Intein-mediated cyclization of bacterial acyl carrier protein stabilizes its folded conformation but does not abolish function

Abstract

Bacterial acyl carrier protein (ACP) is essential for the synthesis of fatty acids and serves as the major acyl donor for the formation of phospholipids and other lipid products. Acyl-ACP encloses attached fatty acyl groups in a hydrophobic pocket within a four-helix bundle, but must at least partially unfold to present the acyl chain to the active sites of its multiple enzyme partners. To further examine the constraints of ACP structure and function, we have constructed a cyclic version of Vibrio harveyi ACP, using split-intein technology to covalently join its closely apposed N and C termini. Cyclization stabilized ACP in a folded helical conformation as indicated by gel electrophoresis, circular dichroism, fluorescence, and mass spectrometry. Molecular dynamics simulations also indicated overall decreased polypeptide chain mobility in cyclic ACP, although no major conformational rearrangements over a 10-ns period were noted. In vivo complementation assays revealed that cyclic ACP can functionally replace the linear wild-type protein and support growth of an Escherichia coli ACP-null mutant strain. Cyclization of a folding-deficient ACP mutant (F50A) both restored its ability to adopt a folded conformation and enhanced complementation of growth. Our results thus suggest that ACP must be able to adopt a folded conformation for biological activity, and that its function does not require complete unfolding of the protein.

FIGURE 1.

A, schematic representation of cyclization constructs. Plasmid pTCYC-L46W encodes a 30-kDa protein (preL46W), which after in vivo cyclization yields the split-intein products I_C and I_N, as well as the cyclic V. harveyi ACP L46W protein (cycL46W). Amino acid residues at both the C- and N-terminal splice junction are indicated in single letter code. The ligation site of the ACP C and N termini in cycL46W is the peptide bond between Gly and Ser. Plasmid pTPRE-L46W-mut serves as an expression control, as it encodes for a 30-kDa protein (preL46Wmut) carrying mutations in I_C (N → A) and I_N (C → A), which prevent intein-mediated cyclization. B, E. coli BL21(DE3)pLysS cells harboring plasmids pTCYC-L46W or pTPRE-L46W-mut were induced with IPTG (+) or grown in the absence of IPTG (−). Total cell lysates were analyzed by SDS-PAGE and Coomassie Blue staining (left) and Western blot analysis using anti-His antibodies (right). Relevant protein species are indicated, and molecular masses are given in kDa. C, native PAGE analysis of soluble protein extracts derived from induced BL21(DE3)pLysS cells harboring indicated plasmids. Gels were either stained with Coomassie Blue or subjected to Western blotting with anti-His antibodies. The control lanes show lysate from BL21(DE3)pLysS cells not overexpressing ACP-related proteins.

FIGURE 2.

Tandem mass spectrometry (MS/MS) analysis of a peptide obtained after trypsin digestion of the protein marked with an asterisk in Fig. 1C. The MS/MS spectrum (top) shows the [M+3H]³⁺ ion, as well as selected y and b ions derived through its fragmentation. The peptide corresponds to the sequence shown below the MS/MS spectrum, with selected y and b ions indicated (bottom).

FIGURE 3.

Migration behavior of purified linL46W and cycL46W in SDS-PAGE (A) and native PAGE (B). Molecular masses in A are given in kDa.

FIGURE 4.

Circular dichroism analysis of linear and cyclic derivatives of L46W and F50A. CD spectra of the indicated proteins were measured in the absence (dashed line) or presence (solid line) of 10 mm Mg²⁺ as described in the text.

FIGURE 5.

Biophysical characterization of cycL46W and linL46W. Tryptophan fluorescence spectra (λ_ex = 296 nm) of linL46W (A) and cycL46W (B) were obtained in the absence (dashed line) and presence (solid line) of 10 mm Mg²⁺. Positive mode nano-ionspray MS spectra of intact linL46W (C) and a mixture of apo- and holo-cycL46W (D) proteins were recorded as described in the text. The charge of relevant peaks are indicated.

FIGURE 6.

Molecular dynamics simulations of linear and cyclic L46W ACP. Panel A shows sausage representations of linL46W (left) and cycL46W (right), where the thickness of the polypeptide chain represents the RMSF of C_α atoms during the final 9 ns of simulation. A scaling factor of ½ was used, and helices I-IV are indicated. Panel B, difference plot of C_α RMSF values of linL46W versus cycL46W as a function of position in the chain. The positions of helical segments of the starting E. coli structure (4) is shown above the plot.

FIGURE 7.

In vivo complementation assay. E. coli CY1861 were transformed with pMAL-derived plasmids encoding the indicated proteins under control of the IPTG-inducible p_tac promoter. Cells were grown in medium containing arabinose (open circles) or IPTG and glucose (closed circles), and the absorbance at 595 nm was recorded over 6 h. Growth curves representative of three individual experiments are shown.