Application of Residual Dipolar Coupling Measurements To Identify Conformational Changes in RNA Induced by Antibiotics

Aminoglycoside antibiotics bind to 16S ribosomal RNA, inducing misreading of the genetic code and inhibiting translocation.1,2 Aminoglycosides bind to short oligonucleotides that contain the ribosomal target; the oligonucleotides roughly mimic the affinity and specificity of the drugs for the ribosome. 3 Threedimensional structures of several oligonucleotide -aminoglycoside complexes have shown that aminoglycosides induce a conformational change on the RNA sequence, supporting an induced fit model for aminoglycoside binding. 4-7 Understanding the structural requirements for specific drug binding requires determination of high resolution structures for many different drugs with different RNA sequences, but unfortunately that process requires the acquisition of numerous NMR experiments followed by structure calculations. Application of a single NMR experiment to test binding of the drug to the RNA and also the conformation of the RNA before and after complex formation would enable one to quickly identify the effect of the drug on the RNA structure. Ligand-macromolecule interactions have been monitored by changes in proton, 13C, or15N chemical shifts, which are sensitive to local environment changes caused by binding; 8-10 1H-15N Heteronuclear Single Quantum Coherence (HSQC) spectra of proteins are ideal for studying binding in a protein -ligand complex because of the resonance dispersion, the short acquisition times required, and the spectral simplicity. Fesik and colleagues have used HSQC spectra to map ligand binding sites during drug screening. 11 In contrast, RNA does not have an equivalent NMR spectrum as indicative of conformation as the proton -amide nitrogen correlation that could be used for a drug screen. Additionally, binding of the drug to an RNA sequence does not necessarily mean the drug is active, since positively charged aminoglycosides will bind negatively charged nucleic acids nonspecifically. Residual dipolar couplings have supplemented NOE and 3 bond J-coupling data to calculate high-resolution structures of proteins and protein-ligand complexes 12-16 and have been used to identify ligand geometry in protein -ligand complexes. 17,18 Dipolar couplings yield information on the orientation of proton -carbon and proton-nitrogen vectors with a molecular alignment axes, which complements short-range distance and torsion angle constraints. 19,20 Dipolar couplings are measured in a nondecoupled 2D single-bond correlation HSQC or HMQC in solution by first measuring theJHC of the RNA in standard conditions then remeasuring theJHC with the RNA suspended in a solution containing the filamentous phage PF1. 12,21 The phage particles align with the magnetic field and induce a partial alignment of the RNA. The scalar coupling is unaffected by the phage, thus, the dipolar coupling ( D) can be determined from the two measurements:D ) JHC(aligned) JHC(not aligned). The dipolar couplings reflect orientation of individual bond vectors with respect to a fixed axes. If binding of aminoglycoside to the RNA induces a conformational change, the structural rearrangement should be observable by comparing dipolar couplings for the RNA in the free form and bound to antibiotic. The structures of the RNA oligonucleotide in its free form, 5