AlphaFold-Multimer predictions for 50 ACP+KS dimers
In the majority of cis -AT PKS modules, the ACP and KS domains are embedded in the same polypeptide; however, modules are also frequently split such that ACP is embedded in the upstream polypeptide and KS in the downstream polypeptide20 . C- and N-terminal docking domain motifs (CDD & NDD) at the polypeptide termini help noncovalently connect the domains from these modules26, 27 . We selected 45 of 50 of the ACP+KS’s in this study from modules naturally embedded in a single polypeptide, since the linker connecting these domains contains fewer than 15 residues and provides a restraint for the folding/docking of these domains20 . Five pairs of ACP’s and KS’s naturally embedded in separate polypeptides were also investigated by connecting them with a 15-residue polyglycine linker (G15). As KS’s are dimeric, 2 copies of each of the 50 ACP+KS sequences were provided to AlphaFold-Multimer (Data File 1, see Table S1 for PKS abbreviations, Figures S1-S3). For each ACP+KS, 5 energy-minimized solutions were obtained (Data Files 2-51)14 .
The ACP’s consistently dock to the same location, with the serine that becomes phosphopantetheinylated facing the entrance to the KS active site (Figure 2a and S2). ACP and KS bind in trans in 76% of the solutions, with the ACP domains almost exclusively making contacts with residues from the KS of the opposite monomer. As the N-terminal end of αI’ of ACP (secondary-structure nomenclature,20, 28 prime used to distinguish ACP from KS, numbering from PikACP6 and PikKS6 used to discuss all ACP/KS pairs) is predicted to be close to the AT domain downstream of KS, the structures of 5 ACP+KS+AT dimers [Ery(ACP1+KS1+AT2), Ery(ACP4+KS4+AT5), Pik(ACP1+KS1+AT2), Pik(ACP6+KS6+AT7), and Rap(ACP1+KS1+AT2)] were also predicted (Figures 2b and S4, Data Files 52-57). All of the solutions show ACP bound to the same site as in the solutions for the ACP+KS dimers with no contact between ACP and AT. Either a rotation about αIII’ or a hinge motion between KS and AT would be necessary for ACP and the downstream AT to make contact.7, 8
The interface between ACP and KS is similar in all solutions (Figures 3 and S2). While contacts with ACP residues in and around αIII’ (residues 73’-80’) are most substantial, contacts with residues in a one-turn helix upstream of αII’ (residues 46’-48’) and residues at the N-terminal end of αII’ (residues 53’-60’) are also significant. KS residues involved in the contacts include those in the structured loop between β8 and α10 (residues 273-283), the N-terminal end of α11 (residues 314-322), and the loop between β11 and β12 (residues 381-388).