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.     

Structure‐Based Design of a Eukaryote‐Selective Antiprotozoal Fluorinated Aminoglycoside

Aminoglycosides (AG) are antibiotics that lower the accuracy of protein synthesis by targeting a highly conserved RNA helix of the ribosomal A‐site. The discovery of AGs that selectively target the eukaryotic ribosome, but lack activity in prokaryotes, are promising as antiprotozoals for the treatment of neglected tropical diseases, and as therapies to read‐through point‐mutation genetic diseases. However, a single nucleobase change A1408G in the eukaryotic A‐site leads to negligible affinity for most AGs. Herein we report the synthesis of 6′‐fluorosisomicin, the first 6′‐fluorinated aminoglycoside, which specifically interacts with the protozoal cytoplasmic rRNA A‐site, but not the bacterial A‐site, as evidenced by X‐ray co‐crystal structures. The respective dispositions of 6′‐fluorosisomicin within the bacterial and protozoal A‐sites reveal that the fluorine atom acts only as a hydrogen‐bond acceptor to favorably interact with G1408 of the protozoal A‐site. Unlike aminoglycosides containing a 6′‐ammonium group, 6′‐fluorosisomicin cannot participate in the hydrogen‐bonding pattern that characterizes stable pseudo‐base‐pairs with A1408 of the bacterial A‐sites. Based on these structural observations it may be possible to shift the biological activity of aminoglycosides to act preferentially as antiprotozoal agents. These findings expand the repertoire of small molecules targeting the eukaryotic ribosome and demonstrate the usefulness of fluorine as a design element.     

Importance of Co-operative Hydrogen Bonding in the Apramycin-Ribosomal Decoding A-Site Interaction

An intramolecular hydrogen bond between the protonated equatorial 7’-methylamino group of apramycin and the vicinal axial 6’-hydroxy group acidifies the 6’-hydroxy group leading to a strong hydrogen bond to A1408 in the ribosomal drug binding pocket in the decoding A site of the small ribosomal subunit. In 6’-epiapramycin, the trans-nature of the 6’-hydroxy group and the 7’-methylamino group results in a much weaker intramolecular hydrogen bond, and a consequently weaker cooperative hydrogen bonding network with A1408, resulting overall in reduced inhibition of protein synthesis and antibacterial activity.     

Knockout of Tobacco Homologs of Arabidopsis Multi-Antibiotic Resistance 1 Gene Confers a Limited Resistance to Aminoglycoside Antibiotics

To explore a possible recessive selective marker for future DNA-free genome editing by direct delivery of a CRISPR/Cas9-single guide RNA (sgRNA) ribonucleoprotein complex, we knocked out homologs of the Arabidopsis Multi-Antibiotic Resistance 1 (MAR1)/RTS3 gene, mutations of which confer aminoglycoside resistance, in tobacco plants by an efficient Agrobacterium-mediated gene transfer. A Cas9 gene was introduced into Nicotiana tabacum and Nicotiana sylvestris together with an sgRNA gene for one of three different target sequences designed to perfectly match sequences in both S- and T-genome copies of N. tabacum MAR1 homologs (NtMAR1hs). All three sgRNAs directed the introduction of InDels into NtMAR1hs, as demonstrated by CAPS and amplicon sequencing analyses, albeit with varying efficiency. Leaves of regenerated transformant shoots were evaluated for aminoglycoside resistance on shoot-induction media containing different aminoglycoside antibiotics. All transformants tested were as sensitive to those antibiotics as non-transformed control plants, regardless of the mutation rates in NtMAR1hs. The NtMAR1hs–knockout seedlings of the T₁ generation showed limited aminoglycoside resistance but failed to form shoots when cultured on shoot-induction media containing kanamycin. The results suggest that, like Arabidopsis MAR1, NtMAR1hs have a role in plants’ sensitivity to aminoglycoside antibiotics, and that tobacco has some additional functional homologs.     

