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firming the plug role of Arg15. The single Arg15Ala substitution had no effects on
the lasso topology, suggesting in this case that the two Phe residues replace Arg15
to fulfil the plug function. Interestingly, a double-substituted Arg15Ala/Phe16Ala
variant was found to be temperature-sensitive. It was hypothesized that the exten-
sion of the β-turn could assist in the unthreading of the tail containing a bulky
Phe18 residue (Knappe et al.
2009
). In addition, the plugs and locks of other lasso
peptides recently discovered were identified by rational mutagenesis followed by
production and structural analysis of the variants. Glutamine or tyrosine at position
16 in caulosegnins I, II, III (Hegemann et al.
2013a
), Phe15 and Tyr16 in astexins 1
and 3 respectively (Zimmermann et al.
2013
) were unambiguously assigned as the
plugs below the ring. Tyrosine and arginine residues were shown to play as the up-
per locks in caulosegnins and astexins respectively. Worth of note, the lower plug in
xanthomonin II, which has a 7-member macrolactam ring was demonstrated to be
Ile12, and substitution of this residue to any amino acid larger than Ser could main-
tain a thermally stable lasso topology (Hegemann et al.
2014
). This observation
indicates clearly that the nature of the plug residue is, at least in part, determined by
the size of the macrolactam ring.
With a length of nine amino acids, the size of the threaded tail below the ring of
astexin 1(23) is at present the longest identified so far (Zimmermann et al.
2013
).
However, shortening of the tail by one to eight residues (1 in MccJ25, 3 in capist-
ruin, 1-3 in caulosegnin I, 1-8 in astexin 1), or lengthening of the tail (1 residue
in MccJ25 or capistruin) appears tolerable for the lasso fold (Knappe et al.
2009
;
Ducasse et al.
2012b
; Hegemann et al.
2013a
; Zimmermann et al.
2013
). Concern-
ing the antibacterial activity exhibited by MccJ25, the lasso topology and the nature
of the C-terminal residue (changing Gly21 to hydrophobic, or negatively charged
or positively charged amino acids) were found as essential (Ducasse et al.
2012b
)
(see Chap. 2).
The size of the macrolactam ring is critical as well. The number of amino acids
involved in the ring ranges between 7 and 9 and the side-chain carboxylate can
come from either Glu or Asp. The diameters of the ring of MccJ25 that is made of
8 amino acids closed by Glu8 (26 atoms) and that of capistruin, which has 9 amino
acids closed by Asp9 (25 atoms) are approximately the same (about 9 Å diameter
in its larger dimension). This is in agreement with the fact that bulkiness of the
side-chains that maintain the tail in the ring are quite similar (i.e. in both cases, Phe
residue can function as a plug residue). The smallest ring of lasso peptides is found
in recently discovered xanthomonins (Hegemann et al.
2014
): It contains 7 residues
and is closed by Glu at position 7 (23 atoms). Given the structural constraints of
[2]rotaxanes that share some basic structural similarities with lasso peptides, it is
likely that 7-residue is the lowest limit of the lasso macrolactam ring. The smallest
macrocycle known of [2]rotaxanes is composed of 20 atoms (Dasgupta et al.
2012
),
suggesting in the case of lasso peptides, a 7-member ring closed by Asp (22 atoms)
could still be possible. Nevertheless, a single Glu7Asp substitution in xanthomonin
II abolished completely the peptide production (Hegemann et al.
2014
).
A total of 25 variants of xanthomonin II were generated to probe different as-
pects and in particular the maintenance of the lasso. The amino acids acting as plugs
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