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Investigating the entire course of telithromycin binding to Escherichia coli ribosomes.

Kostopoulou ON, Petropoulos AD, Dinos GP, Choli-Papadopoulou T, Kalpaxis DL - Nucleic Acids Res. (2012)

Bottom Line: In contrast, mutation Lys63Glu in protein L4 placed on the opposite side of the tunnel, exerts only a minor effect on telithromycin binding.Polyamines disfavor both sequential steps of binding.Our data correlate well with recent crystallographic data and rationalize the changes in the accessibility of ribosomes to telithromycin in response to ribosomal mutations and ionic changes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Medicine, University of Patras, 26504 Patras, Greece.

ABSTRACT
Applying kinetics and footprinting analysis, we show that telithromycin, a ketolide antibiotic, binds to Escherichia coli ribosomes in a two-step process. During the first, rapidly equilibrated step, telithromycin binds to a low-affinity site (K(T) = 500 nM), in which the lactone ring is positioned at the upper portion of the peptide exit tunnel, while the alkyl-aryl side chain of the drug inserts a groove formed by nucleotides A789 and U790 of 23S rRNA. During the second step, telithromycin shifts slowly to a high-affinity site (K(T)* = 8.33 nM), in which the lactone ring remains essentially at the same position, while the side chain interacts with the base pair U2609:A752 and the extended loop of protein L22. Consistently, mutations perturbing either the base pair U2609:A752 or the L22-loop hinder shifting of telithromycin to the final position, without affecting the initial step of binding. In contrast, mutation Lys63Glu in protein L4 placed on the opposite side of the tunnel, exerts only a minor effect on telithromycin binding. Polyamines disfavor both sequential steps of binding. Our data correlate well with recent crystallographic data and rationalize the changes in the accessibility of ribosomes to telithromycin in response to ribosomal mutations and ionic changes.

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Protections in nucleotides of 23S rRNA from chemical probes, caused by binding of telithromycin to complex C prepared from E. coli ribosomes. (A) protections in domain II of 23S rRNA; complex C was incubated in the absence or presence of telithromycin in buffer B (lanes 1–4) or in the same solution also containing 100 μΜ spermine (lanes 5–8). The resulting complexes were then probed with DMS or CMCT. U, A, G and C, dideoxy sequencing lanes; lane 2 and 6, complex C probed in the absence of telithromycin; lanes 3 and 7, complex C pre-incubated with telithromycin for 5 s and then probed; lanes 4 and 8, complex C pre-incubated with telithromycin for 15 min and then probed. (B) protection in the central loop of domain V of 23 S rRNA; incubation of complex C with telithromycin was carried out as in panel A. Samples were then modified with DMS, kethoxal, or CMCT, and analyzed as in panel A. Numbering of nucleotides for the sequencing lanes is indicated at the left of each panel, while nucleotides with reactivity to probes along with reference bands are shown by arrows. Teli, telithromycin.
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gks174-F4: Protections in nucleotides of 23S rRNA from chemical probes, caused by binding of telithromycin to complex C prepared from E. coli ribosomes. (A) protections in domain II of 23S rRNA; complex C was incubated in the absence or presence of telithromycin in buffer B (lanes 1–4) or in the same solution also containing 100 μΜ spermine (lanes 5–8). The resulting complexes were then probed with DMS or CMCT. U, A, G and C, dideoxy sequencing lanes; lane 2 and 6, complex C probed in the absence of telithromycin; lanes 3 and 7, complex C pre-incubated with telithromycin for 5 s and then probed; lanes 4 and 8, complex C pre-incubated with telithromycin for 15 min and then probed. (B) protection in the central loop of domain V of 23 S rRNA; incubation of complex C with telithromycin was carried out as in panel A. Samples were then modified with DMS, kethoxal, or CMCT, and analyzed as in panel A. Numbering of nucleotides for the sequencing lanes is indicated at the left of each panel, while nucleotides with reactivity to probes along with reference bands are shown by arrows. Teli, telithromycin.

Mentions: The first step of telithromycin binding to complex C is presented by a bimolecular reaction. Therefore, to footprint the CT complex, telithromycin at concentration 50 × KT and complex C at 100 nM were incubated at 25°C for 5 s. Since the equilibrium C + T ⇌ CT is established instantaneously while the subsequent isomerization step proceeds slowly, the species mainly produced during this time interval is complex CT (>97%). By longer exposure of complex C to telithromycin (8 × t1/2), the high value, 58.93, for the isomerization constant of the second step favors the formation of C*T complex (>98%). Next to their formation, complexes CT and C*T were probed with DMS, CMCT or kethoxal. Noteworthy, the chemical probes used react with accessible bases within milliseconds (38). Representative autoradiograms obtained by primer extension analysis in helix 35 of domain II and the central loop of domain V of 23S rRNA are shown in Figure 4, while relative reactivities of the modified nucleotides are summarized in Table 3. To assess the footprinting changes relative to the rest of neighboring 23S rRNA areas, larger regions of the scanned sequences are given in Supplementary Figures S1 and S2. Telithromycin in the CT binding state and in the absence of polyamines strongly protects A2058, A2059 and to a lesser degree G2505, C2611, A752, A789 and U790. In contrast, it causes an enhancement in the susceptibility of A2062 to DMS. In the C*T binding state, the protection effects at A789 and U790 soften, the protection at A752 enhances, while a new protection appears on U2609.Figure 4.


