Comparison with other ACP sequences
Because the amino acid sequence of a protein is closely related to its functions, we first compared the amino acid sequence of CaACP with that of the ACPs of various other bacteria (Fig. 1). CaACP showed high sequence similarity with other bacterial ACPs (Fig. 1), sharing the 4′-phosphopantetheine prosthetic (4′-PP) group attached to the conserved serine residue (Ser40 in CaACP) at the beginning of the second helix near the conserved Asp-Ser-Leu (DSL) motif marked in the green box. Distinctively, only CaACP has a unique unconserved Cys50 in helix II, marked in the red box in Fig. 1. Most bacterial ACPs have Leu at the same position. The role of Leu42 and Leu46 in the hydrophobic cavity of E. coli has been investigated extensively using molecular dynamics simulation (Chan et al. 2008). EcACP has two subpockets that use the same entrance. Subpocket I is surrounded by helix II, III, and IV, whereas subpocket II by helix I, II, and IV. When the acyl chain enters subpocket I, both Leu42 and Leu46 switch the dihedral angle to open the pathway for the acyl chains. Leu42 and Leu46 play important roles as gating residues in the opening of two subpockets that can be occupied by the acyl chains by changing the orientation of Leu side chains (Chan et al. 2008).
Proper folding of ACPs requires the hydrophobic interactions in the hydrophobic cavity to include growing acyl chains formed by amino acids such as Leu, Ile, and Val, which are aliphatic branched-chain amino acids. The relative volumes of Leu, Ile, and Val are 63.6%, 63.6%, and 47.7%, respectively, compared to that of Trp (100%) (Bogardt et al. 1980). Cys is an amphipathic amino acid and has a thiol side chain; it occupies lesser space than Leu or Phe does, resulting in reduced hydrophobic interactions in the cavity. Therefore, CaACP might be less stable than the other bacterial ACPs.
Thermal stability of CaACP
To investigate the thermal stability and role of the unique Cys50 in CaACP, we investigated the secondary structure and Tm of CaACP. We mutated Cys50 to Leu (C50L mutant) to obtain a sequence same as that of EcACP, which is conserved in other ACPs (Fig. 1). We performed CD experiments for CaACP and the C50L mutant. The double minima at 205 nm and 222 nm are shown for both proteins from 190 to 260 nm, confirming the characteristics of an α-helix structure (Fig. 2a). The CaACP C50L mutant showed more characteristics of α-helix compared to the wild type. To investigate the thermostability of CaACP in comparison to that of C50L, we measured their Tms using CD; we monitored the mean residue ellipticity at 222 nm, which reflects temperature-induced folding changes of proteins. The Tm of CaACP was 49.6 °C, whereas that of C50L was 55.5 °C, implying that Cys50 is the main factor underlying the low thermal stability of CaACP. Leu, Ile, and Val are typical hydrophobic aliphatic amino acids, whereas Cys is a hydrophilic amino acid. Therefore, the presence of Cys50 in the hydrophobic cavity may loosen the hydrophobic packing, thereby affecting substrate specificity.
H/D exchange experiments
To investigate the structural stability of CaACP in further detail using NMR spectroscopy, we completed the backbone assignment of CaACP spectra using HNCO, HNCACB, and CBCA(CO)NH experiments. In addition, the assignment of Hα was completed using 2D1H–1H nuclear Overhauser effect spectroscopy and total correlation spectroscopy experiments. Using chemical shifts of 13Cα, 13Cβ, and 1Hα, chemical shift indexes (CSIs) were calculated (Wishart and Sykes 1994; Wishart et al. 1992) and used to predict the secondary structure of CaACP with algorithm of the Protein Energetic Conformational Analysis from NMR chemical shifts (PECAN). PECAN predicts the most favorable secondary structure in terms of energy based on amino acid sequence and statistical energy function of each residues (Eghbalnia et al. 2005). CaACP was observed to contain four helical regions: helix I (Lys4–Thr19), helix II (Ser40–Phe54), helix III (Asp60–Asn65), and helix IV (Gly70–His79) (Fig. 3). This suggests that the overall folding of CaACP is very similar to that of other bacterial ACPs having four helix bundles. Therefore, we confirmed that Cys50 is located in the middle of helix II and plays important roles in the stability of the hydrophobic cavity of CaACP.
Because the Tm of CaACP C50L was 6 °C higher than that of CaACP WT, we conducted H/D exchange experiments to understand the role of Cys50 in the structure of CaACP. Figure 4a shows the heteronuclear single quantum coherence (HSQC) spectrum of CaACP with a marking for Ser40, which is the 4′-PP group attachment site, and Cys50, which is crucial in the thermal stability of CaACP. WT CaACP showed an extremely fast H/D exchange rate. In the WT CaACP, amide peaks of only 10 residues––Ile7, Ile14, Val15, Val44, Gly45, Ile47, Ala49, Val73, Ile76, and Ile77––remained after 10 min; however, the peaks disappeared within 50 min. In contrast, the C50L mutant showed a relatively slower H/D exchange rate. As shown in Fig. 4b, c, only Ile47 and Ala49 in helix II of CaACP WT remained after 30 min, whereas more than 10 peaks from residues such as Ile7, Ile14, Val44, Leu50, Val69, and Ile74 in helix I, II, and IV forming a hydrophobic cavity remained after 30 min. However, in the C50L mutant, only three residues––Ile14 in helix I, Ala49 and Leu50 in helix II––remained after 120 min (Fig. 4d). These results imply that the mutated C50L can stabilize a protein by ensuring tight packing in the hydrophobic cavity of CaACP.
Substrate specificity of CaACP as studied by CSPs
Because ACP is an essential cofactor that delivers acyl groups to various fatty acid synthases, the motional property of ACP is very important as a carrier. CaACP is converted to an active holo-form via the attachment of a 4′-PP group to the conserved Ser40, resulting in CSPs due to the hydrophobic interactions between the 4′-PP group and the residues in the hydrophobic cavity. Upon conversion from apo- to holo-CaACP, a large CSP was observed mainly for the residues near the Ser40-binding site and near the entrance of the cavity (Fig. 5a, d).
To clarify the substrate specificity of CaACPs with linear or branched acyl chains, the CSPs in 1H–15N HSQC spectra upon conversion from apo to butyryl and isobutyryl forms of CaACP were investigated. The entrance of acyl chains into the hydrophobic cavity of ACP formed by four helices resulted in CSPs, as compared to that in the apo form. Figure 5d–f provide a structural representation of the CSPs in CaACP on the homology modeled structure. When a linear butyryl chain is attached to Ser40, the residues in helix I and helix III as well as in helix II showed large CSPs, implying that CaACP can accommodate a butyryl group deep in the hydrophobic cavity (Fig. 5b, e). Similar to the FAS in most bacteria, CaACP carries linear fatty acyl chains to fatty acid synthases. However, as shown in Fig. 5c, f, CaACP also showed large CSPs upon conversion from apo to isobutyryl forms in a pattern similar to that of the butyryl form, thereby confirming the substrate specificity of CaACP for BCFAs. Because the buried side chains of the hydrophobic residues form a hydrophobic cavity surrounded by four amphipathic helixes and loops (Roujeinikova et al. 2007), all the residues showing large CSPs were hydrophobic residues forming hydrophobic interactions with hydrophobic acyl chains. Furthermore, Val56 and Glu57 at loop II between helix II and helix III also showed CSPs upon conversion to the holo, butyryl, and isobutyryl forms, implying that flexibility of loop II is important in acyl binding.