in the phosphate-binding loop is 11.1 Å (Cα of Ala-100 in MDD_EF–ATP and Cα of Ala-101 in the MDD_SE complex structure). Second, the γ-thiophosphate group of ATPγS in MDD_SE–FMVAPP–ATPγS is reported to form contacts (hydrogen bonding) with the backbone amide of two residues, Ser-107 and Ala-108 in Helix α2 of MDD_SE(106–121) (supplemental Fig. S1A), which are located in the active site; instead, the γ-phosphate of ATP in our structure of MDD_EF–ATP is facing out and does not interact with any residues in the molecule (Fig. 3D and supplemental Fig. S1B). Third, the α- and β-phosphates in these two structures are located and oriented differently and thus result in the distinct orientations of the phosphate tails in these structures (Fig. 3D). The adenosine group of ATP in our MDD_EF–ATP binds to the ATP-binding pocket with a temperature factor similar to the average B-factor of the protein (Table 1). However, the phosphate tail of ATP does not interact with any residues, consistent with its higher temperature factor (B-factor >60), suggesting ATP does not initially bind to MDD_EF in its favored position. These findings lead us to hypothesize the prerequisite binding of MVAPP to MDD_EF may be important for forming the ATP-binding pocket. To test the hypothesis, we utilized enzymatic and thermodynamic approaches to determine the substrate-binding order of MDD_EF and to examine whether the binding of MVAPP enhances binding of ATP.

Kinetics reveals a compulsory ordered bi-substrate mechanism of MDD_EF with MVAPP binding first

The crystal structure of MDD_EF–ATP described above implied that the binding of MVAPP may affect ATP binding. The previous work on avian MDD suggests the substrate-binding order of MDD from chickens belongs to an ordered bi-substrate mechanism with MVAPP binding first (28). From our crystal structure of MDD_EF–ATP, ATP can bind to the ATP-binding pocket by itself, even if in an unproductive form (Fig. 3, B and D). Therefore, the question arises whether the substrate-binding order is conserved in both eukaryotic and prokaryotic MDD proteins. To address this issue, we performed kinetics studies introducing ATPγS as a competitive inhibitor of ATP to determine the enzyme mechanism of MDD_EF. We utilized the coupled enzyme method (pyruvate kinase/lactate dehydrogenase) to determine the enzymatic activity of MDD_EF at varying concentrations of the two substrates and inhibitors (25, 29, 32). The detailed procedures are described under “Experimental procedures.”

MDD triggers MVAPP decarboxylation coupled with ATP consumption. MVAPP and ATP are its two substrates. To differentiate whether the reaction belongs to either the ping-pong or the sequential bi-substrate mechanism, we have performed enzymatic reactions at varying concentrations of both substrates of MDD_EF, MVAPP, and MgATP (“Experimental procedures”) (Fig. 4, A and C), and we analyzed the kinetic data by the Lineweaver-Burk method (Fig. 4, B and 4D). Each 1/v (reciprocal reaction velocity) was plotted as a function of either 1/[MVAPP] (reciprocal of varying concentrations of MVAPP) (Fig. 4B) or 1/[MgATP] (reciprocal of varying concentrations of MgATP) (Fig. 4D). The data were best represented by a sequential bi-substrate model using the general equation for a bi-substrate enzyme catalysis system as represented in Equation 1,

$$v=\frac{V_{\max }\left[A\right]\left[B\right]}{K_{IA}{K}_B+K_B\left[A\right]+K_A\left[B\right]+\left[A\right]\left[B\right]}$$ (Eq. 1)

where V_max is the maximum velocity; [A] is the varying concentration of MVAPP; [B] is the varying concentration of ATP; K_IA is K_dMVAPP, K_B is K_mMgATP; and K_A is K_mMVAPP (33).

Figure 4.

Kinetic analysis of MDD_EF. A, set of kinetic data at varying concentrations of MVAPP and several fixed concentrations of MgATP ((●) 50, (○) 100, (▾) 200, (▿) 400, (■) 600, (□) 800, and (♦) 1000 μm) is shown as a Michaelis-Menten plot. B, same data set from A is shown as a Lineweaver-Burk plot. C, data set at varying concentrations of MgATP and several fixed concentrations of MVAPP ((●) 10, (○) 15, (▾) 25, (▿) 50, (■) 100, (□)150, (♦) 200, and (♢) 300 μm) is shown as a Michaelis-Menten plot. D, same data set from C is shown as a Lineweaver-Burk plot. Kinetic data are represented by Equation 1 and used for a sequential bi-substrate model. Each data point represents independent triplicate results, and the error bar for each point indicates standard deviations.

