Dynamics of acyl carrier protein in de novo fatty acid synthesis by Enterococcus faecalis based on NMR spectroscopy and molecular dynamics simulation

Abstract

Fatty acid synthesis (FAS) is essential for the production of biological components such as cell membrane building blocks and metabolism-related compounds. There are two types of bacterial FAS: de novo FAS and FAS through the incorporation of external fatty acids. Enterococcus faecalis possesses two distinct acyl carrier proteins (ACPs), AcpA (EfAcpA) and AcpB (EfAcpB), which serve as cofactors in the two types of FAS. We previously showed through NMR spectroscopy that EfAcpA comprises only three long helices, while EfAcpB consists of four helices, including a short α₃ helix, similar to other bacterial ACPs. An increase in melting temperature (T_m) from 64.0 to 76.1 °C confirmed that protein structural stability increased in the presence of calcium ions. Using NMR spectroscopy, two metal binding sites were identified in EfAcpA: site A was located at the start of the α₂ helix while site B was situated near the α₂ helix and α₂α₃ loop. To understand the importance of structural flexibility of EfAcpA in de novo FAS, we investigated its motional properties using backbone spin relaxation and molecular dynamics simulations. The α₂α₃ loop in EfAcpA displayed high flexibility, as indicated by low heteronuclear NOE values. The residues Val51, Glu54, and Gly58 exhibited significant R₂ values, likely due to the movement of this loop. EfAcpA created a novel cavity towards the α₁α₂ loop, in contrast to conventional cavity formation in most bacterial ACPs. This unique behavior was attributed to the flexibility exhibited by the α₂α₃ loop. The structural and motional characteristics of EfAcpA confirmed that its conformational plasticity is a crucial factor influencing acyl chain transfers in de novo FAS. Given the increasing antibiotic resistance observed for E. faecalis in clinical settings, the findings of this study may contribute to the development of more effective pathogen management strategies targeting FAS.

Introduction

Enterococcus faecalis, an important gram-positive bacterium commonly found in the intestines, exhibits remarkable resilience under environmental stresses, such as high temperatures, salinity, and antibiotic exposure (Kuch et al. 2012). E. faecalis exhibits high resistance to oxidative and dry stresses, and thrives under high salinity levels (such as 6.5% NaCl or 40% bile salts) and over a broad pH range (Orr et al. 2002; Wade 1997). Particularly noteworthy is its wide temperature tolerance: E. faecalis can grow at 10–40 °C and survive at 85 °C. This heat resistance enables its persistence in hospital laundry settings (Diarra et al. 2023; Laport et al. 2003). In human hosts, especially in medical settings, E. faecalis has been associated with life-threatening infections. Moreover, E. faecalis can develop resistance to various antibiotics, including vancomycin, cephalosporins, penicillin, and aminoglycosides, through genetic mutations (Hourigan et al. 2024; Nallapareddy et al. 2011; Strobel et al. 2024). Vancomycin-resistant enterococci are of special concern given their highly infectious nature in hospital settings (Eichel et al. 2023).

Fatty acids are fundamental biological components, and fatty acid synthesis (FAS) plays a vital role in the creation of cell membranes, energy storage molecules, and signaling compounds (Beld et al. 2015; Paiva et al. 2021). Additionally, fatty acids regulate post-translational protein function and can influence gene expression (Uauy et al. 2000). Bacteria typically utilize type II FAS systems, which are characterized by separate individually expressed enzymes, unlike the multifunctional complexes in eukaryotes (White et al. 2005). Acyl carrier proteins (ACPs) are key cofactors in FAS and essential for transferring acyl groups to FAS-related enzymes (Chan and Vogel 2010; Singh et al. 2023). The structures of various type II ACPs isolated from different organisms have been extensively examined. The solution structures of ACPs from Escherichia coli, Bacillus subtilis, Mycobacterium tuberculosis, Vibrio harveyi, Helicobacter pylori, Borrelia burgdorferi, and Plasmodium falciparum have been elucidated using NMR spectroscopy (Barnwal et al. 2011; Chan et al. 2010; Choi et al. 2021; Holak et al. 1988; Lee et al. 2020; Park et al. 2004, 2016; Sharma et al. 2006; Wong et al. 2002; Wu et al. 2009; Xu et al. 2001; Yeon et al. 2022). E. coli ACP (EcACP), an extensively studied bacterial ACP (Kim and Prestegard 1989; Roujeinikova et al. 2002), possesses two metal binding sites. ACPs are highly acidic proteins and divalent cations can help stabilize the electrostatic repulsion resulting from these acidic residues.