Mutations in bdcA and valS Correlate with Quinolone Resistance in Wastewater Escherichia coli

Single mutations can confer resistance to antibiotics. Identifying such mutations can help to develop and improve drugs. Here, we systematically screen for candidate quinolone resistance-conferring mutations. We sequenced highly diverse wastewater E. coli and performed a genome-wide association study (GWAS) to determine associations between over 200,000 mutations and quinolone resistance phenotypes. We uncovered 13 statistically significant mutations including 1 located at the active site of the biofilm dispersal gene bdcA and 6 silent mutations in the aminoacyl-tRNA synthetase valS. The study also recovered the known mutations in the topoisomerases gyrase (gyrA) and topoisomerase IV (parC). In summary, we demonstrate that GWAS effectively and comprehensively identifies resistance mutations without a priori knowledge of targets and mode of action. The results suggest that mutations in the bdcA and valS genes, which are involved in biofilm dispersal and translation, may lead to novel resistance mechanisms.     

Targeting a Regulatory Element in Human Thymidylate Synthase mRNA

Thymidylate synthase (TS) is a key enzyme in the biosynthesis of thymidine. The use of TS inhibitors in cancer chemotherapy suffers from resistance development in tumors through upregulation of TS expression. Autoregulatory translation control has been implicated with TS overexpression. TS binding at its own mRNA, which leads to sequestration of the start codon, is abolished when the enzyme forms an inhibitor complex, thereby relieving translation suppression. We have used the protein‐binding site from the TS mRNA in the context of a bicistronic expression system to validate targeting the regulatory motif with stabilizing ligands that prevent ribosomal initiation. Stabilization of the RNA by mutations, which were studied as surrogates of ligand binding, suppresses translation of the TS protein. Compounds that stabilize the TS‐binding RNA motif and thereby inhibit ribosomal initiation might be used in combination with existing TS enzyme‐targeting drugs to overcome resistance development during chemotherapy.     

Crystal structure of the bacterial ribosomal decoding site complexed with a synthetic doubly functionalized paromomycin derivative: a new specific binding mode to an a-minor motif enhances in vitro antibacterial activity

The crystal structure of the complex between oligonucleotide containing the bacterial ribosomal decoding site (A site) and the synthetic paromomycin analogue 1, which contains the gamma-amino-alpha-hydroxybutyryl (L-haba) group at position N1 of ring II (2-DOS ring), and an ether chain with an O-phenethylaminoethyl group at position C2' of ring III, is reported. Interestingly, next to the paromomycin analogue 1 specifically bound to the A site, a second molecule of 1 with a different conformation is observed at the crystal packing interface which mimics the A-minor interaction between two bulged-out adenines from the A site and the codon-anticodon stem of the mRNA-tRNA complex. Improved antibacterial activity supports the conclusion that analogue 1 might affect protein synthesis on the ribosome in two different ways: 1) specific binding to the A site forces maintenance of the "on" state with two bulged out adenines, and 2) a new binding mode of 1 to an A-minor motif which stabilizes complex formation between the ribosome and the mRNA-tRNA complex regardless of whether the codon-anticodon stem is of the cognate or near-cognate type.     

Lyophyllin,a Mushroom Protein from the Peptidase M35 Superfamily Is an RNA N-Glycosidase

Ribosome-inactivating proteins (RIPs) hydrolyze the N-glycosidic bond and depurinate a specific adenine residue (A-4324 in rat 28S ribosomal RNA, rRNA) in the conserved α-sarcin/ricin loop (α-SRL) of rRNA. In this study, we have purified and characterized lyophyllin, an unconventional RIP from Lyophyllum shimeji, an edible mushroom. The protein resembles peptidase M35 domain of peptidyl-Lys metalloendopeptidases. Nevertheless, protein either from the mushroom or in recombinant form possessed N-glycosidase and protein synthesis inhibitory activities. A homology model of lyophyllin was constructed. It was found that the zinc binding pocket of this protein resembles the catalytic cleft of a classical RIP, with key amino acids that interact with the adenine substrate in the appropriate positions. Mutational studies showed that E122 may play a role in stabilizing the positively charged oxocarbenium ion and H121 for protonating N-3 of adenine. The tyrosine residues Y137 and Y104 may be used for stacking the target adenine ring. This work first shows a protein in the peptidase M35 superfamily based on conserved domain search possessing N-glycosidase activity.     

