Structure and dynamics of a predicted ferredoxin-like selenoprotein in Japanese encephalitis virus

Homologues of the selenoprotein glutathione peroxidase (GPx) have been previously identified in poxviruses and in RNA viruses including HIV-1 and hepatitis C virus (HCV). Sequence analysis of the NS4 region of Japanese encephalitis virus (JEV) suggests it may encode a structurally related but functionally distinct selenoprotein gene, more closely related to the iron-binding protein ferredoxin than to GPx, with three highly conserved UGA codons that align with essential Cys residues of ferredoxin. Comparison of the probe JEV sequence to an aligned family of ferredoxin sequences gave an overall 30.3% identity and 45.8% similarity, and was statistically significant at 4.9 S.D. (P < 10-6) above the average score computed for randomly shuffled sequences. A 3-dimensional model of the hypothetical JEV protein (JEV model) was constructed by homology modeling using SYBYL, based upon a high resolution X-ray structure of ferredoxin (PDB code: 1awd). The JEV model and the model from 1awd were subsequently subjected to molecular dynamics simulations in aqueous medium using AMBER 6. The solution structure of the JEV model indicates that it could fold into a tertiary structure globally similar to ferredoxin 1awd, with RMSD between the averaged structures of 1.8 Å for the aligned regions. The modeling and MD simulations data also indicate that this structure for the JEV protein is energetically favorable, and that it could be quite stable at room temperature. This protein might play a role in JEV infection and replication via TNF and other cellular stimuli mediated via redox mechanisms

Structure-based discovery and in-parallel optimization of novelcompetitive inhibitors of thymidylate synthase

AbstractBackground:The substrate sites of enzymes are attractive targets for structurebased inhibitor design. Two difficulties hinder efforts to discover and elaborate new (nonsubstrate-like) inhibitors for these sites. First, novel inhibitors often bind at nonsubstrate sites. Second, a novel scaffold introduces chemistry that is frequently unfamiliar, making synthetic elaboration challenging.Results:In an effort to discover and elaborate a novel scaffold for a substrate site, we combined structure-based screening with in-parallel synthetic elaboration. These techniques were used to find new inhibitors that bound to the folate site of Lactobacillus casei thymidylate synthase (LcTS), an enzyme that is a potential target for proliferative diseases, and is highly studied. The available chemicals directory was screened, using a molecular-docking computer program, for molecules that complemented the three-dimensional structure of this site. Five high-ranking compounds were selected for testing. Activity and clocking studies led to a derivative of one of these, dansyltyrosine (Ki 65 μM. Using solid-phase in-parallel techniques 33 derivatives of this lead were synthesized and tested. These analogs are dissimilar to the substrate but bind competitively with it. The most active analog had a Ki of 1.3 μM. The tighter binding inhibitors were also the most specific for LcTS versus related enzymes.Conclusions:TS can recognize inhibitors that are dissimilar to, but that bind competitively with, the folate substrate. Combining structure-based discovery with in-parallel synthetic techniques allowed the rapid elaboration of this series of compounds. More automated versions of this approach can be envisaged

Pseudoknot structures with conserved base triples in telomerase RNAs of ciliates

Telomerase maintains the integrity of telomeres, the ends of linear chromosomes, by adding G-rich repeats to their 3′-ends. Telomerase RNA is an integral component of telomerase. It contains a template for the synthesis of the telomeric repeats by the telomerase reverse transcriptase. Although telomerase RNAs of different organisms are very diverse in their sequences, a functional non-template element, a pseudoknot, was predicted in all of them. Pseudoknot elements in human and the budding yeast Kluyveromyces lactis telomerase RNAs contain unusual triple-helical segments with AUU base triples, which are critical for telomerase function. Such base triples in ciliates have not been previously reported. We analyzed the pseudoknot sequences in 28 ciliate species and classified them in six different groups based on the lengths of the stems and loops composing the pseudoknot. Using miniCarlo, a helical parameter-based modeling program, we calculated 3D models for a representative of each morphological group. In all cases, the predicted structure contains at least one AUU base triple in stem 2, except for that of Colpidium colpoda, which contains unconventional GCG and AUA triples. These results suggest that base triples in a pseudoknot element are a conserved feature of all telomerases