The patterns in Fig. 4, B and D, show that the kinetic curves in the double-reciprocal plots converge to the x axis. We could then confirm that the enzyme mechanism of MDD_EF belongs to a sequential bi-substrate mechanism instead of a ping-pong mechanism or a rapid-equilibrium sequential ordered mechanism. From the curve fitting, we obtain the following kinetic parameters: V_max = 16.1 ± 0.3 μmol/min/mg; k_cat = 9.8 ± 0.2 s⁻¹; K_mMVAPP = 39.7 ± 2.8 μm; and K_mMgATP = 166 ± 12 μm (Table 2).

Table 2.

Enzyme kinetic parameters

The means and the standard deviation of each data point were obtained from a triplicate test (means ± S.D.).

Vmax	Vmax	kcat	Km, MVAPP	K>m, MgATP	KI, ATPγS	Metal	Inhibition pattern
μmol/min/mg	μm/s	s−1	μm	μm	μm
16.1 ± 0.3	0.75 ± 0.02	9.8 ± 0.2	39.7 ± 2.8	166 ± 12		Mg2+
20.4 ± 0.8	0.94 ± 0.04	12.4 ± 0.6	42.6 ± 3.4		173 ± 16	Mg2+	Uncompetitive
24.0 ± 0.6	1.12 ± 0.2	14.6 ± 0.4		165 ± 9	199 ± 13	Mg2+	Competitive

To determine whether substrates bind to MDD_EF in an ordered or random manner, we introduced ATPγS into the reaction buffer as a dead-end inhibitor of MDD_EF and then determined the substrate-binding order based on the inhibitory patterns of ATPγS (supplemental Table S2) (28, 33). Two sets of inhibitory assays were conducted. The first set of experiments was performed at a constant MVAPP concentration of 40 μm and varying concentrations of MgATP and ATPγS (see under “Experimental procedures”). The kinetic data from the inhibitory assays were best represented by a modified competitive inhibition model as shown in Equation 2,

$$\frac{1}{v}=\frac{1}{0.5\times V_{\max }}\left(1+\frac{K_B\left(1+K_{IA}/K_A\right)/2}{\left[B\right]}+\frac{\left[I\right]}{K_I\left(1+K_{IA}/K_A\right)}\right)$$ (Eq. 2)

where V_max is the maximum velocity; [B] is the varying concentrations of ATP; K_B is K_mMgATP; K_IA is K_dMVAPP; K_A is K_mMVAPP; K_IA/K_A is 0.8, which was derived from the enzymatic reactions described above; [I] is the varying concentration of ATPγS, and K_I is K_IAATPγS (33).

The kinetics data are shown as a Michaelis-Menten plot (Fig. 5A) and a Lineweaver-Burk plot (Fig. 5B). The values of V_max, K_mMgATP, and K_IATPγS were derived from Equation 2 and are summarized in Table 2.

Figure 5.

Inhibitory assays of MDD_EF. A, different fixed concentrations of ATPγS ((●) 0, (○) 100, (▾) 200, or (▿) 400 μm) were added to the reactions at a fixed concentration of MVAPP (40 μm) and varying concentrations of MgATP (100, 150, 200, or 400 μm). The set of inhibitory data is shown as a Michaelis-Menten plot. B, same set of data from A is shown as a Lineweaver-Burk plot. C, different fixed concentrations of ATPγS ((●) 0, (○) 100, (▾) 200, and (▿) 400 μm) were added to the reactions at a fixed concentration of MgATP (200 μm) and varying concentrations of MVAPP (10, 20, 40, and 80 μm). The set of inhibitory data is shown as a Michaelis-Menten plot. D, same set of data from C is shown as a Lineweaver-Burk plot. A and B can be analyzed as a competitive inhibition model; C and D can be analyzed as an uncompetitive inhibition model. The means and the standard deviation of each data point were obtained from a triplicate test. Each data point represents independent triplicate results, and the error bar for each point indicates standard deviations.

