Figure 1.
Three classes of type I iterative polyketide synthases (PKS).
The proposed PKS products are tethered to the ACP domains.
Figure 2.
Residues only from Ala938-Glu1014 are shown, excluding flexible N- and C- terminal tails. (A) Backbone of an ensemble of the lowest energy conformations shown as line representation (B) Mean apo-meACP solution structure is shown as ribbon model.
Table 1.
Experimental restraints and structural statistics for ten lowest-energy NMR structures of apo-meACP out of 100 structures.
Figure 3.
Comparison of meACP with other ACPs.
(A) Multiple sequence alignment of meACP with selected type I and type II ACPs. The secondary structures of meACP (top) and B. subtilis FAS ACP (bottom) are shown. The phosphopantetheine attaching serine site is shaded in red. (B) Phylogenetic analysis of the type I and II ACPs from (A).
Figure 4.
Conformation of the motif harboring the phosphopantetheine attaching serine in meACP and two other ACPs.
The GX(H/D)S(L/I) motif in type I NSAS PKS ACP (PDB ID: 2KR5) (pink) and type II actinorhodin PKS ACP (PDB ID: 1AF8) (orange) and the HMSSI motif in meACP (grey) are shown as ribbons. The conserved Gly and Ser residues in GX(H/D)S(L/I) motif are shown as stick. The His and Ser residues of the HMSSI motif in meACP are also shown as stick.
Figure 5.
Conformation mobility of loop-2.
(A) Type II actinorhodin PKS ACP (PDB ID: 1AF8) that contain mainly small residues in loop-2. (B) Type I NSAS PKS ACP (PDB ID: 2KR5) with the two bulky residues (Phe1768 and Phe1771) highlighted (C) meACP with the two bulky residues (Phe996 and Met992) packed against the hydrophobic pocket highlighted. (D). 1H-15N NOE plot of apo-meACP acquired at an 800 MHz spectrometer at 25°C. The positions of the helices are indicated.
Figure 6.
Overlaid 1H-15N HSQC spectra of apo-meACP and its derivatives.
apo-meACP (black), holo-meACP (A, yellow), hydroxybutyryl-meACP (B, green) and octanoyl-meACP (C, red) acquired at 15°C. The change of chemical shifts for the residues Ser971 and Gly975 from apo, holo, hydroxybutyryl to octanoyl-meACP are indicated by the black arrows.
Figure 7.
Chemical shift perturbations (Δδ) of meACP caused by phosphopantetheinylation and acylation.
Combined chemical shift change plots of apo-meACP versus (A) holo-meACP, (B) hydroxybutyryl-meACP and (C) octanoyl-meACP. Combined chemical shift change plots of (D) hydroxybutyryl-meACP versus holo-meACP, (E) octanoyl-meACP versus holo-meACP and (F) octanoyl-meACP versus hydroxybutyryl-meACP. A combined chemical shift cut-off of 0.05 ppm is shown as the dashed line. The residue number and the positions of the helices (α1, α2 and α3) are also indicated. (G) Ribbon representation of apo-meACP with the positions of the perturbed residues upon phosphopantetheinylation are highlighed (combined chemical shift cut-off >0.05 ppm (yellow sphere) and >0.10 ppm (red sphere)). (H) Ribbon representation of apo-meACP with the positions of the perturbed residues based on (F) upon acylation by octanoyl moiety are highlighted (combined chemical shift cut-off >0.05 ppm (blue sphere)).
Figure 8.
Embedded ribbon representation and electrostatic potential surface of ACPs.
Type I meACP (A, E and I), rat FAS ACP (B, F and J, PDB ID: 2PNG), NSAS PKS ACP (C, G and K, PDB ID: 2KR5) and Type II actinorhodin PKS ACP (D, H and L, PDB ID: 1AF8). The invariant Ser is shown as blue stick and labeled. The protein surfaces are colored as white (hydrophobic), blue (basic) and red (acidic) with the same electrostatic potential scale.
Figure 9.
Ribbon representation of apo-meACP with either hydrophobic or acidic face.
The hydrophobic and Glu-Asp acidic faces are shown with highlighted spheres colored red (Glu) and orange (Asp).