Activating Ly-49d and Inhibitory Ly-49a Natural Killer Cell Receptors Demonstrate Distinct Requirements for Interaction with H2-Dd

The activating Ly-49D receptor and the inhibitory Ly-49A receptor mediate opposing effects on natural killer (NK) cell cytotoxicity after interaction with the same major histocompatibility complex ligand, H2-Dd. To compare Ly-49D and Ly-49A interactions with H2-Dd, we created mutations in H2-Dd and examined the functional ability of these mutants to activate lysis through Ly-49D or to inhibit lysis through Ly-49A. Specific single amino acid changes in either the H2-Dd α1 helix or the α2 helix abrogated Ly-49D–mediated cytotoxicity, but these changes had no significant effect on Ly-49A–dependent inhibition. Each of three α2 domain mutations in the floor of the peptide binding groove reduced functional recognition by either Ly-49D or Ly-49A, but all three were required to fully abrogate inhibition by Ly-49A. Our studies indicate that Ly-49D/H2-Dd interactions require distinct determinants compared with Ly-49A/H2-Dd interactions. These differences have important implications for the integration of activating and inhibitory signals in NK cells

Measles Virus Recognizes Its Receptor, CD46, via Two Distinct Binding Domains within SCR1-2

AbstractMeasles virus (MV) enters cells by attachment of the viral hemagglutinin to the major cell surface receptor CD46 (membrane cofactor protein). CD46 is a transmembrane glycoprotein whose ectodomain is largely composed of four conserved modules called short consensus repeats (SCRs). We have previously shown that MV interacts with SCR1 and SCR2 of CD46. (M. Manchesteret al.(1995)Proc. Natl. Acad. Sci. USA92, 2303–2307) Here we report mapping the MV interaction with SCR1 and SCR2 of CD46 using a combination of peptide inhibition and mutagenesis studies. By testing a series of overlapping peptides corresponding to the 126 amino acid SCR1-2 region for inhibition of MV infection, two domains were identified that interacted with MV. One domain was found within SCR1 (amino acids 37–56) and another within SCR2 (amino acids 85–104). These results were confirmed by constructing chimeras with complementary regions from structurally similar, but non-MV-binding, SCRs of decay accelerating factor (DAF; CD55). These results indicate that MV contacts at least two distinct sites within SCR1-2

Structural studies of glutathione transferases towards the development of novel applications and drug design