A refined model for the active site within the NO decomposition catalyst, Cu-ZSM-5

Static atomistic simulation techniques have been employed to identify a model for the active site configuration and its location within the NO decomposition catalyst, Cu-ZSM-5. We propose that the active site comprises a copper pair, bridged by OH and forming a six membered ring, specifically, {-O-Cu(II)-OH-Cu(I)-O-Al-}, within the zeolite framework. The six-membered ring arises from the strong association of both of the copper species with a single aluminium in the zeolite framework and consequently the ring is strained, reflected in the low (3.1 Å) intercopper distance in the cluster. Indeed, this Cu-Cu distance compares well with the experimentally determined value of 3.0. In addition, it is expected that the strain in the cluster influences the activity of the cluster, which we suggest may be responsible for its unique activity for NO decomposition.     

Decoding the Molecular Effects of Atovaquone Linked Resistant Mutations on Plasmodium falciparum Cytb-ISP Complex in the Phospholipid Bilayer Membrane

Atovaquone (ATQ) is a drug used to prevent and treat malaria that functions by targeting the Plasmodium falciparum cytochrome b (PfCytb) protein. PfCytb catalyzes the transmembrane electron transfer (ET) pathway which maintains the mitochondrial membrane potential. The ubiquinol substrate binding site of the protein has heme bL, heme bH and iron-sulphur [2FE-2S] cluster cofactors that act as redox centers to aid in ET. Recent studies investigating ATQ resistance mechanisms have shown that point mutations of PfCytb confer resistance. Thus, understanding the resistance mechanisms at the molecular level via computational approaches incorporating phospholipid bilayer would help in the design of new efficacious drugs that are also capable of bypassing parasite resistance. With this knowledge gap, this article seeks to explore the effect of three drug resistant mutations Y268C, Y268N and Y268S on the PfCytb structure and function in the presence and absence of ATQ. To draw reliable conclusions, 350 ns all-atom membrane (POPC:POPE phospholipid bilayer) molecular dynamics (MD) simulations with derived metal parameters for the holo and ATQ-bound -proteins were performed. Thereafter, simulation outputs were analyzed using dynamic residue network (DRN) analysis. Across the triplicate MD runs, hydrophobic interactions, reported to be crucial in protein function were assessed. In both, the presence and absence of ATQ and a loss of key active site residue interactions were observed as a result of mutations. These active site residues included: Met 133, Trp136, Val140, Thr142, Ile258, Val259, Pro260 and Phe264. These changes to residue interactions are likely to destabilize the overall intra-protein residue communication network where the proteins’ function could be implicated. Protein dynamics of the ATQ-bound mutant complexes showed that they assumed a different pose to the wild-type, resulting in diminished residue interactions in the mutant proteins. In summary, this study presents insights on the possible effect of the mutations on ATQ drug activity causing resistance and describes accurate MD simulations in the presence of the lipid bilayer prior to conducting inhibitory drug discovery for the PfCytb-iron sulphur protein (Cytb-ISP) complex.     

Photocrosslinkable modified vegetable oil based resin for wood surface coating application

An acrylated epoxidized linseed oil (AELO) was synthesized from epoxidized linseed oil through ring opening of the oxirane group using acrylic acid as ring opening agent. The occurrence of the acrylate group and the ring opening of oxirane group was monitored using FT-IR spectroscopy. The AELO was mixed with three different photoinitiators at two different concentrations. Wood surfaces were coated with the mixtures, subsequently cured under UV light and the resulting surface properties of the coated samples gloss, scratch resistance, solvent resistance, and coating adhesion were characterized. The efficiency of the photoinitiators and the influence of their concentration on the rate and the extent of the curing were studied by curing the AELO mixtures under a monochromatic wavelength of 365 nm and measuring absorption spectra during the cure by real time FT-IR spectroscopy. The decrease of absorption in the measured spectra at 1406 cm⁻¹ was used to calculate the conversion of acrylic double bonds with increasing time of UV light exposure to obtain information on the cure kinetics for each photoinitiator and concentration.     

