Here we have demonstrated that the highly constrained lasso topology of the antimicrobial peptide MccJ25 can be computationally redesigned. Two levels of experimental verification were performed. First, we tested whether the redesigned variants were produced and correctly exported using HPLC analysis of the culture supernatants. This test also implicitly interrogates whether the peptide is correctly cyclized into its lasso conformation since uncyclized peptide likely would not be exported by McjD. Secondly, we tested the variants for retention of antimicrobial activity against the MccJ25-sensitive strain Salmonella newport
. This test of the MccJ25 function is multifaceted; successful killing of this bacterium requires recognition and internalization of the antibiotic by the outer membrane protein FhuA (Salomon and Farias, 1993
), transport of the MccJ25 variant across the cytoplasmic membrane by SbmA (de Cristobal et al., 2006
) and recognition of the peptide by RNAP. It should be noted that an alternative mode of action for MccJ25 has been proposed in which the antibiotic causes an increase in the reactive oxygen species production via disruption of the respiratory chain (Bellomio et al., 2007
). It is possible that our variants kill S.newport
by this mode of action rather than by the canonical killing mechanism of RNAP inhibition. Six of the eight redesigned MccJ25 variants tested in this work were successfully produced and exported by E.coli
, while three of these six were able to kill S.newport
, albeit not as efficaciously as the wild-type peptide.
The finding that the mut1–mut3 and mut5–mut7 peptides were produced demonstrates that the computational redesign algorithm can be successful even on a very stable, highly constrained structure like the lasso architecture. Moreover, this result provides additional information regarding the question of the extent of amino acid sequence space that the lasso fold can occupy. Though the structure of MccJ25 is quite rigid, it can accommodate multiple amino acid substitutions. Of the six variants that are produced, five contain three amino acid substitutions while the sixth has two substitutions. In the case of the triple variants, one in every seven amino acids in the peptide has been changed while still retaining the ability to fold into the lasso conformation. Some of the recurring amino acid changes in the variants that are produced and exported are quite non-conservative, such as the A3Q substitution that occurs in mut3 and mut5–mut7. The Val-6 position is replaced by either tryptophan or tyrosine, both much larger amino acids, in all of the exported variants. In general, our design variants have amino acid changes that result in increases in sidechain volume (Table ). Future redesign efforts of this peptide could focus on the amino acids A3 and V6, both of which are frequently changed in our designed peptides. These results build on previous observations (Pavlova et al., 2008
; Knappe et al., 2009
) that the sequence space that can be occupied by lasso peptides may actually be quite large and diverse. Fig. shows a sequence alignment of the four structurally confirmed lasso peptides and the six exported variants from this study. The fact that the aa substitutions we observe in our successful redesigns are largely not present in known lasso peptides (with the exception of A3Q and V6Y, which are found in lariatin) affirms the use of the redesign technique in probing the set of sequences that can adopt the lasso architecture.
Fig. 6. Sequence alignment of known class II lasso peptides and redesigned MccJ25 variants successfully produced by E.coli in this study. The requisite N-terminal glycine and acidic residue are highlighted in dark gray. Other residues that occur in the variants (more ...)
The computational redesign algorithm functions solely at the structural level to determine a sequence that will fit a given structure or set of structures. In other words, the algorithm does not directly take into account the function of the peptide or protein being designed. Thus, it is fairly remarkable that three of the eight redesigned MccJ25 variants retain some antimicrobial activity. As mentioned above, Pavlova et al
. published a study in which a near-complete set of single amino acid variants of MccJ25 was tested sequentially for production and export, inhibition of RNAP, and for antimicrobial activity against E.coli
or Shigella flexneri
(Pavlova et al., 2008
). Most of the amino acid substitutions we find in our computational redesign of MccJ25 are tolerated substitutions according to this study. One possible exception is the V6Y substitution we observe in the mut5 peptide, which is both exported and has antimicrobial activity. Pavlova et al.
report that V6Y single variant is competent in export, but falls below the threshold (20% of wild-type activity) of inhibiting RNAP activity (Pavlova et al., 2008
). Since variants that fell below this threshold were not tested for antimicrobial activity, it is possible that the V6Y single variant has weak antimicrobial activity on par with the weak activity we observed for mut5. It is also possible that mut5 is an antibiotic that functions via inhibition of respiration. An alternative explanation is that the V6Y substitution does cripple MccJ25, but one of the other aa substitutions in mut5 (A3Q and T15Q) plays a compensatory role and restores some function to the variant. At the opposite end of the spectrum, we also found a variant, mut4, that includes three ‘tolerated’ aa substitutions (A3Q, V6W and V11L) according to Pavlova et al.
but is not even exported.
Lasso peptides represent an intriguing architecture for future drug design (Everts, 2010
), and the stability and protease resistance (Rebuffat et al., 2004
) of these peptides eliminate some major limitations to using peptides as drugs. The first step toward the goal of using these molecules as drugs is an understanding of which sequences can be tolerated in a lasso fold. We demonstrate here that computational redesign is a valuable technique toward this end. Our group is also complementing these computational approaches with purely experimental approaches based on high-throughput screening of random libraries of MccJ25 for antimicrobial activity to determine more sequences that can exist in the lasso fold.