Thermodynamics of Aminogl.–rRNA Recognition
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Transcript of Thermodynamics of Aminogl.–rRNA Recognition
Daniel S. Pilch1,2
Christopher M. Barbieri1
John E. Kerrigan2,31 Department ofPharmacology,
University of Medicine andDentistry of New
JerseyRobert Wood JohnsonMedical School,675 Hoes Lane,
2 The Cancer Institute ofNew Jersey,
New Brunswick,NJ 08901
3 Division of AcademicComputing,
University of Medicine andDentistry of New Jersey,
Received 18 February 2003;accepted 18 February 2003
Abstract: 2-Deoxystreptamine (2-DOS) aminoglycosides are a family of structurally relatedbroad-spectrum antibiotics that are used widely in the treatment of infections caused by aerobicGram-negative bacilli. Their antibiotic activities are ascribed to their abilities to bind a highlyconserved A site in the 16 S rRNA of the 30 S ribosomal subunit and interfere with protein synthesis.The abilities of the 2-DOS aminoglycosides to recognize a specific subdomain of a large RNAmolecule make these compounds archetypical models for RNA-targeting drugs. This article presentsa series of calorimetric, spectroscopic, osmotic stress, and computational studies designed toevaluate the thermodynamics (G, H, S, Cp) of aminoglycosiderRNA interactions, as well asthe hydration changes that accompany these interactions. In conjunction with the current structuraldatabase, the results of these studies provide important insights into the molecular forces thatdictate and control the rRNA binding affinities and specificities of the aminoglycosides. Signifi-cantly, identification of these molecular driving forces [which include binding-linked drug proto-nation reactions, polyelectrolyte contributions from counterion release, conformational changes,hydration effects, and molecular interactions (e.g., hydrogen bonds and van der Waals interac-
Correspondence to: Daniel S. Pilch; email: [email protected] grant sponsor: American Cancer Society (ACS) and
NIHContract grant number: RSG-99-153-04-CDD (ACS) and
RR15959-01 (NIH)Biopolymers, Vol. 70, 5879 (2003) 2003 Wiley Periodicals, Inc.
tions)], as well as the relative magnitudes of their contributions to the binding free energy, couldnot be achieved by consideration of structural data alone, highlighting the importance of acquiringboth thermodynamic and structural information for developing a complete understanding of thedrugRNA binding process. The results presented here begin to establish a database that can beused to predict, over a range of conditions, the relative affinity of a given aminoglycoside oraminoglycoside mimetic for a targeted RNA site vs binding to potential competing secondary sites.This type of predictive capability is essential for establishment of a rational design approach to thedevelopment of new RNA-targeted drugs. 2003 Wiley Periodicals, Inc. Biopolymers 70: 5879,2003
Keywords: drugRNA interactions; calorimetry; major groove binders; binding-induced drugprotonation; heat capacity; molecular dynamics; osmotic stress; hydration; free energy; polyelec-trolyte effect
RNA is a versatile molecule that, like proteins, iscapable of folding into a broad range of differentstructures and conformations, which can serve as spe-cific recognition elements for small molecules. Thetargeting of small molecules to specific RNA struc-tures and sequences offers the potential for modulat-ing the biological activities of the host RNA mole-cules, and in turn, the development of effective phar-maceutical agents. Realizing this potential requires anintimate understanding of small moleculeRNA bind-ing interactions. Such information is critical not onlyfor defining mechanism of action, but also for thedevelopment of a rational approach to the design ofnew compounds that exhibit desired activities. SmallmoleculeRNA interactions are also of interest from amore fundamental point of view in that they offer awell-defined model system for defining the factorsthat are important in the selective recognition ofRNA. Numerous cellular and viral biochemical pro-cesses that are critical for replication and gene expres-sion involve specific proteinRNA interactions. Thus,there is considerable interest in understanding thegeneral principles that contribute to selective RNArecognition. Thermodynamic studies of small mol-eculeRNA interactions can provide insight into themolecular forces that dictate and control RNA bindingaffinity and specificity.