To address the emergence of antibiotic resistance in E. faecalis, we previously determined the solution structures of two E. faecalis ACPs using NMR spectroscopy. E. faecalis AcpA (EfAcpA) is an essential cofactor in de novo FAS, whereas E. faecalis AcpB (EfAcpB) is an essential cofactor in FAS using external fatty acids (Park et al. 2016; Yeon et al. 2022). We found that these two ACPs exhibited differences in the presence of the α₃ helix: EfAcpA possessed only three helices and lacked α₃, which was present in EfAcpB. These ACPs showed a low sequential identity of 28.7%, and the presence of a long α₂α₃ loop due to the lack of α₃ in EfAcpA suggested that the two ACPs serve different functions (Yeon et al. 2022).

In this study, we investigated the influence of divalent cations on the structural stability of EfAcpA and identified its metal-binding sites by measuring the melting temperature (T_m) and chemical shift perturbation (CSP) upon the addition of calcium ions. To understand the role of structural flexibility in acyl chain transfer in EfAcpA due to the absence of the α₃ helix, we explored the dynamic properties of EfAcpA using backbone spin relaxation and molecular dynamics (MD) simulations. This study aimed to elucidate the significance of structural flexibility in determining the distinct roles and functions of EfAcpA, with the ultimately goal of contributing to the development of novel antibiotics against E. faecalis.

Results

Comparative sequence analysis of EfAcp

To identify the key residues associated with ACP functions, we conducted sequence alignments of EfAcpA, EfAcpB, and EcACP as a reference (Fig. 1a). The majority of bacterial ACPs, including EcACP, possess a proline residue at the end of the α₂α₃ loop, only just preceding α₃. In contrast, EfAcpA has Glu54 and EfAcpB has Ser58 at this position. In EcACP, Phe28 played a key role in accessing the acyl chain, a function mirrored by Ile27 and Ile31 in EfAcpA and EfAcpB, respectively. Two residues are absent in the α₂α₃ loop of EfAcpA, a region where most bacterial ACPs typically feature a short α₃ helix. This suggests that this deletion sequence in this region facilitates the lack of α₃ in EfAcpA. All ACPs featured conserved DSL (or DSI) motifs crucial for the binding of 4′-phosphopantetheine prosthetic binding sites, along with a conserved serine at the beginning of α₂. EfAcpA harbored Leu38 and Phe39 at the beginning of α₂, which may facilitate hydrophobic interactions with acyl chains as well as FAS-related enzymes. Leu42 and Leu46 are critical residues in EcACP, with side chains that act as switches and aid in accessing the acyl group within the cavity, whereas EfAcpA has isoleucine at these positions.

Fig. 1

Comparison of sequence and structural features of EfAcpA, EfAcpB, and EcACP. a The DSL motif is highlighted with a green box. Non-conserved residues, such as Ile27, are shown in purple, Leu38 and Phe39 in gray, and Glu54 in brown. Ile41 and Ile45, which correspond to Leu42 and Leu46 in EcACP and act as switches to open the subpocket in EcACP, are shown in blue. Deletion sequence in EfAcpA is marked in red. b Comparison of solution structures for EfAcpA (8GSA) (Yeon et al. 2022), EfAcpB (2N50) (Park et al. 2016), and EcACP (1L0I) (Roujeinikova et al. 2002)