The second set of experiments was performed at a constant MgATP concentration of 200 μm and varying concentrations of MVAPP and ATPγS (“Experimental procedures”). The data set was best represented by a modified uncompetitive inhibition model as shown in Equation 3,

$$\frac{1}{v}=\frac{1}{0.5\times V_{\max }}\left(\left(1+\frac{\left[I\right]}{2\times K_I}\right)+\frac{K_A\left(1+K_{IA}/K_A\right)/2}{\left[A\right]}\right)$$ (Eq. 3)

where V_max is the maximum velocity; [A] is the varying concentrations of MVAPP; K_A is K_mMVAPP; K_IA is K_dMVAPP; K_IA/K_A is 0.8, which was derived from the enzymatic reactions described above; [I] is the varying concentration of ATPγS, and K_I is K_IATPγS (33).

The kinetics data are shown as a Michaelis-Menten plot (Fig. 5C) and a Lineweaver-Burk plot (Fig. 5D). The values of V_max, K_mMVAPP, and K_IATPγS were also derived from Equation 3 and are summarized in Table 2. According to the inhibitory patterns with the addition of ATPγS in the enzymatic reactions under the designed conditions, we suggest that the enzymatic reaction of MDD_EF belongs to a sequential ordered bi-substrate mechanism with MVAPP binding first, and we find the substrate-binding mechanism of MDD to be conserved between the eukaryotic and prokaryotic species in the family of MDD proteins.

Isothermal titration calorimetry (ITC) results suggest an MVAPP-induced ATP-binding mechanism of MDD_EF

From the kinetics study on MDD_EF, MVAPP is identified as the first substrate in the reaction mechanism. Our complex structure of MDD_EF–ATP shows that in the absence of MVAPP, ATP binds the ATP-binding pocket in an unusual configuration (Fig. 3, B and D), and the phosphate-binding loop does not bend down to interact with the ATP molecule (Fig. 3C). These results imply the binding of MVAPP may help form a binding site for ATP in its catalytically favored configuration. In other words, the binding of ATP might be enhanced by the prerequisite binding of MVAPP. To test this, we employed thermodynamic approaches and utilized ITC (34) to determine the dissociation constants of the first substrate, MVAPP, the second substrate, MgATP, and the ATP analogues, ATPγS and AMPPCP.

We have determined the K_d values of substrates or ligands (MVAPP, MgATP, AMPPCP, and ATPγS). The raw data for titrations of MDD_EF are shown in Fig. 6. All the derived thermodynamic parameters, K_d (1/K_a), are listed in Table 3. The K_dMVAPP value is 20.4 ± 9.3 μm (Fig. 6A); the K_dATP value is 288 ± 36 μm (Fig. 6B); and the K_dATPγS value is 215 ± 8 μm (Fig. 6C). Next, we determined K_dATPγS under the condition in which MDD_EF was pre-incubated with MVAPP. MDD_EF was incubated with 1 mm MVAPP for 30 min to ensure that the MVAPP-binding pocket of MDD_EF was nearly fully occupied (about 97.8%), according to the dissociation constant at equilibrium as shown in Equation 4, where [MDD_EF]_total is the total concentration of MDD_EF; [MVAPP]_total is the total concentration of MVAPP; and x is the concentration of the MDD_EF–MVAPP complex.

$$K_d=\frac{\left({\left[{\text{MDD}}_{\text{EF}}\right]}_{\text{total}}-x\right)\left({\left[\text{MVAPP}\right]}_{\text{total}}-x\right)}{x}$$ (Eq. 4)

Figure 6.

Original titration curves from ITC experiments with MDD_EF. A, MDD_EF (100 μm) titrated with MVAPP (2 mm). B, MDD_EF titrated with ATP (3 mm). C, MDD_EF titrated with ATPγS (3 mm). D, MDD_EF pre-incubated with MVAPP (1 mm) and then titrated with ATPγS (2 mm). The protein concentration is adjusted to 100 μm, and all the protein and titrants are dissolved in the buffer containing 100 mm HEPES, pH 7, 100 mm KCl, and 10 mm MgCl₂.

