From the Producer Microorganisms to the Lasso Scaffold - Lasso Peptides: Bacterial Strategies to Make and Maintain Bioactive Entangled Scaffolds - page 27

2.3.4 Stability of the Lasso Scaffold and Modes of Stabilization

Similar to other knotted topologies such as cyclotides (Craik and Malik 2013 ),
lasso peptides are generally described as remarkably stable, being resistant to deg-
radation by most proteases, high temperatures, acidic conditions and to denaturing
agents. As an example, the lasso topology of MccJ25 cannot be destroyed in any
of these conditions, but in strong basic medium (1M NaOH), which leads to open-
ing of the macrolactam ring and thus releasing the tail (Wilson et al. 2003 ). Their
structural characteristics associated to their biological activities have thus attracted
much interest. Characterization of an increasing number of lasso peptides as well
as well-designed variants, is now leading to a better understanding of the modes of
stabilization of lasso peptides. As described above (Sect. 2.3.1), the lasso scaffold
that encompasses a peptide C-terminal tail threaded through and locked inside a
N-terminal macrolactam ring is an extraordinary topology, in that it is difficult to
be acquired and to be maintained. On one hand, threading cannot occur after ring
closure, as it would be prevented by the bulk of certain amino acid side-chains that
would not be able to cope with the ring diameter. On the other hand, keeping the
tail locked into the ring after it is threaded is entropically disfavoured; as a conse-
quence, the threaded structure cannot be preserved without the help of braces or/and
plugs. The lasso topology therefore has two requirements: (i) the right shape of the
peptide chain has to be acquired before ring closure by the lactam bond at a correct
position that permits the tail being blocked within the ring; this can be ensured only
by the bacterial enzymes (detailed in Chap. 3) and makes the chemical synthesis of
lasso peptides a real challenge; (ii) the tail has to be locked into the ring to avoid
unthreading.
In addition to hydrogen bonds and van der Waals interactions between hydropho-
bic amino acids that are generally predominant in lasso peptides, the stabilization
of the lasso topology is mainly ensured by sterically demanding side-chains that act
as locks and plugs or/and by disulfide bonds that reinforce the already constrained
structure by adding braces (see Sect. 2.3.2). Mostly type Il lasso peptides have been
described before 2000. At that time knowledge on lasso peptides was very limited
and all the questions afforded by the lasso topology stability were not investigated.
In order to identify braces and plugs sites, one needs precise knowledge of the 3D
structures, as well as both facile systems to conceive and produce variants by mu-
tagenesis and reliable methods such as combined thermal and enzymatic degrada-
tion studies. Such studies have been performed on MccJ25 (Ducasse et al. 2012b ),
capistruin (Knappe et al. 2008 ), and lately identified type II lasso peptides includ-
ing caulosegnins (Hegemann et al. 2014 ), astexins (Zimmermann et al. 2013 ) and
xanthomonins(Hegemann et al. 2013b ). These studies make it possible to rational-
ize some general requirements for the stability of the lasso topology.
Type I lasso peptides contain a macrolactam ring closed between Cys1 and the
Asp9 side-chain carboxylate, making a 25-atom ring (Table 2.5 ). The type I lasso
peptide topology is described as essentially stabilized by two disulfide bonds in-
volving four cysteines located at conserved positions 1, 7, 13, 19, with the cystine