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Biomedical subjects

Joseph E Wedekind

Publications and source records attributed to Joseph E Wedekind.

At least 19 recordsLinked to original sources
PubMed2006

Functional characterization of APOBEC-1 complementation factor phosphorylation sites.

ApoB mRNA editing involves site-specific deamination of cytidine 6666 producing an in-frame translation stop codon. Editing minimally requires APOBEC-1 and APOBEC-1 complementation factor (ACF). Metabolic stimulation of apoB mRNA editing in hepatocytes is associated with serine phosphorylation of ACF localized to editing competent, nuclear 27S editosomes. We demonstrate that activation of protein kinase C (PKC) stimulated editing and enhanced ACF phosphorylation in rat primary hepatocytes. Conversely, activation of protein kinase A (PKA) had no effect on editing. Recombinant PKC efficiently phosphorylated purified ACF64 protein in vitro, whereas PKA did not. Mutagenesis of predicted PKC phosphorylation sites S154 and S368 to alanine inhibited ethanol-stimulated induction of editing suggesting that these sites function in the metabolic regulation of editing. Consistent with this interpretation, substitution of S154 and S368 with aspartic acid stimulated editing to levels comparable to ethanol treatment in control McArdle RH7777 cells. These data suggest that phosphorylation of ACF by PKC may be a key regulatory mechanism of apoB mRNA editing in rat hepatocytes.

Amino Acid Sequence↗
PubMed2006

Nanostructures of APOBEC3G support a hierarchical assembly model of high molecular mass ribonucleoprotein particles from dimeric subunits.

Human APOBEC3G (hA3G) is a cytidine deaminase that restricts human immunodeficiency virus (HIV)-1 infection in a vif (the virion infectivity factor from HIV)-dependent manner. hA3G from HIV-permissive activated CD4+ T-cells exists as an inactive, high molecular mass (HMM) complex that can be transformed in vitro into an active, low molecular mass (LMM) variant comparable with that of HIV-non-permissive CD4+ T-cells. Here we present low resolution structures of hA3G in HMM and LMM forms determined by small angle x-ray scattering and advanced shape reconstruction methods. The results show that LMM particles have an extended shape, dissimilar to known cytidine deaminases, featuring novel tail-to-tail dimerization. Shape analysis of LMM and HMM structures revealed how symmetric association of dimers could lead to minimal HMM variants. These observations imply that the disruption of cellular HMM particles may require regulation of protein-RNA, as well as protein-protein interactions, which has implications for therapeutic development.

APOBEC-3G Deaminase↗
PubMed2006

APOBEC-1 and AID are nucleo-cytoplasmic trafficking proteins but APOBEC3G cannot traffic.

Human APOBEC3G (hA3G) is a member of the APOBEC-1 related protein (ARP) family of cytidine deaminases. hA3G functions as a natural defense against endogenous retrotransposons and a multitude of retroviruses, most notably human immunodeficiency virus type 1 (HIV-1). Nothing is known about the cellular function of hA3G, however, upon HIV-1 infection hA3G functions as an antiviral factor by mutating viral single-stranded DNA during reverse transcription. Whereas homologous deaminases such as APOBEC-1 and AID act on RNA and DNA, respectively, in the cell nucleus, hA3G mutagenic activity appears to be restricted to the cytoplasm. We demonstrate that hA3G is not a nucleo-cytoplasmic shuttling protein like APOBEC-1 and AID, but is strongly retained in the cytoplasm through a mechanism that involves both the N and C-terminal regions of the protein.

APOBEC-1 Deaminase↗
PubMed2006

Structure-based design of novel HIV-1 protease inhibitors to combat drug resistance.

Structure-based design and synthesis of novel HIV protease inhibitors are described. The inhibitors are designed specifically to interact with the backbone of HIV protease active site to combat drug resistance. Inhibitor 3 has exhibited exceedingly potent enzyme inhibitory and antiviral potency. Furthermore, this inhibitor maintains impressive potency against a wide spectrum of HIV including a variety of multi-PI-resistant clinical strains. The inhibitors incorporated a stereochemically defined 5-hexahydrocyclopenta[b]furanyl urethane as the P2-ligand into the (R)-(hydroxyethylamino)sulfonamide isostere. Optically active (3aS,5R,6aR)-5-hydroxy-hexahydrocyclopenta[b]furan was prepared by an enzymatic asymmetrization of meso-diacetate with acetyl cholinesterase, radical cyclization, and Lewis acid-catalyzed anomeric reduction as the key steps. A protein-ligand X-ray crystal structure of inhibitor 3-bound HIV-1 protease (1.35 A resolution) revealed extensive interactions in the HIV protease active site including strong hydrogen bonding interactions with the backbone. This design strategy may lead to novel inhibitors that can combat drug resistance.

Bridged Bicyclo Compounds, Heterocyclic↗
PubMed2006

Structural phylogenetic analysis of activation-induced deaminase function.