Table 3.

Thermodynamic parameters

Titration experiments were done at 25 °C. NBD indicates no detectable binding.

	Species	Substrate	Kd	ΔGa	ΔH	TΔSb	ΔS
			μm	kcal/mol	kcal/mol	kcal/mol	cal/mol·K
a	MDDEF (100 μm)	MVAPP (2 mm)	20.4 ± 9.3	−6.5 ± 0.3	−0.7 ± 0.1	5.8 ± 0.2	19.5 ± 0.5
b	MDDEF (100 μm)	ATP (3 mm)	288 ± 36	−4.8 ± 0.1	−3.8 ± 0.5	1.0 ± 0.3	3.5 ± 1.0
c	MDDEF (100 μm)	ATPγS (3 mm)	215 ± 8	−5.00 ± 0.02	−6.4 ± 0.3	−1.4 ± 0.2	−4.7 ± 0.6
d	MDDEF (100 μm) + MVAPP (1 mm)	ATPγS (2 mm)	25.4 ± 5.5	−6.3 ± 0.1	−4.8 ± 1.1	1.5 ± 0.6	5.0 ± 2.1
e	MDDEF (100 μm)	AMPPCP (3 mm)	271 ± 43	−4.87 ± 0.09	−3.20 ± 0.50	1.67 ± 0.36	5.61 ± 1.21
f	MDDEF (100 μm) + MVAPP (1 mm)	AMPPCP (5 mm)	NDB	NDB	NDB	NDB	NDB
g	MDDEF (100 μm) +AMPPCP (1 mm)	MVAPP (2 mm)	18.7 ± 7.4	−6.50 ± 0.23	0.91 ± 0.18	7.41 ± 0.20	24.88 ± 0.69

^a The mean of ΔG (μ) is derived from the equation: μ = −RTln(m/√1 + (v/m²)) and the standard deviation of ΔG (σ) is calculated from the equation: σ = RT√ln(1 + (v/m²)), where m is the mean of the association constant (K_a), and v is the variance of K_a derived from each ITC experiment.

^b The mean of TΔS (μ_1–2) is derived from the difference between the means of ΔG (μ₁) and ΔH (μ₂) and the standard deviation (σ_1–2) of TΔS is derived from the equation: σ_1–2 = √(σ₁²/n + σ₂²/n), where σ₁ is the standard deviation of ΔG and σ₂ is the standard deviation of ΔH.

Under these conditions, the K_d value between ATPγS and MDD_EF–MVAPP was determined to be 25.4 ± 5.5 μm (Fig. 6D), which is 10-fold less than the K_d value between ATPγS and MDD_EF alone, supporting our proposed model in which the pre-binding of MVAPP would strengthen ATPγS binding. Our results show that the binding of ATPγS is enhanced by the prerequisite binding of MVAPP to MDD_EF, suggesting the binding of MVAPP would trigger conformational changes of MDD_EF to accommodate the ATP molecule in its catalytically favored position for the subsequent chemical steps in the reaction.

Discussion

GHMP family kinases employ a variety of mechanisms and enzyme order. Previously, the substrate-binding mechanism of MDD from chickens was determined to belong to a sequential bi-substrate mechanism with MVAPP as the first substrate (28); from our study, we determined prokaryotic MDD from E. faecalis also binds MVAPP first during the enzymatic reaction. MDD is characterized as a member GHMK of the kinase family (26). For the galactokinase from pig liver, MgATP is the first substrate of the galactokinase, followed by the binding of galactose (35); however, in E. coli, the galactokinase enzyme performs a random bi-substrate mechanism (36). For the homoserine kinase in E. coli, MgATP is preferred to bind to the enzyme first (37). For the mevalonate kinase from hog liver, a sequential ordered bi-substrate mechanism is performed with mevalonate binding first (38). The enzyme mechanism of the phosphomevalonate kinase from pigs belongs to a sequential ordered bi-substrate mechanism with phosphomevalonate binding first (39). In the case of the MDD, the enzyme mechanism may be conserved among the family of proteins; however, in other cases in the GHMP kinase family, the enzyme mechanisms may vary in species. Although in this study we aim at elucidating the intriguing observations from the structure of MDD_EF–ATP and the structural basis of MVAPP for assisting ATP binding to MDD_EF, alternative substrates or product inhibition strategies at both saturated and unsaturated concentrations of MVAPP and MgATP (40) are also the ways to comprehensively investigate the overall enzyme mechanism of MDD_EF.