The structure of EcACP comprises four helices (Kim and Prestegard 1989; Roujeinikova et al. 2007; Sztain et al. 2021; Wu et al. 2009) (Fig. 1b). The α₃ in ACPs enables the flipping of chains and transfer of the acyl chain enclosed within the hydrophobic cavity to other enzymes. This mechanism is facilitated by the flexibility inherent in α₃ (Cronan 2014; Nguyen et al. 2014). In contrast, the lack of α₃ in EfAcpA creates an atypical structure consisting only of three helices (Fig. 1b). EfAcpA participates in de novo FAS, whereas EfAcpB aids in the utilization of externally acquired fatty acids (Zhu et al. 2019). These distinct functions support the unique sequence alignment and structural features of EfAcpA, prompting further investigation into the dynamic properties associated with these differences.

Effect of calcium ions on the thermal stability of EfAcpA and its metal binding sites

The isoelectric point of EfAcpA was 3.7, reflecting its acidic nature associated with 21 acidic residues. The circular dichroism (CD) spectrum of EfAcpA (Fig. 2a) was examined along with the thermal denaturation curves in the range of 10–98 °C. EfAcpA displayed a typical alpha-helical structure at 25 °C in the presence of calcium as well as in the absence of calcium ion. When comparing the CD spectra of EfAcpA with and without calcium ions at 70 ℃, the protein with calcium ion exhibited a much more defined folding patterns, suggesting that calcium ions enhance the alpha-helicity of EfAcpA even at high temperature, thereby improving its structural stability. Especially, we recorded T_m of 64.0 °C in the absence of calcium and 76.1 °C in the presence of calcium, demonstrating the role of metal ions in enhancing the structural stability of EfAcpA.

Fig. 2

Thermal stability and metal binding sites of EfAcpA. a Secondary structures at 25 ℃ and 70 ℃ and melting temperatures of EfAcpA in the presence and absence of calcium ion. b Chemical shift perturbation in ¹H-¹⁵N HSQC spectra upon titration of EfAcpA with calcium ions from 1:0 to 1:10 ratio c Trace of residue peaks in ¹H-¹⁵N HSQC spectra when EfAcpA is titrated with calcium ions in the following ratios: 1:0 (red), 1:0.5 (orange), 1:1 (yellow), 1:2 (green), 1:5 (blue), 1:10 (purple), 1:50 (pink), and 1:100 (gray)

We investigated the metal binding sites of EfAcpA based on the CSP in the ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) spectra during the titration of calcium ions. Superimposition of the HSQC spectra during titration revealed noticeable shifts in the HSQC peaks for specific residues, indicating the presence of potential metal-binding sites. Residues associated with > 0.13 ppm CSPs near the N-terminal of α₂ included Glu30, Asp32, Ala33, Asp34 and Ser35 (Site A, Fig. 2b). Asp47, Ile 53, Glu54, and Glu56 near the C-terminal of α₂ and the α₂α₃ loop (Site B) exhibited significant CSPs, suggesting the existence of two metal binding sites. Residues affected by metal binding (CSP > 0.13 ppm) are mapped onto the EfAcpA structure in Fig. 2b. Figure 2c illustrates the ¹H–¹⁵N HSQC peak traces of crucial residues in EfAcpA, including Glu30, Asp34, Asp47, and Glu56, during titration of calcium ions. The peaks exhibited gradual directional shifts until reaching saturation.

Backbone dynamics of EfAcpA

To elucidate the functional properties of EfAcpA, we investigated its backbone dynamics in the holo-form attached to the prosthetic group. We conducted longitudinal relaxation rate (R₁), transverse relaxation rate (R₂), and heteronuclear NOE (hNOE) experiments at picosecond to nanosecond timescales. The average rates in EfAcpA were 1.89 ± 0.02 for R₁, 8.02 ± 0.07 for R₂, and 0.79 ± 0.01 s⁻¹ for hNOE (Fig. 3). The relaxation rates were comparable in stable helix regions, whereas those in the N- and C-terminal and α₂α₃ loop regions varied. The absence of a short third helix in EfAcpA generated only three helices and a long α₂α₃ loop, resulting in high flexibility in the α₂α₃ loop and low hNOE values of 0.61 ± 0.01 s⁻¹ (0.18 s⁻¹ lower than the average). Val51, Glu54, and Gly58 in the α₂α₃ loop exhibited higher R₂ values than other residues, suggesting that they may be crucial for shuttling acyl groups.

