NMR solution structure and biophysical characterization of Vibrio harveyi acyl carrier protein A75H: effects of divalent metal ions

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 ∼20 °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.

FIGURE 1.

Protein sequence alignment of VhACP and other related ACPs. The numbering is performed according to the V. harveyi sequence and begins at Ser-1, which is preceded by a four residue extension present because of the cloning protocol. Anionic residues are highlighted in light gray, and cationic residues are shown in dark gray. The phosphopantetheine attachment site is boxed, and a secondary structure diagram is provided above the sequences. The differences in primary structure between the E. coli and V. harveyi ACP sequences are highlighted in bold. The residues mutated for the calcium-binding site A and B elimination mutants are also shown.

FIGURE 2.

NMR solution structure of VhACP A75H. A shows the backbone overlay of the 20 lowest energy structures of VhACP overlaid on the four α-helices, shown in the same orientation as B. B, shows a ribbon diagram of the lowest energy structure, colored in a spectrum from red to blue from the N to the C terminus, respectively. His-75 and Tyr-71 are shown in stick representation to highlight their stacking interaction. The arrow shows the position of Ser-36.

FIGURE 3.

Divalent cation binding to wild-type and A75H V. harveyi ACP. A, natural abundance HSQC spectra of wild-type V. harveyi ACP in the presence of calcium (red) and magnesium (black) ions suggest ACP adopts similar conformations in the presence of either divalent cation. B, VhACP A75H structure showing in orange where the residues display the largest chemical shift differences in the presence of the two cations. C, HSQC spectra of VhACP A75H collected at 0, 0.3, 0.5, 0.9, and 4.0 mm CaCl₂ (black, red, blue, cyan, magenta, respectively) indicate that calcium binding occurs on the fast NMR timescale for both sites (see arrows). Only a portion of the HSQCs are shown for clarity. D, residues affected the most upon addition of calcium are highlighted in green, spatially matching the E. coli ACP divalent cation-binding sites (arrows).

FIGURE 4.

A, residues observed after 30 min of H/D exchange in calcium-free VhACP A75H are shown in gold, while additional residues observed in the Ca²⁺-bound protein are shown in blue. B, H/D exchange rates as observed in Ca²⁺-bound (black) and Ca²⁺-free (gray) VhACP A75H, with lower values indicative of slower exchange. Note the vastly slower rates of the Ca²⁺-bound protein in the helical regions. Residues denoted by stars indicate peaks were present in too few spectra to reliably determine any rates. Binding of Mg²⁺ has a similar effect on the H/D exchange rates as Ca²⁺ (not shown).

FIGURE 5.

Electrostatic potential plots of various ACPs. Isocontour surfaces are shown at ± 3 kT/e, with the blue surface being cationic, while the red shows the anionic surface. VhACP A75H is shown in A, with the surface plot in the same orientation in i as in the ribbon diagram in ii and rotated about the y axis by 180° in iii. Aii highlights the position of charged residues on the structure of ACP with helices II and IV labeled with roman numerals. The divalent cation-binding sites A and B are highlighted by the upper and lower dotted circles, respectively. B shows the surface of wild-type VhACP, while C shows ACP A75H with two +2 charges placed at the calcium-binding sites. D displays the electrostatic surface plot of wild-type EcACP. B–D are oriented the same as in Ai.