Investigating the entire course of telithromycin binding to Escherichia coli ribosomes.

Kostopoulou ON, Petropoulos AD, Dinos GP, Choli-Papadopoulou T, Kalpaxis DL - Nucleic Acids Res. (2012)

Protections in nucleotides of 23S rRNA from chemical probes, caused by binding of telithromycin to complex C prepared from E. coli ribosomes. (A) protections in domain II of 23S rRNA; complex C was incubated in the absence or presence of telithromycin in buffer B (lanes 1–4) or in the same solution also containing 100 μΜ spermine (lanes 5–8). The resulting complexes were then probed with DMS or CMCT. U, A, G and C, dideoxy sequencing lanes; lane 2 and 6, complex C probed in the absence of telithromycin; lanes 3 and 7, complex C pre-incubated with telithromycin for 5 s and then probed; lanes 4 and 8, complex C pre-incubated with telithromycin for 15 min and then probed. (B) protection in the central loop of domain V of 23 S rRNA; incubation of complex C with telithromycin was carried out as in panel A. Samples were then modified with DMS, kethoxal, or CMCT, and analyzed as in panel A. Numbering of nucleotides for the sequencing lanes is indicated at the left of each panel, while nucleotides with reactivity to probes along with reference bands are shown by arrows. Teli, telithromycin.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367204&req=5

gks174-F4: Protections in nucleotides of 23S rRNA from chemical probes, caused by binding of telithromycin to complex C prepared from E. coli ribosomes. (A) protections in domain II of 23S rRNA; complex C was incubated in the absence or presence of telithromycin in buffer B (lanes 1–4) or in the same solution also containing 100 μΜ spermine (lanes 5–8). The resulting complexes were then probed with DMS or CMCT. U, A, G and C, dideoxy sequencing lanes; lane 2 and 6, complex C probed in the absence of telithromycin; lanes 3 and 7, complex C pre-incubated with telithromycin for 5 s and then probed; lanes 4 and 8, complex C pre-incubated with telithromycin for 15 min and then probed. (B) protection in the central loop of domain V of 23 S rRNA; incubation of complex C with telithromycin was carried out as in panel A. Samples were then modified with DMS, kethoxal, or CMCT, and analyzed as in panel A. Numbering of nucleotides for the sequencing lanes is indicated at the left of each panel, while nucleotides with reactivity to probes along with reference bands are shown by arrows. Teli, telithromycin.
Mentions: The first step of telithromycin binding to complex C is presented by a bimolecular reaction. Therefore, to footprint the CT complex, telithromycin at concentration 50 × KT and complex C at 100 nM were incubated at 25°C for 5 s. Since the equilibrium C + T ⇌ CT is established instantaneously while the subsequent isomerization step proceeds slowly, the species mainly produced during this time interval is complex CT (>97%). By longer exposure of complex C to telithromycin (8 × t1/2), the high value, 58.93, for the isomerization constant of the second step favors the formation of C*T complex (>98%). Next to their formation, complexes CT and C*T were probed with DMS, CMCT or kethoxal. Noteworthy, the chemical probes used react with accessible bases within milliseconds (38). Representative autoradiograms obtained by primer extension analysis in helix 35 of domain II and the central loop of domain V of 23S rRNA are shown in Figure 4, while relative reactivities of the modified nucleotides are summarized in Table 3. To assess the footprinting changes relative to the rest of neighboring 23S rRNA areas, larger regions of the scanned sequences are given in Supplementary Figures S1 and S2. Telithromycin in the CT binding state and in the absence of polyamines strongly protects A2058, A2059 and to a lesser degree G2505, C2611, A752, A789 and U790. In contrast, it causes an enhancement in the susceptibility of A2062 to DMS. In the C*T binding state, the protection effects at A789 and U790 soften, the protection at A752 enhances, while a new protection appears on U2609.Figure 4.

Bottom Line: In contrast, mutation Lys63Glu in protein L4 placed on the opposite side of the tunnel, exerts only a minor effect on telithromycin binding.Polyamines disfavor both sequential steps of binding.Our data correlate well with recent crystallographic data and rationalize the changes in the accessibility of ribosomes to telithromycin in response to ribosomal mutations and ionic changes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Medicine, University of Patras, 26504 Patras, Greece.

ABSTRACT
Applying kinetics and footprinting analysis, we show that telithromycin, a ketolide antibiotic, binds to Escherichia coli ribosomes in a two-step process. During the first, rapidly equilibrated step, telithromycin binds to a low-affinity site (K(T) = 500 nM), in which the lactone ring is positioned at the upper portion of the peptide exit tunnel, while the alkyl-aryl side chain of the drug inserts a groove formed by nucleotides A789 and U790 of 23S rRNA. During the second step, telithromycin shifts slowly to a high-affinity site (K(T)* = 8.33 nM), in which the lactone ring remains essentially at the same position, while the side chain interacts with the base pair U2609:A752 and the extended loop of protein L22. Consistently, mutations perturbing either the base pair U2609:A752 or the L22-loop hinder shifting of telithromycin to the final position, without affecting the initial step of binding. In contrast, mutation Lys63Glu in protein L4 placed on the opposite side of the tunnel, exerts only a minor effect on telithromycin binding. Polyamines disfavor both sequential steps of binding. Our data correlate well with recent crystallographic data and rationalize the changes in the accessibility of ribosomes to telithromycin in response to ribosomal mutations and ionic changes.

Show MeSH
Related in: MedlinePlus