An ACP Structural Switch: Conformational Differences between the Apo and Holo Forms of the Actinorhodin Polyketide Synthase Acyl Carrier Protein

Abstract

The actinorhodin (act) synthase acyl carrier protein (ACP) from Streptomyces coelicolor plays a central role in polyketide biosynthesis. Polyketide intermediates are bound to the free sulfhydryl group of a phosphopantetheine arm that is covalently linked to a conserved serine residue in the holo form of the ACP. The solution NMR structures of both the apo and holo forms of the ACP are reported, which represents the first high resolution comparison of these two forms of an ACP. Ensembles of twenty apo and holo structures were calculated and yielded atomic root mean square deviations of well-ordered backbone atoms to the average coordinates of 0.37 and 0.42 A, respectively. Three restraints defining the protein to the phosphopantetheine interface were identified. Comparison of the apo and holo forms revealed previously undetected conformational changes. Helix III moved towards helix II (contraction of the ACP), and Leu43 on helix II subtly switched from being solvent exposed to forming intramolecular interactions with the newly added phosphopantetheine side chain. Tryptophan fluorescence and S. coelicolor fatty acid synthase (FAS) holo-synthase (ACPS) assays indicated that apo-ACP has a twofold higher affinity (K(d) of 1.1 muM) than holo-ACP (K(d) of 2.1 muM) for ACPS. Site-directed mutagenesis of Leu43 and Asp62 revealed that both mutations affect binding, but have differential affects on modification by ACPS. Leu43 mutations in particular strongly modulate binding affinity for ACPS. Comparison of apo- and holo-ACP structures with known models of the Bacillus subtilis FAS ACP-holo-acyl carrier protein synthase (ACPS) complex suggests that conformational modulation of helix II and III between apo- and holo-ACP could play a role in dissociation of the ACP-ACPS complex.

Probing the Interactions of Early Polyketide Intermediates with the Actinorhodin ACP from S. coelicolor A3(2)

“…8 The results presented above indicate that the polyketide ACP has the inherent ability to undergo large conformational changes that can be modulated by the chain length and polarity of the attached intermediate. The binding of substrate by a FAS or PKS ACP may serve the dual purpose of providing some degree of sequestration/stabilisation of the attached intermediate whilst inducing structural changes that can modulate protein-protein interactions.…”

“…Figure 3d shows a superposition of acetyl, malonyl, and 3-oxobutyl ACP with the holo ACP structure reported recently. 8 Helix II can clearly be seen to remain essentially static, whereas helix III is shifted away from its position in holo ACP for both acetyl and malonyl ACPs. In 3-oxobutyl ACP, helix III is essentially superimposable with that of holo ACP.…”

“…Samples of 13 C/ 15 N-labelled act apo ACP were prepared as reported previously 8 and derivatised by utilising the relaxed specificity of S. coelicolor holo ACP synthase (ACPS) for modified coenzyme As (CoA). 32 Although a polyketide ACP may also be derivatised via chemical methods, 32,33 the use of ACPS is preferable for study by solution NMR as it offers a useful spectroscopic means of discriminating between the non-isotopically enriched derivatised 4'-PP cofactor and uniformly 13 C/ 15 N-labelled protein using filter/editing of 12 C/ 13 C and 14 N/ 15 N nuclei (see below) ( Fig.…”

“…The function of this sequestration is not clear, but it may serve to protect the thiol ester from hydrolysis or confer allosteric regulation of the ACP; that is, its interaction with other enzymes may be modulated and controlled through conformational changes. 8,29,30 In the following study, we have prepared seven derivatised PKS ACPs with different chain lengths and polarities, which include stabilised mimics for di-and triketide intermediates. We have utilised the actinorhodin (act) PKS ACP from Streptomyces coelicolor, 31 which has become a model system for in vitro polyketide studies.…”

Probing the interactions of an acyl carrier protein domain from the 6‐deoxyerythronolide B synthase

“…These findings are also in agreement with recent studies on ACP6 of DEBS 25 and the actinorhodin ACP. 26,27 Influence of the growing polyketide chain on protein-protein interactions during intermodular chain translocation…”