MDD proteins have been known to trigger the ATP-dependent decarboxylation of MVAPP, but with a requirement for metal ions (Mg²⁺) to perform catalysis under physiological conditions (29). In our crystal structure of MDD_EF–ATP, the ATP molecule does not have magnesium binding to the α- and β-phosphate group. In the crystallization condition, we have 5 mm magnesium chloride and 10 mm ATP in the solution. The buffer condition is 50 mm sodium acetate at pH 4.6, and the protonation state of ATP is mainly HATP³⁻ under this condition (41). The dissociation constant between HATP³⁻ and magnesium is about 1.58 mm (log K_a = 2.79 ± 0.15) (42), and the MgATP concentration under this condition is about 4 mm, suggesting the concentration of free HATP³⁻ is about 6 mm. The reason for not seeing magnesium-bound ATP in the crystal structure of MDD_EF–ATP could be due to the competitive binding of free HATP³⁻ to the ATP-binding pocket of MDD_EF. It is also possible that a higher average temperature factor of the phosphate tail of ATP results in poorly defined electron density in this region so that we cannot identify a magnesium ion in this structure. In 2012, Barta et al. (27) reported experimental findings on the substrate-binding order of MDD_SE. However, they do not identify any metal-binding sites in their crystal structures of MDD_SE with substrate analogues. Based on the observations above, we suggest the current structure models of MDD proteins in the PDB do not necessarily represent the final conformation of MDD proteins upon substrate binding and enzyme catalysis. This could also be a problem for structure-based drug design, which is heavily dependent on the structural details in atomic models of macromolecules. To study the structure and function of the MDD family of proteins remains an important but challenging field.

The open conformation of the phosphate-binding loop and the unusual configuration of ATP in our crystal structure of MDD_EF–ATP suggest the importance of MVAPP binding in forming the productive ATP-binding site. We proposed that MVAPP could induce ATP binding to MDD_EF, and the thermodynamic results are in agreement with our proposal. In addition to the examination of changes of K_dATPγS in the presence of excess amounts of MVAPP in the solution, we also tested whether this enhanced binding occurs in the titration experiments using AMPPCP. The K_dAMPPCP value was 271 ± 43 μm (Table 3, row e); however, in the presence of MVAPP, we could not detect heat changes and so could not obtain the thermodynamic parameters in this case (Table 3, row f). If instead MDD_EF is pre-incubated with excess amounts of AMPPCP (10-fold, MDD_EF–AMPPCP complex formation rate: 77.4%) and then the mixture is titrated with MVAPP (Table 3, row g), the K_dMVAPP value in this case is 18.7 ± 7.4 μm, which is a similar value as from the MDD_EF–MVAPP titration (Table 3, row a), indicating AMPPCP may have little effect on MVAPP binding. We have also done a preliminary test on ATPγS. MDD_EF was pre-incubated with excess amounts of ATPγS (10-fold, MDD_EF–ATPγS complex formation rate: 81.1%) and then the mixture was titrated with MVAPP (data not shown). The thermogram shows an unusual two-process binding event and reaches saturation with no further heat change after the 6th injection, suggesting a tight binding event occurs. K_dMVAPP was roughly determined to be 76 nm (data not shown). Although there are no other experimental results to explain the two-process binding, a quick saturation after the 6th injection indicates that MVAPP binds MDD_EF–ATPγS much tighter than MVAPP binds MDD_EF alone (Table 3, row a). It also implies that AMPPCP might not be able to access a catalytically-favored conformation for “locking” MVAPP in the active site, which might need, for example, an aid from the conformational changes of MDD_EF upon substrate binding. The conserved phosphate-binding loop of the MDD family of proteins functions to interact with the phosphate tail of ATP. It has been shown that AMPPCP has poor inhibitory activity (K_i > 1 mm) against MDD from chickens (28) and MDD_EF in our preliminary enzymatic study (data not shown). AMPPCP has a replacement of the bridging oxygen with the methyl group (CH₂–) between the β- and γ-phosphorus atoms. We therefore propose that the β-,γ-bridging oxygen of ATP serves as a key checkpoint for the molecular recognition of ATP by MDD_EF during the enzymatic reaction.

