Jiro Kondo

Crystal Structure of Metallo DNA Duplex Containing Consecutive Watson-Crick-like T-Hg(II) -T Base Pairs

The metallo DNA duplex containing mercury-mediated T-T base pairs is an attractive biomacromolecular nanomaterial which can be applied to nanodevices such as ion sensors. Reported herein is the first crystal structure of a B-form DNA duplex containing two consecutive T-Hg(II)-T base pairs. The Hg(II) ion occupies the center between two T residues. The N3-Hg(II) bond distance is 2.0 Å. The relatively short Hg(II)-Hg(II) distance (3.3 Å) observed in consecutive T-Hg(II)-T base pairs suggests that the metallophilic attraction could exist between them and may stabilize the B-form double helix. To support this, the DNA duplex is largely distorted and adopts an unusual nonhelical conformation in the absence of Hg(II). The structure of the metallo DNA duplex itself and the Hg(II)-induced structural switching from the nonhelical form to the B-form provide the basis for structure-based design of metal-conjugated nucleic acid nanomaterials.

The structure of metallo-DNA with consecutive thymine–HgII–thymine base pairs explains positive entropy for the metallo base pair formation

We have determined the three-dimensional (3D) structure of DNA duplex that includes tandem HgII-mediated T–T base pairs (thymine–HgII–thymine, T–HgII–T) with NMR spectroscopy in solution. This is the first 3D structure of metallo-DNA (covalently metallated DNA) composed exclusively of ‘NATURAL’ bases. The T–HgII–T base pairs whose chemical structure was determined with the 15N NMR spectroscopy were well accommodated in a B-form double helix, mimicking normal Watson–Crick base pairs. The Hg atoms aligned along DNA helical axis were shielded from the bulk water. The complete dehydration of Hg atoms inside DNA explained the positive reaction entropy (ΔS) for the T–HgII–T base pair formation. The positive ΔS value arises owing to the HgII dehydration, which was approved with the 3D structure. The 3D structure explained extraordinary affinity of thymine towards HgII and revealed arrangement of T–HgII–T base pairs in metallo-DNA.

High‐Resolution Crystal Structure of a Silver(I)–RNA Hybrid Duplex Containing Watson–Crick‐like CSilver(I)C Metallo‐Base Pairs

Metallo-base pairs have been extensively studied for applications in nucleic acid-based nanodevices and genetic code expansion. Metallo-base pairs composed of natural nucleobases are attractive because nanodevices containing natural metallo-base pairs can be easily prepared from commercially available sources. Previously, we have reported a crystal structure of a DNA duplex containing T-Hg(II)-T base pairs. Herein, we have determined a high-resolution crystal structure of the second natural metallo-base pair between pyrimidine bases C-Ag(I)-C formed in an RNA duplex. One Ag(I) occupies the center between two cytosines and forms a C-Ag(I)-C base pair through N3-Ag(I)-N3 linear coordination. The C-Ag(I)-C base pair formation does not disturb the standard A-form conformation of RNA. Since the C-Ag(I)-C base pair is structurally similar to the canonical Watson-Crick base pairs, it can be a useful building block for structure-based design and fabrication of nucleic acid-based nanodevices.

A metallo-DNA nanowire with uninterrupted one-dimensional silver array

The double-helix structure of DNA, in which complementary strands reversibly hybridize to each other, not only explains how genetic information is stored and replicated, but also has proved very attractive for the development of nanomaterials. The discovery of metal-mediated base pairs has prompted the generation of short metal–DNA hybrid duplexes by a bottom-up approach. Here we describe a metallo-DNA nanowire—whose structure was solved by high-resolution X-ray crystallography—that consists of dodecamer duplexes held together by four different metal-mediated base pairs (the previously observed C–Ag–C, as well as G–Ag–G, G–Ag–C and T–Ag–T) and linked to each other through G overhangs involved in interduplex G–Ag–G. The resulting hybrid nanowires are 2 nm wide with a length of the order of micrometres to millimetres, and hold the silver ions in uninterrupted one-dimensional arrays along the DNA helical axis. The hybrid nanowires are further assembled into three-dimensional lattices by interactions between adenine residues, fully bulged out of the double helix. A metallo–DNA hybrid nanowire composed only of silver-mediated base pairs has been prepared and its crystal structure resolved by X-ray diffraction. The nanowire, which is 2 nm wide and whose length reaches the μm to mm scale, holds silver ions into uninterrupted one-dimensional arrays along the DNA helical axis.

Identification of the molecular attributes required for aminoglycoside activity against Leishmania.

Leishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, affects millions of people worldwide. Aminoglycosides are mostly known as highly potent, broad-spectrum antibiotics that exert their antibacterial activity by selectively targeting the decoding A site of the bacterial ribosome, leading to aberrant protein synthesis. Recently, some aminoglycosides have been clinically approved and are currently used worldwide for the treatment of leishmaniasis; however the molecular details by which aminoglycosides induce their deleterious effect on Leishmaina is still rather obscure. Based on high conservation of the decoding site among all kingdoms, it is assumed that the putative binding site of these agents in Leishmania is the ribosomal A site. However, although recent X-ray crystal structures of the bacterial ribosome in complex with aminoglycosides shed light on the mechanism of aminoglycosides action as antibiotics, no such data are presently available regarding their binding site in Leishmania. We present crystal structures of two different aminoglycoside molecules bound to a model of the Leishmania ribosomal A site: Geneticin (G418), a potent aminoglycoside for the treatment of leishmaniasis at a 2.65-Å resolution, and Apramycin, shown to be a strong binder to the leishmanial ribosome lacking an antileishmanial activity at 1.4-Å resolution. The structural data, coupled with in vitro inhibition measurements on two strains of Leishmania, provide insight as to the source of the difference in inhibitory activity of different Aminoglycosides. The combined structural and physiological data sets the ground for rational design of new, and more specific, aminoglycoside derivatives as potential therapeutic agents against leishmaniasis.

Inhibition of aminoglycoside-deactivating enzymes APH(3')-IIIa and AAC(6')-Ii by amphiphilic paromomycin O2''-ether analogues.

Aminoglycoside antibiotics have been in clinical use for the last sixty years. Following the discovery of streptomycin in 1944, many other broad-spectrum aminoglycosides have been discovered (Figure 1). 2] Typically, aminoglycosides are classified according to the substitution pattern of the deoxystreptamine unit that forms the core of these antimicrobial compounds. 4,5-Disubstituted deoxystreptamine compounds are comprised in class A aminoglycosides (Figure 1 a), whereas 4,6disubstituted derivatives are in class B (Figure 1 b). The isolation of new aminoglycosides declined rapidly in the early seventies, when efforts were diverted to the preparation of semisynthetic analogues intended to counteract increasing bacterial resistance to these useful drugs. This led to dibekacin (11), a deoxygenated analogue of kanamycin B (7), and to arbekacin (12), by modifying the N1 group of dibekacin with the 2S-4-amino-2-hydroxybutanoyl moiety originally found in butirosin (2). Although it has been known for decades that aminoglycosides interfere with protein biosynthesis by binding to the prokaryotic ribosome, the lack of precise structural information hampered the identification of beneficial drug modification until the late nineties. A better understanding of the mode of action of aminoglycosides, exemplified by paromomycin (3), was obtained from biochemical and spectroscopic approaches, as well as by mass spectrometry and nuclear magnetic resonance. Definitive confirmation was provided by X-ray structures of the 30S ribosomal subunit bound to aminoglycosides, as well as kinetic studies of protein biosynthesis. 10] This long-awaited information led to an increase in structurebased modifications of aminoglycosides, leading to many of semisynthetic analogues from our laboratory and elsewhere. 12] Aminoglycoside therapy is usually limited to a clinical environment since parenteral injection of these highly hydrophilic drugs is required to obtain the desired plasma concentration in a patient. Their use is also limited by their otoand nephrotoxicity. Since well-studied dosage strategies are used to maximize their antibiotic potential while minimizing their toxicity, the future of these antibiotics will eventually be compromised by the emergence of bacterial resistance. In order to overcome this threat to human health, the structures of some other classes of antibiotics have also been substantially modified. For example, the b-lactam family has “evolved” remarkably since the first report of penicillin resistance. 14] However, clinically effective aminoglycosides have been only minimally modified since their first use. 2] Bacteria have developed two general strategies to resist aminoglycosides: 1) diminution of intracellular concentration of the antibiotic, mainly by efflux; and 2) chemical modification of the drug itself or its biological target. Fortunately, bacterial responses influencing aminoglycoside intracellular concentration, as well as the chemical modification of the ribosomal A-site, are still not widespread. However, modification of aminoglycosides by deactivating enzymes is a major threat to the continued clinical efficacy of these antibiotics. Aminoglycoside deactivating enzymes can be divided into three categories: nucleotidyltransferases (ANTs), acetyltransferases (AACs), and phosphotransferases (APHs). Once adenylated, acetylated or phosphorylated, the affinity of an aminoglycoside for its biological target is drastically attenuated. There are multiple ANTs, AACs and APHs, that can each target different amino or hydroxy groups on the various aminoglycosides. APH(3’)-IIIa mediates the phosphorylation of aminoglycosides at their 3’-OH position by a sequential mechanism where ATP binds first and ADP is the last species to leave the active site. X-ray structures of APH(3’)-IIIa bound to kanamycin A or neomycin B are available. The Enterococcus faecium enzyme AAC(6’)-Ii catalyzes the acetylation of most aminoglycosides at the 6’-N position. This isoform proceeds via an ordered bi bi mechanism, with acetyl coenzyme A (AcCoA) binding first. Crystal structures have been reported for AAC(6’)-Ii in complex with AcCoA, CoA, and some inhibitors. A number of inhibitors of AAC(6’)-Ii have been reported. Based on mechanistic and structural information regarding the mode of action of aminoglycosides as well as the enzymes that deactivate them, aminoglycoside analogues have recently been prepared in an attempt to overcome bacterial resistance. In this regard, we reported the preparation of paromomycin analogues with hydrophobic substituents at the O2’’ position. 23] The persistent antimicrobial activities of some of these amphiphilic O2’’ analogues compared to the parent pa[a] Dr. J. Szychowski, Prof. S. Hanessian, Prof. J. W. Keillor Department of Chemistry, Universit de Montr al C. P. 6128, Succ. Centre-Ville, Montr al, QC, H3C 3J7 (Canada) [b] Dr. J. Kondo, Prof. E. Westhof Architecture et R activit de l’ARN, IBMC-CNRS, Universit de Strasbourg 15 rue Ren Descartes, 67084 Strasbourg Cedex (France) [c] Dr. J. Kondo Department of Materials and Life Sciences Faculty of Science and Technology, Sophia University 7–1 Kioi-cho, Chiyoda-ku, Tokyo 102-8554 (Japan) [d] O. Zahr, Prof. K. Auclair Department of Chemistry, McGill University 801 Sherbrooke Street West, Montreal, QC, H3A 2K6 (Canada) [e] Prof. J. W. Keillor Current address : Department of Chemistry, University of Ottawa 10 Marie-Curie, Ottawa, ON, K1N 6N5 (Canada) E-mail : jkeillor@uottawa.ca Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201100346.

