Clindamycin binding to ribosomes revisited: foot printing and computational detection of two binding sites within the peptidyl transferase center.
Clindamycin is a semi-synthetic lincosamide, active against most Gram-positive bacteria and some protozoa. It binds to the 50S ribosomal subunit and inhibits early peptide chain elongation. By kinetic analysis it has been shown that clindamycin (I) competitively interacts with the A-site of translating ribosomes (C) to form the encounter complex CI, which then slowly isomerizes to a tighter complex, termed C*I. As the final complex is capable of synthesizing peptide bonds with decreased velocity, it was assumed that in C*I complex the drug is fixed near the P-site of the ribosome. In the present study, two series of chemical foot printing experiments were carried out. In the first series, clindamycin and ribosomal complex C were incubated for 1 s and then DMS or kethoxal was added (CI probing). In the second series, complex C was preincubated with clindamycin for 1 min before the addition of DMS or kethoxal (C*I probing). It was found that clindamycin in CI complex protects A2451 and A2602 from chemical probing, both located within the A-site of the catalytic center. In contrast, it strongly protects G2505 in C*I complex, which is a discrete foot print of peptidyl-tRNA bound to the P-site. In both CI and C*I complexes, clindamycin also protects nucleotides A2058 and A2059, located next to the entrance of the exit-tunnel where the nascent peptide leaves the ribosome. Polyamines negatively affect the protection of G2505, but favor the protection of A2451 and A2602 nucleotides. Structure modeling confirms the kinetic and chemical foot printing results and suggests that clindamycin mode of action is more complex than a simple competitive inhibition of peptide bond formation.
Department of Biochemistry, School of Medicine, University of Patras, Greece.
O N
ON
Kostopoulou
G
G
Papadopoulos
E C
EC
Kouvela
D L
DL
Kalpaxis
D000465:Algorithms; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001665:Binding Sites; D002981:Clindamycin / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D007202:Indicators and Reagents; D007700:Kinetics; D008958:Models, Molecular; D010458:Peptidyl Transferases / Q000378:metabolism*; D012338:RNA, Ribosomal, 23S / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D007202:Indicators and Reagents; D012338:RNA, Ribosomal, 23S; D002981:Clindamycin; D010458:Peptidyl Transferases