To summarize our results, we suggest that an induced substrate-binding mechanism of MDD proteins occurs during the enzymatic reaction (Fig. 7), in which the binding of the first substrate, MVAPP, to MDD will trigger the conformational changes of the enzyme to accommodate ATP binding to the ATP-binding pocket in its catalytically favored position, followed by enzyme catalysis and product release. The observation that ATP can bind by itself but in a catalytically unfavorable position may also provide an alternative configuration of the MDD_EF enzyme that provides a new structural basis for antibacterial design for pathogenic enterococci.

Figure 7.

Induced substrate-binding mechanism of MDD proteins. Left panel (green), apo-form of MDD in which two pockets for substrate binding are empty; middle panel (orange), the MVAPP-bound MDD in which the binding of MVAPP (shown in a red triangle) triggers conformational changes of the enzyme and reshapes the ATP-binding pocket, which allows the binding of ATP (shown in a blue arrow) to its catalytically favored position; right panel (purple), two-substrate-bound MDD. Step 1, the binding of MVAPP; step 2, the binding of ATP; step 3, enzyme catalysis and product release. Products in different shapes are shown in gray.

Experimental procedures

Cloning, overexpression, and purification of the recombinant form of MDD_EF

A gene fragment encoding MDD from E. faecalis (MDD_EF) was amplified via PCR and subcloned into the expression plasmid pET30a (43). Upon confirmation of the DNA sequence, the construct was transformed into E. coli BL21 (DE3). Transformed cells were cultured in LB broth supplemented with kanamycin (50 mg/ml) at 37 °C to an A_{600 nm} of 1.0. Protein expression of MDD_EF was induced by addition of isopropyl 1-thio-β-d-galactopyranoside (0.1 mm) for another 4 h at 37 °C.

Cells were harvested by centrifugation at 8000 rpm, resuspended in binding buffer (50 mm sodium phosphate, pH 7.4, 300 mm NaCl, and 10 mm imidazole), and lysed to homogeneity by a French press. His-tagged MDD_EF protein was soluble in the supernatant after centrifugation and trapped on a Ni²⁺-NTA column followed by the elution with increasing concentrations of elution buffer (50 mm sodium phosphate, pH 7.4, 300 mm NaCl, and 300 mm imidazole). Eluted fractions were pooled and desalted against dialysis buffer (25 mm Tris-HCl, pH 8.0, 100 mm NaCl, and 10 mm MgSO₄) twice, the first time with β-mercaptoethanol (2-ME) (20 mm) and the second time without 2-ME. The N-terminal His tag was removed from MDD_EF by treatment with recombinant TEV protease in dialysis buffer containing 1 mm DTT and 0.5 mm EDTA overnight at room temperature for 4 h followed by a final dialysis procedure at 4 °C in the dialysis buffer without DTT and EDTA. His-tagged TEV and residual His-tagged MDD_EF were removed by passing the protein mixture through a nickel affinity resin. The artificial sequence NA remains at the N terminus of MDD_EF after the TEV treatment. The purified MDD_EF protein solution was concentrated to 8–10 mg/ml by ultrafiltration and stored at −20 °C.

Protein crystallization

The co-crystal of MDD_EF in complex with ATP (MDD_EF–ATP) grew under the crystallization condition (10 mm ATP, 26% PEG 3350, 50 mm sodium acetate, pH 4.6, and 5 mm MgCl₂) by sitting drop vapor diffusion. ATP was added to the protein solution to a final concentration of 10 mm. 1 μl of MDD_EF–ATP solution was mixed with 1 μl of the reservoir. Crystals formed after a 2-day equilibrium period. Cryo-protectant (26% PEG 3350, 17% PEG 400, 50 mm sodium acetate, pH 4.6, and 5 mm MgCl₂) was prepared and gradually added into the protein/reservoir mixture to prevent ice formation when flash-freezing the crystal in liquid nitrogen.

