Complementarity at the transacylation site
The interactions observed in the top solution for PikMod6 are representative of interactions observed in each of the 17 top solutions with the rotated ACP (L315 contacting αII’) (Table S2). As solutions with an unrotated ACP (24 with the α-conformation and 8 with the β-conformation) generally do not permit the thioester connecting the phosphopantetheinyl arm and the polyketide to approach the KS reactive cysteine, they are not considered here. All contacts are made between residues in a 36-aa region of PikACP6 (positions 45’-80’) and in a 109-aa region of PikKS6 (positions 275-383) (Figure 3a).
The solution from PikMod6 shows the T74’ methyl group from its ACP occupying a hydrophobic pocket formed by P318, I319, and Q322 on the surface of PikKS6 (Figures 3c and 5a-b, Table S2). A threonine plays this role in 15 out of the 17 top solutions with the rotated ACP. The T74’ hydroxyl group and the D78’ carboxylate are observed making a hydrogen bond in each solution. The sidechains of residues at positions 72’ and 73’ also make contact with KS. A proline most commonly at position 72’ usually makes Van der Waals contact with the sidechain of the residue in position 322, and an alanine most commonly at position 73’ fills space adjacent to the backbone carbonyl of position 383. When a serine or threonine is in position 73’, its side chain usually shares a hydrogen bond with backbone carbonyl of the residue in position 383 (EbeMod7, CpkMod4, E837Mod2, EryMod3). An unusual leucine in position 73’ of BafMod3 makes greater van der Waals contact with the residue in position 383.
In the PikMod6 solution, D78’ interacts with the N-terminal end of α10. A hydrogen bond is present between its carboxylate and the NH of G282, while the nonpolar portion of D78’ makes van der Waals interactions with P283. A hydrogen bond between the carboxylate of the aspartate in position 78’ with the NH of a glycine at position 282 is present in the 17 top solutions with the rotated ACP. The backbone carbonyl of D78’ most commonly forms a hydrogen bond with the side chain of an asparagine in position 281.
In 16 of the 17 top solutions with the rotated ACP, a phenylalanine occupies position 77’, while in EryMod1 a tyrosine is present. In all of the solutions, the benzyl group is equivalently oriented, with its methylene in contact with the δ-methyl group of the isoleucine at position 319. When the phosphopantetheinyl arm is present, itsgem -dimethyl moiety can hydrophobically interact with the aromatic ring. In each of the 25 top solutions with the α-conformation, it is the gem -dimethyl moiety of L315 that interacts with the aromatic ring.
The C-terminal end of αIII’ and the N-terminal end of a one-turn helix (residues 46’-49’) upstream of αII’ in ACP make contact with the KS surface residue at position 275 and nearby residues in the structured loop between β8 and α10. Van der Waals contacts between a proline in position 80’ and residues in positions 274 and 275 are substantial. This proline is much less conserved in the ACP’s of thioesterase (TE)-containing modules in which KS is replaced by a TE (Figure S5). Usually, 1 or 2 hydrogen bonds are formed between the carbonyls of residues in positions 76’ and 77’ and the NH2 of an asparagine highly conserved at position 275. In the absence of these hydrogen bonds, one is usually formed between the asparagine sidechain carbonyl and the backbone NH of the residue in position 47’. The residue in position 47’ is usually an arginine or lysine, whose positively-charged group is most often observed in contact with the sidechain oxygen of a threonine conserved at position 278. In contrast, the residue in position 47’ is usually hydrophobic in the ACP’s of TE-containing modules. In PikMod3, the residue at position 275 is a serine rather than an asparagine. The serine sidechain makes shape-complementary interactions with ACP and its backbone carbonyl forms a hydrogen bond with the backbone NH of an unusual leucine in position 47’. The A279 side chain and the sidechain of this leucine also make hydrophobic contact.
Although αIII’ of ACP makes the most contact with the KS surface, αII’ is also involved in significant interactions. In the PikMod6 solution, the methylene of S53’ and the sidechains of L54’ and V57’ on the ACP create a hydrophobic pocket for L315 from the KS TxLGDP motif. Residues in αII’ also make contact with the residue in position 383, most often with its sidechain, but also with its backbone carbonyl. The residue in position 57’, usually a valine, often makes contact with the sidechain of the residue in position 383, most commonly through van der Waals interactions with a histidine or glutamine. In 7 of the 17 top solutions with the rotated ACP the sidechain of a conserved arginine at position 70’ in the loop between αII’ and αIII’ makes contact with residues in a 310 helix on KS (positions 385-387), especially the aspartate conserved in position 385.