DISCUSSION
Among the many docking events within a polyketide assembly line, those
between KS’s and their upstream ACP’s are of high significance because
they enable KS’s to gatekeep for properly processed intermediates and
thus ensure the fidelity of the synthase18 .
Although modern structural biology techniques, such as cryo-EM, have
advanced our knowledge of the interactions between PKS domains, they are
still resource-intensive and low throughput.7,
8 In contrast, modern in silico methods require few resources
and are high throughput. Thus, we sought to identify the transacylation
site using the most advanced folding/docking algorithms.
Alphafold-Multimer uses restraints derived from covariation analysis,
both within and between domains, to guide
folding/docking.15 In half of the 50 top
solutions for the ACP+KS dimers, a structural feature is present at the
interface that has not been previously observed - the TxLGDP motif of KS
forms a type I β-turn rather than the α-helical turn present in reported
structures and the other 25 top solutions. The β-conformation is
correlated with a closer approach between the ACP and KS domains. The
distance between the side-chain oxygen of the conserved ACP serine and
the reactive KS cysteine is greater than 18.4 Å in 24 of the 25 top
solutions with the α-conformation (20.8 Å, on average). The exception is
AjuMod3 (15.8 Å), a module containing non-ribosomal peptide synthetase
(NRPS) domains,36 in which an unusual
phenylalanine in position 47’ makes contact with an unusual valine at
position 275. The 17 top solutions that possess the β-conformation and a
relatively rotated ACP possess distances between the
phosphopantetheinylated serine and the reactive cysteine short enough
for polyketides to transfer from the acyl-ACP to the KS (17.2 Å, on
average).
The TxLGDP motif has not been experimentally observed in the
β-conformation. However, these studies suggest that acyl chains are not
readily transferred between ACP and KS when this motif is in the
α-conformation. This raises the possibility that favorable interactions
between the polyketide intermediate and the KS substrate tunnel
facilitate the transition from the α-conformation to the β-conformation,
which enables a closer association of the acyl-ACP and KS, productive
interactions between KS and the phosphopantetheinyl arm (such as the
coordination of H309 NεH and T311 OH with the distal
carbonyl), and polyketide transfer through the approach of the thioester
and the reactive cysteine. This may be part of the KS gatekeeping
mechanism by which properly processed intermediates are selected for
transfer to the reactive cysteine.18
During the reaction cycle of a module, ACP associates with the upstream
AT and processing domains as well as the downstream KS (Figure 1). While
it must be complementary to each of these cognate enzymes, how well
ACP’s dock with noncognate domains introduced through natural
recombination events or genetic engineering is unknown. As the domains
of one module are able to evolutionarily co-migrate separately from the
domains of other modules, their interfaces could diverge. If this is the
case, engineering efforts should focus on module-swapping rather than
domain-swapping. In a recent KS gatekeeping study, AmpKS15 was swapped
for PikKS3 in PikMod3 ofP1 -P2 -P3 -P7 , a tetraketide synthase
composed of PikMod1, PikMod2, PikMod3, and
PikMod7.37 A triketide rather than the expected
tetraketide was produced by the resulting synthase, indicating that the
hybrid third module was completely skipped. An analysis of natural
ACP/KS interfaces of AmpMod15 and PikMod3 reveals large differences in
how their ACP’s interact with the KS surface residue in position 275
(asparagine in AmpKS15 and serine in PikKS3).
The docking solutions presented here show interfaces between KS’s and
their upstream ACP’s that are quite distinct from one another (Figure 5,
Table S2). The most dramatic example is the interface between AjuACP3
and AjuKS3, in which a valine occupies position 275 of AjuKS3 and
AjuACP3 is rotated 17º relative to the consensus orientation. As
mentioned above, KS’s containing a serine at position 275, such as
PikKS3 and VstKS7, may not be recognized by most ACP’s since they
associate with KS’s containing an asparagine at this position.
The transacylation site may be under selective pressure to diversify,
whereas the extension site may be under selective pressure not to. The
ordered assembly of PKS polypeptides could be jeopardized if the
interfaces between KS’s and their upstream ACP’s were too similar. CDD’
and NDD’s do not make the only contact between PKS polypeptides, since
ACP’s and KS’s also associate, at least while mediating the
transacylation reaction and likely even when ACP is
unacylated.27 The surface area for the
transacylation site is larger than that observed for CDD/NDD interfaces
(1338 Å2 for PikACP6 with PikKS6 vs. 1043
Å2 for one DEBS2 CDD helix with the dimeric DEBS3
NDD).26 If all the KS’s and ACP’s at the
polypeptide termini equally docked with one another, the synthase could
assemble incorrectly. In contrast, the evolution of polyketide assembly
lines may benefit from the interface between KS’s and downstream ACP’s
not diverging, such that a module still functions when moved to a new
location in the same or a different synthase.7,
8 In the new location, the ACP and KS domains must respectively
collaborate with a new upstream KS and downstream ACP to catalyze
extension reactions.
The proposed transacylation site is consistent with the observed losses
in activity from the 20 P1 -P6 -P7 point
mutants (Figure 5b). That mutations to the conserved KS surface residues
N275 and L315 resulted in the least active synthases agrees with the
interactions between PikACP6 and PikKS6 proposed to enable the
transacylation reaction. The AlphaFold-Multimer predictions show the
sidechain of N275 making substantial contact with PikACP6, through Van
der Waals contact with P80’ and hydrogen bond contact with both the
C-terminal end of αIII’ and the N-terminal end of the one-turn helix
upstream of αII’. The AlphaFold-Multimer predictions also show the
invariant leucine in the TxLGDP motif at position 315 making significant
hydrophobic contact with F77’ of PikACP6 when the motif is in the
α-conformation and with a hydrophobic pocket on αII’ next to the
phophospantheinylated serine when the motif is in the β-conformation.
Each of the other mutations also resulted in losses of activity compared
to unmutated P1 -P6 -P7 , usually moreso when
located at or near the putative interface (e.g. , I319A, H281A,
Q286A, D382A, D385A, Q330A). The relatively small decreases in activity
observed for the R314A and Q322A point mutants might be surprising given
the proximity of these residues to the interface; however, in the
Alphafold-Multimer predictions the arginine and the glutamine sidechains
make minimal Van der Waals contact with PikACP6. The low activity of the
T140A point mutant control was expected from the importance of this
residue at the PikKS6 extension site.7, 8
AlphaFold has apparently helped discover the transacylation site on the
surface of KS and provided 50 examples of how upstream ACP’s dock at
that interface. Such a feat through experimental structural biology
techniques would require many more resources and much more time.
AlphaFold will likely help predict other domain-domain interactions
within PKS’s, although these should be experimentally tested within
functional assembly lines. Our burgeoning understanding of how ACP
domains dock with cognate enzymes is illuminating the dynamics and
architectures of PKS’s and will help realize the potential of these
assembly lines in producing designer molecules and medicines.