A stable condition (1.6 m ammonium sulfate, 50 mm sodium acetate, pH 4.6) for growing crystals of the apo-form of MDD_EF was obtained and optimized from a commercialized crystallization screening kit (Qiagen, class II, condition A2: 0.1 m sodium acetate, pH 4.5, 2 m ammonium sulfate). Protein solution (8 to 9 mg/ml) was mixed with the reservoir solution at a 1:1 ratio and equilibrated by vapor diffusion of sitting drops at 20 °C for 2 days. In a 96-well plate, there were about 20 wells containing single cuboid crystals with a size of 0.2 to 0.3 mm. Cryo-protectant (25% glycerol, 1.6 m ammonium sulfate, 50 mm sodium acetate, pH 4.6) was prepared and gradually added into the protein/reservoir mixture to prevent ice formation when flash-freezing the crystal in liquid nitrogen.

Data collection and structure determination, refinement analysis, and refine statistics

The diffraction data from crystals of MDD_EF co-crystallized with ATP (MDD_EF–ATP) and an apo-form of MDD_EF (MDD_EF) were collected at the 23-ID-D and 19-ID-BM beamlines at the Advanced Photon Source (APS) at Argonne National Laboratory in Chicago. The HKL2000 software was used for data integration, data reduction, and data scaling (44), generating a scale pack reflection file. The space group of the crystal was determined as P2₁2₁2 (MDD_EF–ATP: a, b, c = 82.0, 97.7, 46.3 Å; α, β, γ = 90°; MDD_EF: a, b, c = 81.9, 98.1, 46.1 Å; α, β, γ = 90°) by the software Pointless in CCP4 and phenix.phaser (45, 46). The software in CCP4, scalepack2mtz, was then used to convert the scale pack reflection file (.sca) to an MTZ format (.mtz) with R-free flag assigned (5–5.5%) (47).

One molecule was expected in an asymmetric unit by analyzing the Matthews coefficient and the water content in each case (MDD_EF–ATP: cell volume,: 370710.5 ų, molecular mass: 36,489.5 Da, Matthews coefficient: 2.54, % solvent: 51.6; MDD_EF: cell volume: 370,279.3 ų, molecular mass: 36489.5 Da, Matthews coefficient: 2.54, % solvent: 51.6). Phenix.phaser was used to solve the phases and determine the structure of MDD_EF. MDD_EF and MDD_SE share 60% sequence identity. A modified search model (polyalanine) generated from MDD from S. epidermidis (MDD_SE, PDB code 3QT5) using CHAINSAW in CCP4 (48) was used to produce initial phases for the co-crystal structure of MDD_EF–ATP in molecular replacement with phenix.phaser (45). In each step of molecular replacement, only one solution was found. The rotation function with log-likelihood gain greater than 0, the translation function with Z-score greater than 8, and no violations after the packing analysis also suggest the success of the molecular replacement approach. The phases for determining the crystal structure of the apo-form of MDD_EF were produced by molecular replacement with Phenix.phaser (45) using a polyalanine-modified MDD_EF–ATP structure (described above) as a search model.

After phase determination, the structure refinement was performed in phenix.refine (45). During structure refinement, strategies for refining the model geometry (XYZ coordinates), atomic positions (Real space), and atomic B-factors (individual B-factors) were chosen, and after the refinement, the missing side chains of the residues in the structural model were manually rebuilt in the graphics program Coot (49) based on observation of the electron density map (2F_o − F_c) and the difference map (F_o − F_c). The simulated-annealing (Cartesian) option was also employed in the first few runs of structure refinement. In phenix, we used the phenix.composit_omit_map software to generate the omit map and used the maps to evaluate the refined structural models. Target function optimization was also chosen for refining the weight between X-ray data and the structural model (optimize X-ray/stereochemistry weight, optimize X-ray/ADP weight). Water molecules were built by either phenix.refine or Coot and inspected in the Coot interface.

The crystallographic information file (.cif) and the PDB format file (.pdb) of ATP were generated using phenix. eLBOW (45). After a few runs of structure refinement without ligands, ATP was manually placed and fitted into the weighted difference electron density maps in Coot (49). Geometry and rotamer outliers were inspected and evaluated after each structure refinement. Finally, ATP was omitted by setting the ligand occupancy to zero or simply removing it from the structure for calculating a simulated-annealing-ligand-omit map in phenix.composite_omit_map, and the ligand-omit map and its corresponding structure were examined in PyMOL (50).

