Investigation on PGIP sequence-function correlation
Although PvPGIP1 and PvPGIP2 are highly similar in amino acid sequence
(Figure 1c), they exhibit distinct activities against different PGs,
such as BcPG2 (Figure 2). Deciphering the sequence-function correlation
of PvPGIP1 and PvPGIP2 to PG recognition may provide insights for the
engineering of PvPGIP2 for enhanced activity or spectrum. One previous
docking-based study demonstrated the importance of the Val172Gly
(located in LRR5) and found that replacing PvPGIP2’s Val with PvPGIP1’s
Gly decreased inhibition activity against BcPG1 (Manfredini et al.,
2005; Sicilia et al., 2005). However, in our study, we found that that
PvPGIP1 and PvPGIP2 had similar levels of interaction with BcPG1, with
PvPGIP2 only displaying a slightly higher growth rate in the exponential
phase (Figure 2). This is likely because the system lacks sensitivity,
which could be due to some steric hinderance from the fused yeast
reporter proteins or some post-translational modifications of the PGIP.
There are ten different residues between the full length PvPGIP1 and
PvPGIP2, but most have previously been found to have little to no effect
on PG recognition, with the exception of those found within the ODA
(Sicilia et al., 2005). Three of these residues are located in the
truncated region of LRR5 to LRR8 (Figure 5a). Our finding that the
truncated forms possess a similar activity profile to their full-length
counterparts provides a unique opportunity to clearly elucidate the
sequence-function correlation of PG recognition (Figure S2). To
determine which residue(s) are essential for the inhibitory activity, we
constructed six tPvPGIP_5-8 mutants that shuffled the sequences between
tPvPGIP1_5-8 and tPvPGIP2_5-8. Each mutant contains a combination of
Val172Gly (LRR5), Ser198Ala (LRR6), or Gln244Lys (LRR8), named in that
order, with each mutation designated as 1 or 2 depending on if the amino
acid was from PvPGIP1 or PvPGIP2 respectively. The mutants are
tPvPGIP_5-8 “221” (Figure 5d), tPvPGIP_5-8 “212” (Figure 5e),
tPvPGIP_508 “211” (Figure 5f), tPvPGIP_5-8 “122” (Figure 5g),
tPvPGIP_5-8 “112” (Figure 5h), and tPvPGIP_5-8_”121” (Figure 5i).
The activities of the six tPvPGIP_5-8 mutants were examined using the
Y2H assay by mating PJ69-4A containing the different tPvPGIP_5-8 with
the same controls as previously described. The growth of the six diploid
yeast strains in -HTL medium were compared with the controls. When the
chimeric tPGIP_5-8 contained Val from PvPGIP2 instead of Gly from
PvPGIP1 at position 172, it retained PG interaction ability similar to
tPvPGIP2_5-8 regardless of mutations at other sites. If residue 244
contains Gln from PvPGIP2, a slight increase in FmPG activity is found
compared to pure PvPGIP1, indicating the importance of 244Gln towards
the recognition of FmPG. Residue 198 was not noted to have any impact on
PG-PGIP interaction (Figure 5). Furthermore, we conducted saturated
site-directed mutagenesis at residue 172 of tPvPGIP2_5-8 (Figure 6).
All 20 amino acids were compared using Y2H assay. While most mutants
showed decreased levels of interaction with the tested PGs compared to
the wild type tPvPGIP2_5-8, several mutants displayed a different
inhibition profile against different PGs.
tPvPGIP2_5-8V172M showed a similar level of
interaction with FmPG3 and BcPG2, a slightly higher interaction with
BcPG1, but decreased interactions with AnPG2, compared to the wild type
tPvPGIP2_5-8 (Figure 6). In addition,
tPvPGIP2_5-8V172L showed slightly higher interactions
than wild type tPvPGIP2_5-8 against BcPG1 (Figure 6b) but not the other
three PGs (Figure 6). However, no amino acid substitution showed a
statistically significant improvement in PG interactions compared to the
wild type tPvPGIP2_5-8, which indicates that natural evolution has
already selected for the optimal amino acids for interacting with the
tested PGs.