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

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 α 3 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 α 2 helix while site B was situated near the α 2 helix and α 2 α 3 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 α 2 α 3 loop in EfAcpA displayed high flexibility, as indicated by low heteronuclear NOE values. The residues Val51, Glu54, and Gly58 exhibited significant R 2 values, likely due to the movement of this loop. EfAcpA created a novel cavity towards the α 1 α 2 loop, in contrast to conventional cavity formation in most bacterial ACPs. This unique behavior was attributed to the flexibility exhibited by the α 2 α 3 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. 2004Park et al. , 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 α 3 helix: EfAcpA possessed only three helices and lacked α 3 , which was present in EfAcpB.These ACPs showed a low sequential identity of 28.7%, and the presence of a long α 2 α 3 loop due to the lack of α 3 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 α 3 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.

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 α 2 α 3 loop, only just preceding α 3 .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 α 2 α 3 loop of EfAcpA, a region where most bacterial ACPs typically feature a short α 3 helix.This suggests that this deletion sequence in this region facilitates the lack of α 3 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 α 2 .EfAcpA harbored Leu38 and Phe39 at the beginning of α 2 , 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.
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 α 3 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 α 3 (Cronan 2014;Nguyen et al. 2014).In contrast, the lack of α 3 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.
We investigated the metal binding sites of EfAcpA based on the CSP in the 1 H-15 N 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 α 2 included Glu30, Asp32, Ala33, Asp34 and Ser35 (Site A, Fig. 2b).Asp47, Ile 53, Glu54, and Glu56 near the C-terminal of α 2 and the α 2 α 3 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 1 H-15 N 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 (R 1 ), transverse relaxation rate (R 2 ), and heteronuclear NOE (hNOE) experiments at picosecond to nanosecond timescales.The average rates in EfAcpA were 1.89 ± 0.02 for R 1 , 8.02 ± 0.07 for R 2 , 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 α 2 α 3 loop regions varied.The absence of a short third helix in EfAcpA generated only three helices and a long α 2 α 3 loop, resulting in high flexibility in the α 2 α 3 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 α 2 α 3 loop exhibited higher R 2 values than other residues, suggesting that they may be crucial for shuttling acyl groups.
We compared the backbone dynamics of EfAcpA with that previously reported for EfAcpB (Park et al. 2016).Ser58 at the beginning of α 3 in EfAcpB exhibited extremely high R 2 values (39.86 ± 1.04 s −1 ) (Park et al. 2016).In contrast, Glu54 in EfAcpA had a much lower R 2 value (9.65 ± 0.01 s −1 ) than that of Ser58 in EfAcpB.Generally, EfAcpA did not display high conformational exchange; however, its α 2 α 3 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 α 2 α 3 loop in the cavity formation of EfAcpA.In EfAcpA, 4′-phosphopantetheine predominantly occupied cavity 1 due to the motion of the α 2 α 3 loop (Fig. 4a).Cavity 1 was formed by Leu38, Ile42 in α 2 , Ile53, Thr55 in the α 2 α 3 loop, and Leu65, Val69 in α 3 .While most type II bacterial ACPs possess one cavity, formed by the α 2 , α 3 , and α 2 α 3 loop, EfAcpA exhibited a novel cavity 2 in the direction of the α 1 α 2 loop.The entrance of cavity 2 was delineated by Ile27 in the α 1 α 2 loop and Leu38 in α 2 .Cavity 2 was formed by Ile6, Ile10, and Leu14 in α 1 ; Thr25, Ile27, Leu31 in the α 1 α 2 loop; Leu38, Ile41 in α 2 and Val62 in α 3 .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 α 2 α 3 loop was highly flexible (Fig. 4c).Based on the MD simulations of EfAcpA, the highly flexible α 2 α 3 loop did not facilitate the stable formation of the entrance to cavity 1, leading to the continuous shuttling of 4′-phosphopantetheine (Fig. 4d).
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 α 2 α 3 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 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 α 3 (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 R 2 and hNOE values at the beginning of α 3 due to the proline providing a rigid structure, while Ser58 of EfAcpB displayed exceptionally high R 2 rates (Park et al. 2016).These findings imply that EfAcpB undergoes faster exchanges in α 3 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 α 2 α 3 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 α 2 , α 3 , and the α 2 α 3 loop, whereas subpocket 2 is closer to α 1 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.
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 α 2 α 3 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 α 2 α 3 loop.Unlike most bacterial ACPs, EfAcpA lacks a short helix and is characterized by high flexibility in the α 2 α 3 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.

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 15 NH 4 Cl 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).

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 (cm 2 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 CaCl 2 .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 R 1 , R 2 , and hNOE values of each backbone amide proton in EfAcpA using the Bruker Avance spectrometer.R 1 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 R 2 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 R 1 and R 2 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 protonsaturation 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 α 2 α 3 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 310.15K and isotropic pressure of 1 bar for 800 ns.

Fig. 1
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)

Fig. 3
Fig. 3 Spin relaxation rates of EfAcpA.R 1 , R 2 , 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

Fig. 4
Fig.4MD simulations on EfAcpA.a Representative illustration of cavities 1 and 2 within EfAcpA.b Distance between Ile27 C α in the α 1 α 2 loop and Leu38 C α in α 2 .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 α 2 α 3 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 α 2 α 3 loop is stabilized by calcium ions.The yellow spheres represent calcium ions (See figure on next page.)

Fig. 5
Fig. 5 Comparison of cavities in EcACP and EfAcpA.Cavities 1 and 2 are highlighted with red arrows