Fig. 3

Spin relaxation rates of EfAcpA. R₁, R₂, and hNOE values for EfAcpA were measured at 298 K. The values are plotted in relation to the residue sequence number for EfAcpA, the bars above the plots indicate the helices of EfAcpA

We compared the backbone dynamics of EfAcpA with that previously reported for EfAcpB (Park et al. 2016). Ser58 at the beginning of α₃ in EfAcpB exhibited extremely high R₂ values (39.86 ± 1.04 s⁻¹) (Park et al. 2016). In contrast, Glu54 in EfAcpA had a much lower R₂ value (9.65 ± 0.01 s⁻¹) than that of Ser58 in EfAcpB. Generally, EfAcpA did not display high conformational exchange; however, its α₂α₃ loop exhibited high structural flexibility, as evidenced by the low hNOE values.

MD simulations of EfAcpA

We performed 800 ns MD simulations of two EfAcpA replicates to investigate the role of the α₂α₃ loop in the cavity formation of EfAcpA. In EfAcpA, 4′-phosphopantetheine predominantly occupied cavity 1 due to the motion of the α₂α₃ loop (Fig. 4a). Cavity 1 was formed by Leu38, Ile42 in α₂, Ile53, Thr55 in the α₂α₃ loop, and Leu65, Val69 in α₃. While most type II bacterial ACPs possess one cavity, formed by the α₂, α₃, and α₂α₃ loop, EfAcpA exhibited a novel cavity 2 in the direction of the α₁α₂ loop. The entrance of cavity 2 was delineated by Ile27 in the α₁α₂ loop and Leu38 in α₂. Cavity 2 was formed by Ile6, Ile10, and Leu14 in α₁; Thr25, Ile27, Leu31 in the α₁α₂ loop; Leu38, Ile41 in α₂ and Val62 in α₃. When the distance between Ile27 C_α and Leu38 C_α increased, cavity 2 opened up (the red circled area in Fig. 4b). However, entry seemed to be hindered by the short length of 4′-phosphopantetheine. The distance between Ile27 C_α and Leu38 C_α remained constant otherwise, indicating that 4′-phosphopantetheine primarily resided in cavity 1. Consistent with the hNOE values observed in the backbone dynamics analysis, the root mean square fluctuations (RMSF) estimated for both EfAcpA replicates demonstrated that the α₂α₃ loop was highly flexible (Fig. 4c). Based on the MD simulations of EfAcpA, the highly flexible α₂α₃ loop did not facilitate the stable formation of the entrance to cavity 1, leading to the continuous shuttling of 4′-phosphopantetheine (Fig. 4d).

Fig. 4

MD simulations on EfAcpA. a Representative illustration of cavities 1 and 2 within EfAcpA. b Distance between Ile27 C_α in the α₁α₂ loop and Leu38 C_α in α₂. c Root mean square fluctuation (RMSF) for two EfAcpA replicates. d Snapshot showing the motion of Ile27, Leu38 forming the entrance to cavity 2 and the α₂α₃ loop in 800 ns. At 122.2 ns, the entrance to cavity2 was briefly opened, but 4'-phosphopantentein was unable to enter and remained primarily within cavity 1. e Snapshot showing the calcium binding site and demonstrating that the movement of the α₂α₃ loop is stabilized by calcium ions. The yellow spheres represent calcium ions

We also conducted MD simulations to investigate the effect of calcium ions in stabilizing the structure of EfAcpA. The calcium ions binding near two metal binding sites of EfAcpA resulted in the stabilization of the flexible α₂α₃ loop, as depicted in Fig. 4e.