A Structural Basis for the Antibiotic Resistance Conferred by an A1408G Mutation in 16S rRNA and for the Antiprotozoal Activity of Aminoglycosides

Resistance explained: The crystal structures of the ribosomal decoding A site with an A1408G antibiotic-resistance mutation were solved in the presence and absence of the aminoglycoside geneticin (see structure, geneticin carbon framework in yellow). These structures show how bacteria acquire high-level resistance against aminoglycosides by the mutation.

Structure Determination of an Ag I -Mediated Cytosine–Cytosine Base Pair within DNA Duplex in Solution with 1 H/ 15 N/ 109 Ag NMR Spectroscopy

The structure of an Ag(I) -mediated cytosine-cytosine base pair, C-Ag(I) -C, was determined with NMR spectroscopy in solution. The observation of 1-bond (15) N-(109) Ag J-coupling ((1) J((15) N,(109) Ag): 83 and 84 Hz) recorded within the C-Ag(I) -C base pair evidenced the N3-Ag(I) -N3 linkage in C-Ag(I) -C. The triplet resonances of the N4 atoms in C-Ag(I) -C demonstrated that each exocyclic N4 atom exists as an amino group (-NH2 ), and any isomerization and/or N4-Ag(I) bonding can be excluded. The 3D structure of Ag(I) -DNA complex determined with NOEs was classified as a B-form conformation with a notable propeller twist of C-Ag(I) -C (-18.3±3.0°). The (109) Ag NMR chemical shift of C-Ag(I) -C was recorded for cytidine/Ag(I) complex (δ((109) Ag): 442 ppm) to completed full NMR characterization of the metal linkage. The structural interpretation of NMR data with quantum mechanical calculations corroborated the structure of the C-Ag(I) -C base pair.

Crystal structure and specific binding mode of sisomicin to the bacterial ribosomal decoding site.

Sisomicin with an unsaturated sugar ring I displays better antibacterial activity than other structurally related aminoglycosides, such as gentamicin, tobramycin, and amikacin. In the present study, we have confirmed by X-ray analyses that the binding mode of sisomicin is basically similar but not identical to that of the related compounds having saturated ring I. A remarkable difference is found in the stacking interaction between ring I and G1491. While the typical saturated ring I with a chair conformation stacks on G1491 through CH/π interactions, the unsaturated ring I of sisomicin with a partially planar conformation can share its π-electron density with G1491 and fits well within the A-site helix.

Crystal Structures of a Bioactive 6'-Hydroxy Variant of Sisomicin Bound to the Bacterial and Protozoal Ribosomal Decoding Sites

Parasitic infections recognized as neglected tropical diseases are a source of concern for several regions of the world. Aminoglycosides are potent antimicrobial agents that have been extensively studied by biochemical and structural studies in prokaryotes. However, the molecular mechanism of their potential antiprotozoal activity is less well understood. In the present study, we have examined the in vitro inhibitory activities of some aminoglycosides with a 6′‐hydroxy group on ring I and highlight that one of them, 6′‐hydroxysisomicin, exhibits promising activity against a broad range of protozoan parasites. Furthermore, we have conducted X‐ray analyses of 6′‐hydroxysisomicin bound to the target ribosomal RNA A‐sites in order to understand the mechanisms of both its antibacterial and antiprotozoal activities at the molecular level. The unsaturated ring I of 6′‐hydroxysisomicin can directly stack on G1491, which is highly conserved in bacterial and protozoal species, through π–π interaction and fits closer to the guanidine base than the typically saturated and hydroxylated ring I of other structurally related aminoglycosides. Consequently, the compound adopts a lower energy conformation within the bacterial and protozoal A‐sites and makes pseudo pairs to either A or G at position 1408. The A‐site‐selective binding mode strongly suggests that 6′‐hydroxysisomicin is a potential lead for the design of next‐generation aminoglycosides targeting a wide variety of infectious diseases.