Glutathione transferases (GSTs) represent a ubiquitous large family of multifunctional enzymes which protect cellular macromolecules from reactive electrophiles. GSTs catalyse the nucleophilic addition of glutathione to electrophilic groups of a large variety of hydrophobic molecules, thereby increasing their solubility to facilitate their excretion from the cell. GSTs play a key role in detoxification, drug metabolism, and multiple-herbicide resistance in weed species. However, the structural basis for the activity of several classes of GSTs remains unknown. Multiple-herbicide resistance (MHR) is a global threat to weed control in cereal crops. MHR weeds express specific phi class glutathione transferases (MHR-GSTF) that confer resistance against multiple herbicides. MHR-GSTFs, therefore, represent a promising target against MHR weeds. On the other hand, the mammalian GSTs, such as the mu-class GSTs, are linked to the development of resistance to a variety of anti-cancer drugs, resulting in the failure of the treatment. The mu-class GSTs are also associated with Parkinson´s disease as well as other illnesses related to oxidative stress. The present work investigates the structure of the MHR-GSTFs from different grass weeds and crops, in particular Alopecurus myosuroides and Lolium rigidum. The work also presents structural and functional insights into the mu-class GSTs from Camelus dromedarius and Homo sapiens. The crystal structures of MHR-GSTF from Alopecurus myosuroides (AmGSTF) and Lolium rigidum (LrGSTF) were determined by molecular replacement at 1.33 Å and 1.90 Å resolution, respectively. The structure of AmGSTF was resolved with a bound glutathione sulfenic acid (GSOH) and succinic acid (SIN) at the enzyme´s active site whereas the LrGSTF structure was determined in complex with the inhibitor S-(4-nitrobenzyl) glutathione. Both enzymes showed conserved structural features compared with other phi class glutathione transferases. However, differences were observed at the C-terminal _8-helix and the H-site _4-helix that may affect the substrate specificity. Moreover, the structural analysis of GSTFs revealed an induced-fit mechanism and a decisive role of conserved Tyr118 and Phe122 in ligand binding. The results presented here provide new knowledge on the enzymology of phi class glutathione transferases and may be used to derive strategies to combat MHR weeds. The structures of a mu-class GST from Camelus dromedarius (CdGSTM1-1) with a bound substrate (GSH) or the reaction product, S-p-nitrobenzyl-glutathione (Nb-GSH) were determined by X-ray crystallography at 2.55 Å and 2.05 Å resolution respectively. The H-site of CdGSTM1-1 is variable and lined by Met35, Arg43, Tyr116, Phe209, Leu210, and Met212, which govern the recognition and binding of substrate in the active site. A noticeable 4 Å move of the β2-α2 loop region upon Nb-GSH binding presents snapshots of an induced-fit mechanism that facilitates the binding of various substrates. The studies will improve our understanding of camelid GSTs detoxification mechanisms and their contribution to abiotic stress adaptation in the desert environment. Besides, the structure of ligand-free Homo sapiens mu-class GST (hGSTM1-1) was determined at 1.59 Å resolution. The high-resolution hGSTM1-1 structure allowed the study of the induced-fit mechanism operated by hGSTM1-1 and the binding of Nb-GSH in detail.Glutationitransferaasien rakennetutkimuksilla kohti uusien sovellusten kehitystä ja lääkeaineiden suunnittelua Glutationitransferaasit (GST:t) ovat tärkeä ryhmä entsyymejä, joilla on useita biokemiallisia funktioita ja ne suojaavat solun makromolekyylejä reaktiivisilta elektrofiileiltä. GST:t katalysoivat glutationin nukleofiilistä lisäystä useiden hydrofobisten molekyylien elektrofiilisiin ryhmiin, mikä lisää niiden liukoisuutta helpottaen niiden poistamista soluista. GST:eilla on keskeinen rooli rikkakasvilajien myrkkyjen poistamisessa, lääkeaineenvaihdunnassa ja useiden rikkakasvien torjunta-aineiden vastustuskyvyssä. Useiden GST-luokkien toiminnan rakenteellinen perusta on kuitenkin tuntematon. Moniherbisidiresistenssi (MHR) on maailmanlaajuinen uhka viljakasvien rikkakasvien torjumiselle. MHR-rikkakasvit ekspressoivat