The high-resolution structural database of drugRNA complexes has grown considerably in recentyears, due, in part, to advances in NMR and crystal-lographic methodologies as well as in both enzymaticand chemical synthesis of RNA. The structures ofmore than 40 drugRNA complexes have now beendeposited in the Nucleic Acid Data Base and ProteinData Bank.1,2 This wealth of structural data has pro-vided enormous insight into the molecular basis ofobserved binding specificities. One of the features toemerge from the structural database is that many
small moleculeRNA interactions are accompaniedby conformational changes,316 a feature that compli-cates attempts to correlate structure with binding af-finity. The contributions of hydration effects to bind-ing affinity and specificity are also difficult to ascer-tain from structural information alone. Whileextremely valuable, high-resolution structural infor-mation does not provide a complete understanding ofthe small moleculeRNA interaction. Such an under-standing also requires detailed kinetic and thermody-namic information about the complexes. In this con-nection, recent attempts at structure-based drug de-sign have met with only modest success, due, in largepart, to a poor understanding of the thermodynamicforces governing the binding process. Using amino-glycoside antibiotics as models, one of the goals ofthis article is to illustrate how thermodynamic andstructural information can be integrated to provide amore complete understanding of the molecular driv-ing forces for drugRNA complex formation.
The aminoglycosides are a structurally relatedgroup of broad-spectrum antibiotics that are usedwidely in the treatment of infections caused by aero-bic Gram-negative bacilli.17 They have predictablepharmacokinetic properties and often act in synergywith other antibiotics, two appealing characteristicsthat bolster their clinical value.17,18 The aminoglyco-sides target a highly conserved sequence in the 16 SrRNA of the 30 S ribosomal subunit in pro-karyotes.19,20 This conserved RNA sequence formsthe site, termed the A site, at which the interactionbetween the anticodon of the aminoacyltRNA andthe mRNA codon occurs.21 The antibiotic activities ofthe aminoglycosides are attributed to their abilities tointerfere with this crucial step in the translation pro-cess.20 The deleterious impact of aminoglycosides onprotein synthesis includes both a reduction in transla-tional fidelity as well as inhibition of the translocationstep.20,22,23 The abilities of the aminoglycosides torecognize a specific subdomain of a large RNA mol-
AminoglycosiderRNA Recognition 59
ecule make these compounds archetypical models forRNA-targeting drugs. In this connection, the amino-glycosides represent the paradigm for drugribosomeinteractions, and information gleaned from their studyhas relevance to other ribosome-directed antibiotics ofacute clinical importance.
Aminoglycosides derive their name from theirstructures, which consist of amino sugars and cycli-tols that are linked glycosidically. Reported pKa val-ues for aminoglycoside amino groups range fromapproximately 5.7 to 9.5.2428 Thus, aminoglycosidesexist as oligocations at physiologically relevant valuesof pH. All of the clinically useful aminoglycosidescontain a highly substituted aminocyclitol as either acentral or terminal ring. This ring is streptidinein streptomycin, while being 2-deoxystreptamine(2-DOS) in the other aminoglycosides. There are twomajor classes of 2-DOS aminoglycosides, the 4,5-disubstituted 2-DOS class, which includes neomycinB, paromomycin I, lividomycin A, and ribostamycin(see structures in Figure 1), and the 4,6-disubstituted
2-DOS class, which includes tobramycin, kanamycinsA and B, amikacin, and the gentamicins.
The central potion of the 16 S rRNA A site con-tains an asymmetric internal loop formed by nucleo-tides A1408, A1492, and A1493 (see Figure 2). Foot-printing studies have indicated that this region of 16 SrRNA is essentially free of contacts with ribosomalproteins,29 an observation later confirmed by the re-cently reported crystal structure of the 30 S ribosomalsubunit of Thermus thermophilus.30,31 In the aggre-gate, these results suggested that appropriately de-signed RNA oligonucleotides might be able to reca-pitulate the local structure and/or conformation thatexists in the decoding region A site within the ribo-some. The Puglisi group took such a reductionistapproach by designing a 27-mer hairpin oligonucleo-tide (whose sequence and secondary structure areshown in Figure 2) intended to mimic the decodingregion A site of Escherichia coli 16 S rRNA.9,32
Significantly, they demonstrated that the pattern ofaminoglycoside-induced protection of the RNA bases
FIGURE 1 Structures of paromomycin I, neomycin B, lividomycin A, and ribostamycin in theirfully protonated cationic states, with the atomic and ring numbering systems denoted in Arabic andRoman numerals, respectively. In each of the drugs depicted, ring I is 2-DOS.
60 Pilch et al.
from methylation by dimethylsulfate (DMS) was vir-tually identical in the oligonucleotide construct as itwas in the 30 S ribosomal subunit.9,32
To date, only a handful of structural studies prob-ing the interactions of aminoglycosides with rRNAsequences have been reported.9,11,1416,33 In the firsts