Biological Activities of Lasso Peptides and Structure–Activity Relationships - Lasso Peptides: Bacterial Strategies to Make and Maintain Bioactive Entangled Scaffolds - page 61

Fig. 3.7 Structure-activity relationship of MccJ25. Stick and surface representation of the three-
dimensional structure of MccJ25 (from Rosengren et al. 2003 ), showing the residues involved in
the FhuA-mediated uptake (in blue ), histidine involved in both FhuA and SbmA-mediated uptake
(in green ) and RNAP inhibition (in red )
topoisomer of MccJ25 (containing the macrolactam ring but without interlocked
topology) revealed no antibacterial activity (Ducasse et al. 2012b ). This illustrates
that the lasso scaffold is a prerequisite for the activity. The lasso fold is maintained
thanks to optimized size of the ring and stabilization of the tail within the ring by
bulky amino acids and disulfide bridges (see Chap. 2). MccJ25 cleaved by ther-
molysin in the loop region (Rosengren et al. 2004 ) did not bind FhuA and revealed
much less activity than the native peptide, but showed unaltered propensity to inhib-
it RNAP (Destoumieux-Garzón et al. 2005 ; Semenova et al. 2005 ). Therefore, the
loop region was identified as the key region for FhuA binding, and distinct regions
were proposed to be involved in peptide uptake and RNAP inhibition. The amida-
tion of the C-terminus of MccJ25 reduced importantly the antibacterial activity and
RNAP inhibition (Bellomio et al. 2003 ; Vincent et al. 2005 ), showing that this part
of MccJ25 is a key element for RNAP binding.
In 2008, a systematic structure-activity relationship study of MccJ25 has been
performed by Pavlova et al. (Pavlova et al. 2008 ). Three hundred and eighty one sin-
gly substituted variants generated by saturation mutagenesis permitted delineating
the positions critical for the biosynthesis and antibacterial properties of MccJ25. Of
the 242 variants successfully biosynthesized and exported, 155 were competent for
RNAP inhibition in vitro, 70 of which revealed antibacterial activity. This permitted
to decipher the residues involved in MccJ25 uptake and RNAP inhibition activity,
respectively. Residues Tyr9 (located upstream the macrolactam ring), Gly4 and Pro
7 (within the ring) and Phe19 and Tyr 20 (plug residues straddling the ring) revealed
particularly important for RNAP inhibition. These residues form a continuous sur-
face on one face on the three-dimensional structure of MccJ25, suggesting that they
constitute the RNAP binding site (Fig. 3.7 ). Multiple-site mutagenesis in the loop
region permitted to obtain variants with enhanced antibacterial activity (such as
MccJ25 [Gly12His, Ile13Phe, Thr15Ile]; Pan and Link 2011 ). In the latter study, an
elegant strategy permitted to screen the active/inactive character of MccJ25 variants.