Solutions shows a KS motif in two conformations at the interface
In half of the 50 top solutions, the KS TxLGDP motif (residues 313-318) adopts a type I β-turn distinct from the α-helical conformation observed in solved structures and the other solutions (Figure 4).7, 8, 29-32 In 17 of these 25 solutions (Data Files 6, 8, 11, 12, 16, 18, 22, 24, 25, 27, 36, 37, 43, 44, 45, 46, 50), the β-turn conformation is associated with a relative rotation of ACP about αIII’ such that L315 of the TxLGDP motif makes contact with the serine that becomes phosphopantetheinylated, S53’ (L315 Cγ - S53’ Cβ < 5.0 Å). To investigate whether formation of the β-turn is associated with ACP binding, the sequences of the KS’s from the 25 top solutions with the β-conformation were provided to AlphaFold-Multimer. None of the predictions from the predicted KS dimers contain the TxLGDP motif in the β-conformation.
For 21 of the ACP+KS’s, the 5 predictions made by AlphaFold include both a solution in which the TxLGDP motif is in the α-conformation and a solution in which the motif is in the β-conformation and ACP is relatively rotated (Data Files 2, 6, 11, 12, 13, 14, 16, 18, 22, 27, 31, 32, 33, 36, 39, 43, 44, 45, 48, 50, 51, 58-78). Thus, the differences between the conformations of this motif as well as the relative orientations of ACP and KS can be directly compared (Figures 3-4). In the β-conformation of the TxLGDP motif of PikKS6, a hydrogen bond is present between the carbonyl of T313 and the NH of G316, rather than the NH of D317, as in the α-conformation. The D317 carboxylate associates with the NH’s of T313 and an invariant glycine (G312) immediately upstream of the motif as well as the T313 hydroxyl. The oxygen of the T313 hydroxyl and the D317 NH form another hydrogen bond. P318 adopts the γ-endo conformation rather than the γ-exo conformation in the α-conformation. The β-conformation is stabilized through van der Waals interactions with a structured loop (A232-G234) as well as a hydrogen bond between the L315 carbonyl and the hydroxyl group of a serine or threonine at position 381. The conformational shift is largest for L315 and G316, their Cα’s shifting 4.7 Å and 7.0 Å, respectively.
The conformational change could be important in allowing the approach of the KS reactive cysteine and the thioester of the acylated phosphopantetheinyl arm. In the 17 top solutions containing the β-conformation and the rotated ACP, the arm can move closer to the KS reactive cysteine. While the average distance between the ACP serine oxygen and the KS cysteine sulfur is 20.9 Å for the solutions containing the α-conformation (excluding AjuMod3, vide infra ), it is 17.2 Å (3.7 Å closer, on average) for these 17 solutions (Table S2). Since the distance between the side chain oxygen of the phosphopantetheinylated serine and the carbon of the thioester carbonyl of an acyl-ACP is maximally 19.2 Å, the β-conformation may be more relevant to the transacylation reaction. An acetyl-phosphopantetheinyl moiety modeled with the program Coot33 into a PikMod6 solution with the β-conformation shows that its gem -dimethyl moiety can hydrophobically interact with P280, that its distal carbonyl can coordinate with the H309 NεH and the T311 hydroxyl group, and that its thioester carbonyl can insert into the oxyanion hole formed by the NH’s of C174 and V217 (Figure 3c) (Data File 79). The conformational change of the TxLGDP motif provides space for the phosphopantetheinyl arm and allows the approach of the thioester and the reactive cysteine to enable the transacylation reaction.