From the Producer Microorganisms to the Lasso Scaffold - Lasso Peptides: Bacterial Strategies to Make and Maintain Bioactive Entangled Scaffolds - page 24

first analytical method that allowed characterizing the early known lasso peptides
(Wyss et al. 1991 ; Frechet et al. 1994 ; Constantine et al. 1995 ; Katahira et al. 1995 ;
Iwatsuki et al. 2006 ). Moreover, the NMR data used for this first step paved the way
for further structural analyses that are required for unambiguous demonstration of
the lasso topology.
The methods of choice to unambiguously characterize the lasso scaffold are
NMR, as reviewed by Xie and Marahiel ( 2012 ) and X-ray diffraction crystallog-
raphy (Nar et al. 2010 ). NMR, using conventional 2D NMR 1 H pulse sequences
(COSY, TOCSY, NOESY), accompanied with H/D exchange experiments to point
H-bonds stabilizing the structure, and sometimes with the help of 13 C techniques
(HSQC, HMBC) proved previously is particularly useful for deciphering the struc-
tures of complex, unusual and entangled peptide scaffolds, exemplified by the cy-
clic cystine knot peptides named cyclotides (Craik and Daly 2007 ). Similar to lasso
peptides, cyclotides contain a threaded ring. The particular stability of such entan-
gled topologies makes the structural elucidation independent of the solvent used for
acquiring NMR data. This is the case of lasso peptides, as exemplified by MccJ25,
which has the same lasso structure in water, methanol or dimethylsulfoxide. It is
not necessarily the case while comparing the 3D structures from NMR and X-ray
analyses, as described for BI-32169 (Nar et al. 2010 ) (see below).
Biochemical methods, such as enzyme and thermal stability studies, and MS
fragmentation under different ionization modes are valuable for assigning the inter-
locked topology. They are particularly useful when NMR or X-ray methods cannot
apply due to low amounts of peptides or absence of crystals. Sensitivity to carboxy-
peptidases, which are exoproteases that release amino acids from the C-terminus of
unfolded peptides or proteins, can be used to distinguish the lasso topology from the
macrolactam topological isomer, where the tail is not trapped in the ring (branched-
cyclic peptide). The C-terminal tail of lasso peptides is less accessible, and as such
is partially or completely resistant to carboxypeptidase cleavage depending on the
length of the segment below the ring. In contrast, branched-cyclic peptides are
prone to exoproteolysis, leaving only the macrolactam ring intact (Iwatsuki et al.
2006 ; Knappe et al. 2011 ; Ducasse et al. 2012b ; Hegemann et al. 2013a , b ; Zimmer-
mann et al. 2013 ). Therefore, the use of exoproteases (mainly carboxypeptidases Y
and P) is a useful and rapid tool to distinguish between the folded (lasso) and un-
folded (branched-cyclic) topologies. Thermal degradation studies constitute a com-
plementary approach for lasso topology identification. It was applied successfully
to MccJ25 and its variants, capistruin, caulosegnins, astexins and xanthomonins
(Blond et al. 2002 ; Knappe et al. 2008 ; Hegemann et al. 2014 ). Some lasso peptides
are particularly stable to high temperature. MccJ25 can retain its conformation up
solution. For each peptide, a representative structure showing the backbone and side chains (the
ring and tail regions are shown in orange and blue , respectively, and the bulky amino-acid side
chains involved in the stabilization of the lasso topology are colored in magenta ) together with a
surface representation (with basic, acidic and polar residues shown in blue , red and green , respec-
tively) are shown. We are grateful to Pr Mohamed A. Mohamed (Marburg University, Germany),
Dr Hiroaki Gouda (Showa University, Japan), Pr Sunghyouk Park and Dr Dong-Chan Oh (Seoul
National University, Korea) for providing the pdb files of the published structures