spesifisiä phi-luokan glutationitransferaaseja (MHR-GSTF), jotka antavat vastustuskyvyn useita herbisidejä vastaan. Siksi MHR-GSTF:t edustavat lupaavaa kohdetta MHR-rikkakasveja vastaan. Toisaalta nisäkkäiden GST:t, kuten mu-luokan GST:t, liittyvät resistenssin kehittymiseen useille syöpälääkkeille, mikä johtaa hoidon epäonnistumiseen. Mu-luokan GST:t liittyvät myös Parkinsonin tautiin sekä muihin oksidatiiviseen stressiin liittyviin sairauksiin. Tässä työssä tutkittiin eri ruohorikkakasvien ja -kasvien, erityisesti Alopecurus myosuroidesin ja Lolium rigidumin, MHR-GSTF:ien rakennetta. Tutkimuksessa käsiteltiin myös rakenteellisia ja toiminnallisia eroja Camelus dromedariuksen ja Homo sapiensin mu-luokan GST:ien välillä. Alopecurus myosuroidesin (AmGSTF) ja Lolium rigidumin (LrGSTF) MHR-GSTF:n kiderakenteet määritettiin templaattirakenteen avulla 1,33 Å:n ja 1,90 Å:n resoluutiolla. AmGSTF:n rakenteessa oli aktiiviseen kohtaan sitoutuneena glutationisulfeenihappo (GSOH) ja meripihka-happo (SIN), kun taas LrGSTF:n rakenne määritettiin kompleksina inhibiittorin S-(4-nitroben-tsyyli)glutationin kanssa. Molemmilla entsyymeillä oli konservoituneita rakenteellisia piirteitä verrattuna muihin phi-luokan glutationitransferaaseihin. Kuitenkin eroja havaittiin C-term-naalisessa _8-heliksissä ja H-alueen _4-heliksissä, jotka voivat vaikuttaa substraattispesifi-syyteen. Lisäksi GSTF:n rakenneanalyysi paljasti indusoidun konformaatiomuutoksen ja konser-voituneiden aminohappojen Tyr118:n ja Phe122:n ratkaisevan roolin ligandin sitoutumisessa. Tässä esitetyt tulokset tarjoavat uutta tietoa phi-luokan glutationitransferaasi-entsyymien toiminnasta, ja tuloksia voidaan hyödyntää uusien MHR-rikkakasvien torjunta-aineiden kehittämiksessä. Camelus dromedariuksen (CdGSTM1-1) mu-luokan GST:n rakenteet sidotun substraatin (GSH) tai reaktiotuotteen S-p-nitrobentsyyliglutationin (Nb-GSH) kanssa määritettiin röntgenkristallografialla 2,55 Å ja 2,05 Å resoluutiolla. CdGSTM1-1:n H-kohta on aminohapposekvenssiltään vaihteleva ja sitä reunustavat Met35, Arg43, Tyr116, Phe209, Leu210 ja Met212 säätelevät substraatin tunnistamista ja sitoutumista aktiivisessa kohdassa. Huomattava 4 Å:n liike β2-α2-silmukka-alueella Nb-GSH:n sitoutumisen yhteydessä esittää tilannekuvia indusoiduista konformaatiomuutoksista, jotka vaikuttavat erilaisten substraattien sitoutumiskykyyn. Tutkimukset parantavat ymmärrystämme kamelien GST:n detoksifikaatio-mekanismeista ja niiden vaikutuksesta abioottiseen stressiin sopeutumiseen aavikko-ympäristössä. Lisäksi ligandittoman Homo sapiens mu-luokan GST:n (hGSTM1-1) rakenne määritettiin 1,59 Å:n resoluutiolla. Korkean resoluution hGSTM1-1-rakenne mahdollisti hGSTM1-1:n indusoidun konformaatiomuutoksen (engl. induced-fit mechanism) ja Nb-GSH:n sitoutumisen yksityiskohtaisen tutkimuksen

Long-Residency Hydration, Cation Binding, and Dynamics of Loop E/Helix IV rRNA-L25 Protein Complex

Molecular dynamics simulations of RNA-protein complex between Escherichia coli loop E/helix IV (LE/HeIV) rRNA and L25 protein reveal a qualitative agreement between the experimental and simulated structures. The major groove of LE is a prominent rRNA cation-binding site. Divalent cations rigidify the LE major groove geometry whereas in the absence of divalent cations LE extensively interacts with monovalent cations via inner-shell binding. The HeIV region shows bistability of its major groove explaining the observed differences between x-ray and NMR structures. In agreement with the experiments, the simulations suggest that helix-alpha1 of L25 is the least stable part of the protein. Inclusion of Mg2+ cations into the simulations causes perturbation of basepairing at the LE/HeIV junction, which does not, however, affect the protein binding. The rRNA-protein complex is mediated by a number of highly specific hydration sites with long-residing water molecules and two of them are bound throughout the entire 24-ns simulation. Long-residing water molecules are seen also outside the RNA-protein contact areas with water-binding times substantially enhanced compared to simulations of free RNA. Long-residency hydration sites thus represent important elements of the three-dimensional structure of rRNA