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Influence of Substitutions in the Binding Motif of Proline-Rich Antimicrobial Peptide ARV-1502 on 70S Ribosome Binding and Antimicrobial Activity

Proline-rich antimicrobial peptides (PrAMPs) are promising candidates to treat bacterial
infections. The designer peptide ARV-1502 exhibits strong antimicrobial effects against Enterobacteriaceae
both in vitro and in vivo. Since the inhibitory effects of ARV-1502 reported for the 70 kDa
heat-shock protein DnaK do not fully explain the antimicrobial activity of its 176 substituted analogs,
we further studied their effect on the bacterial 70S ribosome of Escherichia coli, a known target of
PrAMPs. ARV-1502 analogues, substituted in positions 3, 4, and 8 to 12 (underlined) of the binding
motif D3KPRPYLPRP12 with aspartic acid, lysine, serine, phenylalanine or leucine, were tested in a
competitive fluorescence polarization (FP) binding screening assay using 5(6)-carboxyfluoresceinlabeled
(Cf-) ARV-1502 and the 70S ribosome isolated from E. coli BW25113. While their effect on
ribosomal protein expression was studied for green fluorescent protein (GFP) in a cell-free expression
system (in vitro translation), the importance of known PrAMP transporters SbmA and MdtM was
investigated using E. coli BW25113 and the corresponding knockout mutants. The dissociation constant
(Kd) of 201 16 nmol/L obtained for Cf-ARV-1502 suggests strong binding to the E. coli 70S
ribosome. An inhibitory binding assay indicated that the binding site overlaps with those of other
PrAMPs including Onc112 and pyrrhocoricin as well as the non-peptidic antibiotics erythromycin
and chloramphenicol. All these drugs and drug candidates bind to the exit-tunnel of the 70S ribosome.
Substitutions of the C-terminal fragment of the binding motif YLPRP reduced binding. At the same
time, inhibition of GFP expression increased with net peptide charge. Interestingly, the MIC values of
wild-type and DsbmA and DmdtM knockout mutants indicated that substitutions in the ribosomal
binding motif altered also the bacterial uptake, which was generally improved by incorporation of
hydrophobic residues. In conclusion, most substituted ARV-1502 analogs bound weaker to the 70S
ribosome than ARV-1502 underlining the importance of the YLPRP binding motif. The weaker ribosomal
binding correlated well with decreased antimicrobial activity in vitro. Substituted ARV-1502
analogs with a higher level of hydrophobicity or positive net charge improved the ribosome binding,
inhibition of translation, and bacterial uptake.

Identiferoai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:89136
Date18 January 2024
CreatorsBrakel, Alexandra, Krizsan, Andor, Itzenga, Renke, Kraus, Carl N., Otvos Jr., Laszlo, Hoffmann, Ralf
PublisherMDPI
Source SetsHochschulschriftenserver (HSSS) der SLUB Dresden
LanguageEnglish
Detected LanguageEnglish
Typeinfo:eu-repo/semantics/publishedVersion, doc-type:article, info:eu-repo/semantics/article, doc-type:Text
Rightsinfo:eu-repo/semantics/openAccess
Relation3150

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