Novel mutant human fibronectin FIII9-10 domain pair with increased conformational stability and biological activity

The ninth and tenth type III domains (FIII9–10) in thecentral cell binding domain of human fibronectin contain integrinreceptor binding sites, including RGD in FIII10 and a synergysite, PHSRN, in FIII9. The specific amino acids that contributeto cell binding have been identified by the use of wild-typeand mutant fragments of human fibronectin containing the FIII9–10domain pair. At high concentrations FIII9–10 mimics, toa large extent, the biological activity of the full-length fibronectinmolecule. However, FIII9 is conformationally unstable, evenin the context of the FIII9–10 pair. Here we report theconstruction of a series of hybrid mouse–human FIII9–10pairs that confer varying degrees of conformational stabilityto FIII9. The conformational stability of the human FIII9 modulewas increased 2–3-fold by substitution of Leu1408 withPro. We demonstrate that the biological activity of this mutantis enhanced. The resulting FIII9–10 mutant has good solutionproperties and will provide a template into which further mutationscan be incorporated in order to probe the structure–functionrelationship of the cell binding module of fibronectin.     

Substrate binding and catalysis in trichosanthin occur in different sites as revealed by the complex structures of several E85 mutants

Trichosanthin (TCS) is a type I ribosome-inactivating protein(RIP) which possesses rRNA N-glycosidase activity. In recentyears, its immunomodulatory, anti-tumor and anti-HIV propertieshave been revealed. Here we report the crystal structures ofseveral E85 mutant TCS complexes with adenosine-5'-monophosphate(AMP) and adenine. In E85Q TCS/AMP and E85A TCS/AMP, near theactive site of the molecule and parallel to the aromatic ringof Tyr70, an AMP molecule is bound to the mutant without beinghydrolyzed. In the E85R TCS/adenine complex, the hydrolyzedproduct adenine is located in the active pocket where it occupiesa position similar to that in the TCS/NADPH complex. Significantly,AMP is bound in a position different to that of adenine. Incomparison with these structures, we suggest that there areat least two subsites in the active site of TCS, one for initialsubstrate recognition as revealed by the AMP site and anotherfor catalysis as represented by the NADPH site. Based on thesecomplex structures, the function of residue 85 and the mechanismof catalysis are proposed. Received December 10, 2002; accepted May 20, 2003.     

Acquisition of Streptomycin Resistance by Oxidative Stress Induced by Hydrogen Peroxide in Radiation-Resistant Bacterium Deinococcus geothermalis

Streptomycin is used primarily to treat bacterial infections, including brucellosis, plague, and tuberculosis. Streptomycin resistance easily develops in numerous bacteria through the inhibition of antibiotic transfer, the production of aminoglycoside-modifying enzymes, or mutations in ribosomal components with clinical doses of streptomycin treatment. (1) Background: A transposable insertion sequence is one of the mutation agents in bacterial genomes under oxidative stress. (2) Methods: In the radiation-resistant bacterium Deinococcus geothermalis subjected to chronic oxidative stress induced by 20 mM hydrogen peroxide, active transposition of an insertion sequence element and several point mutations in three streptomycin resistance (SmR)-related genes (rsmG, rpsL, and mthA) were identified. (3) Results: ISDge6 of the IS5 family integrated into the rsmG gene (dgeo_2335), called SrsmG, encodes a ribosomal guanosine methyltransferase resulting in streptomycin resistance. In the case of dgeo_2840-disrupted mutant strains (S1 and S2), growth inhibition under antibiotic-free conditions was recovered with increased growth yields in the presence of 50 µg/mL streptomycin due to a streptomycin-dependent (SmD) mutation. These mutants have a predicted proline-to-leucine substitution at the 91st residue of ribosomal protein S12 in the decoding center. (4) Conclusions: Our findings show that the active transposition of a unique IS element under oxidative stress conditions conferred antibiotic resistance through the disruption of rsmG. Furthermore, chronic oxidative stress induced by hydrogen peroxide also induced streptomycin resistance caused by point and frameshift mutations of streptomycin-interacting residues such as K43, K88, and P91 in RpsL and four genes for streptomycin resistance.     

Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region

Type 1 secretion systems (T1SS) have a relatively simple architecture compared to other classes of secretion systems and therefore, are attractive to be optimized by protein engineering. Here, we report a KnowVolution campaign for the hemolysin (Hly) enhancer fragment, an untranslated region upstream of the hlyA gene, of the hemolysin T1SS of Escherichia coli to enhance its secretion efficiency. The best performing variant of the Hly enhancer fragment contained five nucleotide mutations at five positions (A30U, A36U, A54G, A81U, and A116U) resulted in a 2-fold increase in the secretion level of a model lipase fused to the secretion carrier HlyA1. Computational analysis suggested that altered affinity to the generated enhancer fragment towards the S1 ribosomal protein contributes to the enhanced secretion levels. Furthermore, we demonstrate that involving a native terminator region along with the generated Hly enhancer fragment increased the secretion levels of the Hly system up to 5-fold.     

Hedycaryol Synthase in Complex with Nerolidol Reveals Terpene Cyclase Mechanism

The biosynthesis of terpenes is catalysed by class I and II terpene cyclases. Here we present structural data from a class I hedycaryol synthase in complex with nerolidol, serving as a surrogate for the reaction intermediate nerolidyl diphosphate. This prefolded ligand allows mapping of the active site and hence the identification of a key carbonyl oxygen of Val179, a highly conserved helix break (G1/2) and its corresponding helix dipole. Stabilising the carbocation at the substrate's C1 position, these elements act in concert to catalyse the 1,10 ring closure, thereby exclusively generating the anti‐Markovnikov product. The delineation of a general mechanistic scaffold was confirmed by site‐specific mutations. This work serves as a basis for understanding carbocation chemistry in enzymatic reactions and should contribute to future application of these enzymes in organic synthesis.     

Minimal substrate features for Erm methyltransferases defined by using a combinatorial oligonucleotide library

Erm methyltransferases are prevalent in pathogenic bacteria and confer resistance to macrolide, lincosamide, and streptogramin B antibiotics by specifically methylating the 23S ribosomal RNA at nucleotide A2058. We have identified motifs within the rRNA substrate that are required for methylation by Erm. Substrate molecules were constructed in a combinatorial manner from two separate sets (top and bottom strands) of short RNA sequences. Modifications, including LNA monomers with locked sugar residues, were incorporated into the substrates to stabilize their structures. In functional substrates, the A2058 methylation target (on the 13- to 19-nucleotide top strand) was displayed in an unpaired sequence immediately following a conserved irregular helix, and these are the specific structural features recognized by Erm. Erm methylation was enhanced by stabilizing the top-strand conformation with an LNA residue at G2056. The bottom strand (nine to 19 nucleotides in length) was required for methylation and was still functional after extensive modification, including substitution with a DNA sequence. Although it remains possible that Erm makes some unspecific contact with the bottom strand, the main role played by the bottom strand appears to be in maintaining the conformation of the top strand. The addition of multiple LNA residues to the top strand impeded methylation; this indicates that the RNA substrate requires a certain amount of flexibility for accommodation into the active site of Erm. The combinatorial approach for identifying small but functional RNA substrates is a step towards making RNA-Erm complexes suitable for cocrystal determination, and for designing molecules that might block the substrate-recognition site of the enzyme.     

Inhibitors of the Large Ribosomal Subunit from Haloarcula marismortui

The crystal structures that have been obtained for 23 different inhibitors bound to the large ribosomal subunit from Haloarcula marismortui are reviewed here. These structures provide important insights into how anti-ribosomal antibiotics inhibit protein synthesis, how species specificity arises, and the relationship between ribosomal mutations and antibiotic resistance. These structural studies also provide compelling evidence that the conformation of the peptidyl transferase center of the large ribosomal subunit is intrinsically variable, and that conformational equilibria play a role in determining its functional properties.