Negamycin Binds to the Wall of the Nascent Chain Exit Tunnel of the 50S Ribosomal Subunit

Abstract

Negamycin, a small-molecule inhibitor of protein synthesis, binds the Haloarcula marismortui 50S ribosomal subunit at a single site formed by highly conserved RNA nucleotides near the cytosolic end of the nascent chain exit tunnel. The mechanism of antibiotic action and the function of this unexplored tunnel region remain intriguingly elusive.Negamycin is a translation inhibitor that prevents the release of complete proteins from the ribosome and thus causes an accumulation of polysomes. Negamycin also induces miscoding (1, 15, 23, 26) but does not interfere with peptide initiation or elongation (7,25,28). Because the inhibition of peptide release and miscoding vary independently in negamycin derivatives (27), negamycin may interfere with ribosome function in two distinct ways. Here we report the results of crystallographic experiments which show that negamycin binds to the wall of the peptide exit tunnel of the large ribosomal subunit. This interaction may account for negamycin's inhibition of peptide release.The 50S ribosomal subunits were prepared from wild-type Haloarcula marismortui cells and crystallized as described previously (3). Negamycin was soaked into the crystals following previously described protocols (11,16,20,21). Five millimolar negamycin is approximately five times the MIC of negamycin for this organism. Crystals were equilibrated at least twice with solutions containing 5 mM negamycin dissolved in buffer B (12% [wt/vol] polyethylene glycol 6000, 20% [vol/vol] ethylene glycol, 1.7 M NaCl, 0.5 M NH 4 Cl, 1 mM CdCl 2 , 100 mM potassium acetate, 6.5 mM acetic acid [pH 6.0], 30 mM MgCl 2 or 100 mM SrCl 2 , 5 mM ␤-mercaptoethanol) at 4°C over the course of 1 to 4 h, during which SrCl 2 was included in soak solutions, and over 16 to 24 h, during which MgCl 2 was included in soak solutions.Data collected at the Advanced Light Source (ALS), using beamline 8.2.2, were processed as previously described (3). Unbiased-difference electron density maps were computed for each drug-ribosome complex, using as amplitudes [ԽF o (hkl)Խ drug/ribosome Ϫ ԽF o (hkl)Խ best native ], where the ԽF o (hkl)Խs are measured amplitudes of the (hkl) reflection in the data collected from the two crystals whose structures were being compared, and phases obtained by rigid-body refinement of the native structure (Protein Data Bank code 1S72; 12) into the drug-ribosome data set. Structure refinements were done with CNS software (4) and included rigid-body, energy minimization, and B-factor refinement. In the final rounds of refinement, only those atoms within a 25-to 35-Å-diameter sphere centered on the antibiotic binding site were allowed to vary in position. The coordinates and structure factors for the negamycin complex described here may be found in the Protein Data Bank (code 2QEX; http://www.rcsb.org/pdb/home /home.do).The functional significance of the negamycin binding site identified in the crystal structure was investigated by site-directed mutagenesis and random targeted mutagenesis and by the selection of naturally o...

Negamycin induces translational stalling and miscoding by binding to the small subunit head domain of the Escherichia coli ribosome

“…In contrast to the structure of the archaeal 50S subunit in complex with negamycin (14), no evidence was found for difference electron density in the vicinity of the exit tunnel, even at a signal-to-noise cutoff of 2.5σ. Positive difference density, however, was identified through a broader search of the "classical"-state ribosome in the crystallographic asymmetric unit (Fig.…”

“…Uncertainties regarding negamycin's inhibition mechanism, its highly polar physicochemical properties, and the lack of streptomycin cross-resistance led some to propose that it may act through multiple binding sites (9). Crystallographic data led to the speculation that negamycin binds in the peptide exit tunnel of the Haloarcula marismortui large ribosomal subunit (14). This site of interaction, however, was difficult to reconcile with its inhibitory activities given its distance (∼100 Å) from known functional centers of the ribosome located at the interface of the large (50S) and small (30S) subunits.…”

Sense from nonsense: therapies for premature stop codon diseases

“…Negamycin has been shown to suppress the dystrophin stop mutation in the mdx mouse model [55]. Like aminoglycosides, negamycin binds the ribosomal A site [55], but it has also recently been reported to bind the polypeptide exit tunnel in the large ribosomal subunit [56]. It is not known which of these binding sites is responsible for its PTC suppression activity.…”

Strategies for the Design of Rna-Binding Small Molecules

“…Group I Intron 20 µM [202] 79 Paromamine Human A-site >100 µM 203w [203][204][205] 80 Paromomycin A-site 12 µM 1n32 and others [145] 81…”