Discussion

ACPs are small acidic proteins comprising four helices and three loops. ACPs sequester acyl chains in a cavity to facilitate their transfer to other enzymes while preventing their hydrolysis. However, EfAcpA possesses a distinctive structure and cavity characterized by only three helices and two loops. Therefore, we explored the structural flexibility of EfAcpA and the effect of divalent cations on the stability of EfAcpA using CD and NMR spectroscopy as well as MD simulations.

The T_m and metal binding site experiments showed that the presence of calcium ions increased thermal stability by 12.1 degree. Similarly, the T_m of EfAcpB and EcACP increased in the presence of calcium ions (EfAcpB: 72.5 °C → 78.8 °C, EcACP: 55.0 °C → 67.2 °C) (Park et al. 2016), highlighting the importance of metals in the structural stability of ACPs. V. harveyi ACP exhibited protein unfolding at a neutral pH in the absence of divalent metal ions, which was attributed to strong electrostatic repulsion and a lack of histidine residues (Chan et al. 2010). Similarly, calcium ions bound to the two metal-binding sites of EfAcpA enhanced its structural and thermal stability.

Most mesophilic ACPs typically feature a rigid proline residue at the beginning of α₃ (Fig. 1a). In contrast, EfAcpA contains a negatively charged glutamine residue, and EfAcpB features a polar serine residue with relatively short polar hydroxymethyl side chains. Previous backbone dynamics studies on EcACP revealed reported uniform R₂ and hNOE values at the beginning of α₃ due to the proline providing a rigid structure, while Ser58 of EfAcpB displayed exceptionally high R₂ rates (Park et al. 2016). These findings imply that EfAcpB undergoes faster exchanges in α₃ compared to the canonical EfAcpA, enabling the generation of various conformations for the swinging motion of the 4′-phosphopantetheine arm and shuttling of long exogeneous acyl chains.

The backbone spin relaxation and MD simulation experiments on EfAcpA demonstrated the flexibility of the α₂α₃ loop. This flexibility prevents the formation of a stable entrance to cavity 1, which leads to its collapse. Consequently, cavity 2 was formed with a more stable entrance structure. Two subpockets have been identified in EcACP accommodating the hexanoyl acyl chain (Chan et al. 2008). Subpockets 1 and 2 in EcACP share the same entrance but are situated at different positions (Fig. 5). Subpocket 1, commonly observed in the NMR and crystal structures of acyl-EcACPs, comprises α₂, α₃, and the α₂α₃ loop, whereas subpocket 2 is closer to α₁ than subpocket 1. However, the cavities of EfAcpA do not share a common entrance; instead, they form separate cavities that facilitate stable accommodation of acyl chains.

Fig. 5

Comparison of cavities in EcACP and EfAcpA. Cavities 1 and 2 are highlighted with red arrows

In the EcACP, Leu42 and Leu46 act as switches that allow entry to the hexanoyl acyl chain (Chan et al. 2008). However, in contrast to EcACP, Ile41 and Ile45 in EfAcpA, corresponding to Leu42 and Leu46 in EcACP, do not appear to serve as switches. Furthermore, the cavities in EfAcpA were positioned differently. The flexibility of the α₂α₃ loop in EfAcpA plays a role in forming a new cavity 2, contributing to a distinctive cavity structure in EfAcpA.

Conclusions

We elucidated the structural stability and flexibility of EfAcpA, an essential cofactor in de novo FAS by E. faecalis. We identified two metal binding sites of EfAcpA in the presence of calcium ions along with enhanced thermal stability facilitated by reduced electrostatic repulsion, particularly in the region surrounding the elongated and flexible long α₂α₃ loop. Unlike most bacterial ACPs, EfAcpA lacks a short helix and is characterized by high flexibility in the α₂α₃ loop and the formation of the unique cavity 2. These structural features may play a role in interacting with FAS-related enzymes and the shuttling of acyl chains. Further research is required to understand potential correlations and underlying mechanisms. Our findings provide valuable insights into the mechanism by which EfAcpA facilitates acyl chain transfer in de novo FAS.

