Interdomain insertions
Repetitive sequences in the loops between domains occur with the greatest frequency at the updated module boundary, where KS connects with the flanking subdomain (FSD, also referred to as the KS-AT adapter) (Figure 3, Table I)[20, 23]. This loop tolerates significant increases in length, as in NcyB_1c where it is 137 residues. Repetitive sequences occur least frequently in the loops upstream and downstream of DH and in the loop connecting ER and KRc. Unless DH η1 is absent, the loop connecting the end of the AT region (the LPTYxFx5W motif) to DH is strictly 5-7 residues. The composition and length of the loop connecting DH to KRsare not significantly altered through the two observed insertions (SpiE_2c and TcaA4_1c). The ER-KRc loop is usually 2-3 residues long, as observed in the crystal structure of KRs-ER-KRc from the third module of the spinosyn PKS (PDB 3SLK; although it is significantly longer in 3% of δ-modules, including StiF_1d where it is 16 residues)[21].
To determine the length of flexible interdomain loops, their interfaces with structured elements were identified. Absences of optional features such as DH η1, DH α4 (the terminal helix observed in a cremimycin DH, PDB 6K97), and ACP α1 were considered [24-26]. Which residues are unstructured in the loop between KR and ACP in β-modules is apparent; however, which residues are unstructured in the loop between these domains in γ- and δ-modules is less clear. Since 2 consecutive arginines are conserved 4 residues downstream of what was structurally-observed in KRs-ER-KRc from the third module of the spinosyn PKS (PDB 3SLK), this region was considered to be structured [21].
The loops upstream and downstream of ACP are of particular interest in determining how much translational and rotational freedom an ACP domain possesses to access each of its cognate enzymes. Thus, the lengths of the loops upstream and downstream of ACP were analyzed in β-, γ-, and δ-modules not containing DDs. In β-modules, their lengths are 11 ± 4 and 14 ± 4 residues (median ± standard deviation), respectively; in γ-modules, 18 ± 3 and 13 ± 8 residues; and in δ-modules, 8 ± 1 and 14 ± 2 residues. Insertions, some of which likely resulted from slipped-strand mispairing, infrequently do increase the length of these loops; the longest observed upstream and downstream ACP linkers are, respectively, 26 and 33 residues (SlgA2_1b and BafA4_1b) in β-modules, 25 and 50 residues (Pik1_3c and PldA2_2c) in γ-modules, and 30 and 25 residues (FosA_2d and RapC_3d) in δ-modules. For comparison, the average length of linkers in the related trans -AT assembly lines, in which ACPs access separately-encoded trans -ATs, is 50 residues[2]. Instead of a flexible loop between ACP and KS, FscC_5c contains a fused Class 1b docking domain[7].