Selection of peptides that target the aminoacyl-tRNA site of bacterial 16S ribosomal RNA.
For almost five decades, antibiotics have been used successfully to control infectious diseases caused by bacterial pathogens. More recently, however, two-thirds of bacterial pathogens exhibit resistance and are continually evolving new resistance mechanisms against almost every clinically used antibiotic. Novel efforts are required for the development of new drugs or drug leads to combat these infectious diseases. A number of antibiotics target the bacterial aminoacyl-tRNA site (A site) of 16S rRNA (rRNA). Mutations in the A-site region are known to cause antibiotic resistance. In this study, a bacterial (Escherichia coli) A-site rRNA model was chosen as a target to screen for peptide binders. Two heptapeptides, HPVHHYQ and LPLTPLP, were selected through M13 phage display. Both peptides display selective binding to the A-site 16S rRNA with on-bead fluorescence assays. Dissociation constants (Kd's) of the amidated peptide HPVHHYQ-NH2 to various A-site RNA constructs were determined by using enzymatic footprinting, electrospray ionization mass spectrometry (ESI-MS), and isothermal titration calorimetry (ITC) under a variety of buffer and solution conditions. HPVHHYQ-NH2 exhibits moderate affinity for the A-site RNA, with an average Kd value of 16 microM. In addition, enzymatic footprinting assays and competition ESI-MS with a known A-site binder (paromomycin) revealed that peptide binding occurs near the asymmetric bulge at positions U1495 and G1494 and leads to increased exposure of residues A1492 and A1493.
Li
Mei
M
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.
Duc
Anne-Cécile E
AC
Klosi
Edvin
E
Pattabiraman
Srividya
S
Spaller
Mark R
MR
Chow
Christine S
CS
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D004352:Drug Resistance, Microbial / Q000235:genetics*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010303:Paromomycin / Q000737:chemistry / Q000494:pharmacology*; D010455:Peptides / Q000008:administration & dosage* / Q000737:chemistry; D011485:Protein Binding; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S / Q000737:chemistry*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000900:Anti-Bacterial Agents; D010455:Peptides; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012346:RNA, Transfer, Amino Acyl; D010303:Paromomycin