Methods

Expression and purification of EfAcpA

The acpA gene from E. faecalis was cloned into the pET-28a vector. After converting the recombinant vector into E. coli BL21 (DE3), the cells were grown in 500 mL M9 minimal medium with ¹⁵NH₄Cl to express EfAcpA. EfAcpA was purified as previously described (Yeon et al. 2022; Yoo et al. 2024). To produce holo-EfAcpA, apo-EfAcpA, coenzyme A, and holo-ACP synthase, E. coli was added and reacted at 25 °C for 16 h, and the final purification was done with HiTrap Q FF (GE Healthcare).

Detecting CSPs upon calcium ion (Ca²⁺) titration

We identified the metal binding sites of EfAcpA through CSP analysis of ¹H-¹⁵N HSQC spectra detected during calcium ion titration (Choi et al. 2021). This experiment was performed using a 700 MHz Bruker Avance spectrometer at the Korea Basic Science Institute (Ochang, Korea). EfAcpA samples were prepared in a buffer containing 25 mM 2-(N-morpholino)ethanesulfonic acid (MES), 5 mM dithiothreitol (DTT), 10% D₂O, and 0.02% NaN3 (pH 6.1). HSQC spectra were acquired by the successive addition of calcium ions at 0 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 50 mM, and 100 mM.

CD experiment to measure the melting temperatures of EfAcpA

We assessed the thermal stability of EfAcpA by measuring its T_m using a J1500 spectropolarimeter (Jasco, Tokyo, Japan). CD spectra were recorded from 200 to 250 nm from 10 to 98 °C, with 0.1 nm intervals, and accumulation was repeated three times. The CD spectra showed the average residual ellipticity (θ) in degrees (cm² dmol⁻¹). EfAcpA samples (50 µM) were prepared in 25 mM MES, 5 mM DTT and 25 mM MES, or 5 mM DTT, with and without 5 mM CaCl₂. The T_m was calculated from the CD spectra at a wavelength of 222 nm to determine the midpoint between the lowest and highest θ values.

Spin relaxation experiments

We estimated the R₁, R₂, and hNOE values of each backbone amide proton in EfAcpA using the Bruker Avance spectrometer. R₁ rates were obtained from the decay of peak heights in the HSQC spectra over 10 relaxation delays of 0.01 (× 2), 0.05, 0.1, 0.2, 0.3 (× 2), 0.5, 0.8 and 1.2 s. The R₂ rates were determined from 10 relaxation delays at 0 (× 2), 0.01696, 0.03392, 0.05088 (× 2), 0.0848, 0.13568, 0.22048 and 0.32224 s. The recycle delays were 2.3 s and 2.0 s in the R₁ and R₂ experiments, respectively. The hNOE values were obtained from the ratios of the peak heights of the interleaving pulse sequences with and without proton saturation. The recycle delay and proton-saturation pulse were 4.0 s and 3.0 s, respectively.

MD simulations

We utilized the previously reported structure of EfAcpA (8GSA) to perform MD simulations and examine the dynamics of the α₂α₃ loop. Using the Ligand Reader & Modeler tool in CHARMM-GUI, we computed the bonding of 4′-phosphopantetheine groups to serine residues. This involved the integration of acetylated N-termini (ACE) and methyl-amidated C-termini (CT3) with parameters configured according to the CGENFF protocol (version interface 1.0.0, force field 3.0.1) (Jo et al. 2008; Vanommeslaeghe et al. 2010; Yu et al. 2012). The holo-EfAcpA system was prepared using the solution builder in CHARMM-GUI by incorporating 0.15 M KCl or 0.01 M calcium ions into the TIP3P water cube model (Lee et al. 2016). We performed MD simulations with a CHARMM36m force field using OpenMM (version 7.7.0). Equilibration was performed in six consecutive steps using CHARMM-GUI under the NPT ensemble for 1.25 s. The simulations were performed at a constant temperature of 310.15 K and isotropic pressure of 1 bar for 800 ns.