Enzyme kinetics of wild-type MDD_EF

Enzymatic activity of MDD_EF was determined using an ATP/NADH enzyme-coupled assay. In this assay, ADP produced from the MDD reaction is converted back to ATP by pyruvate kinase, coupled with the conversion of phosphoenolpyruvate to pyruvate, and the pyruvate is then converted to lactate by lactate dehydrogenase, coupled with the oxidation of NADH to NAD⁺. The oxidation of NADH to NAD⁺ results in an absorbance decrease at 340 nm. Thus, the ADP production can be detected by measuring the decrease in A_{340 nm}. The MDD enzymatic activity would be proportional to the slope of the continuously declining value of A_{340 nm}. We have utilized this method to determine the enzymatic activity of MDD_EF under different conditions for obtaining kinetic parameters of MDD_EF. Each reaction was performed at 30 °C under the buffer condition (100 mm HEPES, pH 7.0, 100 mm KCl, 10 mm MgCl₂, 0.2 mm NADH, 0.4 mm phosphoenolpyruvate, 4 units of pyruvate kinase, 4 units of lactate dehydrogenase, and 100 nm MDD_EF) (27). Initial velocity of each reaction was determined at a range of concentrations of MgATP (50, 100, 150, 200, 400, 600, 800, and 1000 μm) and MVAPP (10, 15, 25, 50, 100, 150, 200, and 300 μm). Final enzymatic parameters, K_m and V_max, were determined by fitting kinetic data to a sequential bi-substrate mechanism model using SigmaPlot12.5/Enzyme Kinetic Module 1.3 (Systat Software, Inc.).

Inhibition assays

ATPγS was used as a competitive inhibitor of ATP for determining the inhibition kinetics of MDD_EF. Different fixed concentrations of ATPγS (0, 100, 200 and 400 μm) were added into the reactions versus varying MgATP (50, 100, 200, and 400 μm) and fixed MVAPP (40 μm) or varying MVAPP (10, 20, 40, 80 μm) and fixed MgATP (200 μm). Assays were performed under the conditions as described above. The kinetic data with a fixed MVAPP concentration were fit with a competitive inhibition model; the kinetic data with a fixed MgATP concentration were fit with an uncompetitive inhibition model using SigmaPlot as described previously.

ITC experiments

The preparation of TEV-treated MDD_EF was described above. The protein solution was dialyzed against the same buffer as used in the enzymatic reactions described previously (100 mm HEPES, pH 7.0, 100 mm KCl and 10 mm MgCl₂). All the buffer solutions in ITC experiments were filtered through an 0.45-μm filter and degassed for 1 h at room temperature. The protein concentration was adjusted to 100 μm (260 μl). Each ligand (MVAPP, ATP, AMPPCP, and ATPγS) was prepared in the same dialysis buffer to avoid buffer mismatch. The concentration of each titrant was optimized in different experiments based on the experimental designs simulated using the MicroCal Origin 7.0 software package, and the final concentration of each ligand was adjusted to 2 or 3 mm. The ITC instrument, MicroCal iTC200, was employed for isothermal titrations in this study, and the reference cell was filled with double-distilled H₂O containing 0.01% sodium azide. The experimental temperature was set at 25 °C. Each experimental profile was composed of the addition of an initial aliquot of 0.4 μl, followed by 22 aliquots of 1.8 μl of the substrate or ligand solution. The time interval between two consecutive injections was 180 s. The data were further processed with NITPIC (51) and analyzed using an one-site model in SEDPHAT (52). Figures were generated using GUSSI in SEDPHAT.

Author contributions

C. C.-L. and C. V. S. designed the experiments. J. C. M. cloned the MDD protein. C. C.-L. purified the MDDEF protein for the study on enzymology, thermodynamics, and crystallization. C. C.-L. analyzed data and determined the substrate-binding order and the crystal structures of the target protein. L. N. P. designed and performed the ITC experiments. C. N. S. assisted in solving the MDD X-ray crystal structure. C. C.-L. and C. V. S. collaborated in writing the article.