NMR Solution Structure and Biophysical Characterization of Vibrio harveyi Acyl Carrier Protein A75H

Abstract

Bacterial acyl carrier protein (ACP) is a highly anionic, 9 kDa protein that functions as a cofactor protein in fatty acid biosynthesis. Escherichia coli ACP is folded at neutral pH and in the absence of divalent cations, while Vibrio harveyi ACP, which is very similar at 86% sequence identity, is unfolded under the same conditions. V. harveyi ACP adopts a folded conformation upon the addition of divalent cations such as Ca 2؉ and Mg 2؉ and a mutant, A75H, was previously identified that restores the folded conformation at pH 7 in the absence of divalent cations. In this study we sought to understand the unique folding behavior of V. harveyi ACP using NMR spectroscopy and biophysical methods. The NMR solution structure of V. harveyi ACP A75H displays the canonical ACP structure with four helices surrounding a hydrophobic core, with a narrow pocket closed off from the solvent to house the acyl chain. His-75, which is charged at neutral pH, participates in a stacking interaction with Tyr-71 in the far C-terminal end of helix IV. pH titrations and the electrostatic profile of ACP suggest that V. harveyi ACP is destabilized by anionic charge repulsion around helix II that can be partially neutralized by His-75 and is further reduced by divalent cation binding. This is supported by differential scanning calorimetry data which indicate that calcium binding further increases the melting temperature of V. harveyi ACP A75H by ϳ20°C. Divalent cation binding does not alter ACP dynamics on the ps-ns timescale as determined by 15 N NMR relaxation experiments, however, it clearly stabilizes the protein fold as observed by hydrogen-deuterium exchange studies. Finally, we demonstrate that the E. coli ACP H75A mutant is similarly unfolded as wild-type V. harveyi ACP, further stressing the importance of this particular residue for proper protein folding.

Novel Structural Components Contribute to the High Thermal Stability of Acyl Carrier Protein from Enterococcus faecalis

“…In comparison with the B. subtilis ACP structure, the distinctive 55 , showed uniform R 2 value because proline had a tendency to form a rigid bent structure in the loop in our previous study (59). These results imply that the Ile 57 and Ser 58 loop region might have a conformational exchange to release and accept the acyl chain and generate enough room to accommodate the longer acyl chains.…”

“…However, both results confirmed that the T m of Ec-ACP in the presence of Ca 2ϩ was Ͼ12°C higher than that of Ec-ACP in the absence of metal ions. Also, it has been reported that V. harveyi ACP has an ␣-helical structure with a T m value of 66.4°C in the presence of Ca 2ϩ , whereas this protein is unfolded in the absence of Ca 2ϩ (58). The measurement of melting temperatures implied that Ef-ACP has much higher thermal stability than does other ACPs.…”

“…1). There are proline residues in the ␣ 2 ␣ 3 loops connecting helix II and helix III in most ACPs, including Ec-ACP (Pro 55 ), whereas in Ef-ACP, this is replaced with a serine residue (Ser 58 ), providing more flexibility (marked with a yellow box in Fig. 1).…”

“…In previous studies, Ec-ACP has been reported to exhibit higher melting temperatures (64.3°C) in the presence of calcium ion compared with Ec-ACP in the absence of calcium ion (52.7°C) (57). In addition, in the presence of calcium ion, V. harveyi ACP has an ␣-helical structure with a T m of 66.4°C; however, in the absence of calcium ion, this protein is unfolded (58). As shown in Fig.…”

“…In the case of Ec-ACP, His 75 at helix IV retained a stable native conformation by shielding the electrostatic repulsion between negatively charged residues (59). Furthermore, V. harveyi ACP, which has an alanine instead of histidine at the C terminus, becomes unfolded, whereas the A75H mutation restores proper folding of the ␣-helical structures by reducing the electrostatic repulsion (58,61,62).…”