In mammals, activation-induced deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) of Ig genes. SHM and CSR activities require separate regions within AID. A chromosome region maintenance 1 (CRM1)-dependent nuclear export signal (NES) at the AID C terminus is necessary for CSR, and has been suggested to associate with CSR-specific cofactors. CSR appeared late in AID evolution, during the emergence of land vertebrates from bony fish, which only display SHM. Here, we show that AID from African clawed frog (Xenopus laevis), but not pufferfish (Takifugu rubripes), can induce CSR in AID-deficient mouse B cells, although both are catalytically active in bacteria and mammalian cell systems, albeit at decreased level. Like mammalian AID, Takifugu AID is actively exported from the cell nucleus by CRM1, and the Takifugu NES can substitute for the equivalent region in human AID, indicating that all the CSR-essential NES motif functions evolutionarily predated CSR activity. We also show that fusion of the Takifugu AID catalytic domain to the entire human noncatalytic domain restores activity in mammalian cells, suggesting that AID features mapping within the noncatalytic domain, but outside the NES, influence its function.

Amino Acid Sequence↗
PubMed2006

Water in the active site of an all-RNA hairpin ribozyme and effects of Gua8 base variants on the geometry of phosphoryl transfer.

The hairpin ribozyme requires functional group contributions from G8 to assist in phosphodiester bond cleavage. Previously, replacement of G8 by a series of nucleobase variants showed little effect on interdomain docking, but a 3-250-fold effect on catalysis. To identify G8 features that contribute to catalysis within the hairpin ribozyme active site, structures for five base variants were determined by X-ray crystallography in a resolution range between 2.3 and 2.7 A. For comparison, a native all-RNA "G8" hairpin ribozyme structure was refined to 2.05 A resolution. The native structure revealed a scissile bond angle (tau) of 158 degrees, which is close to the requisite 180 degrees "in-line" geometry. Mutations G8(inosine), G8(diaminopurine), G8(aminopurine), G8(adenosine), and G8(uridine) folded properly, but exhibited nonideal scissile bond geometries (tau ranging from 118 degrees to 93 degrees) that paralleled their diminished solution activities. A superposition ensemble of all structures, including a previously described hairpin ribozyme-vanadate complex, indicated the scissile bond can adopt a variety of conformations resulting from perturbation of the chemical environment and provided a rationale for how the exocyclic amine of nucleobase 8 promotes productive, in-line geometry. Changes at position 8 also caused variations in the A-1 sugar pucker. In this regard, variants A8 and U8 appeared to represent nonproductive ground states in which their 2'-OH groups mimicked the pro-R, nonbridging oxygen of the vanadate transition-state complex. Finally, the results indicated that ordered water molecules bind near the 2'-hydroxyl of A-1, lending support to the hypothesis that solvent may play an important role in the reaction.

Binding Sites↗
PubMed2005

Crystallization and X-ray diffraction analysis of the Trp/amber editing site of hepatitis delta virus (+)RNA: a case of rational design.

RNA editing by mammalian ADAR1 (Adenosine Deaminase Acting on RNA) is required for the life cycle of the hepatitis delta virus (HDV). Editing extends the single viral open reading frame to yield two protein products of alternate length. ADARs are believed to recognize double-stranded RNA substrates via a ;structure-based' readout mechanism. Crystals of 10-mer duplexes representing the HDV RNA-editing site diffracted to 1.35 A resolution, but suffered from merohedral twinning and averaging of the base registry. Expansion of the construct to include two flanking 3 x 1 internal loops yielded crystals in the primitive tetragonal space group P4(1)2(1)2 or P4(3)2(1)2. X-ray diffraction data were collected to 2.8 A resolution, revealing a unit cell with parameters a = 62.5, c = 63.5 A. The crystallization and X-ray analysis of multiple forms of the HDV RNA-editing substrate, encounters with common RNA crystal-growth defects and a strategy to overcome these problems are reported.

Base Sequence↗
PubMed2005

Conformational heterogeneity at position U37 of an all-RNA hairpin ribozyme with implications for metal binding and the catalytic structure of the S-turn.

The hairpin ribozyme is an RNA enzyme that performs site-specific phosphodiester bond cleavage between nucleotides A-1 and G+1 within its cognate substrate. Previous functional studies revealed that the minimal hairpin ribozyme exhibited "gain-of-function" cleavage properties resulting from U39C or U39 to propyl linker (C3) modifications. Furthermore, each "mutant" displayed different magnesium-dependence in its activity. To investigate the molecular basis for these gain-of-function variants, crystal structures of minimal, junctionless hairpin ribozymes were solved in native (U39), and mutant U39C and U39(C3) forms. The results revealed an overall molecular architecture comprising two docked internal loop domains folded into a wishbone shape, whose tertiary interface forms a sequestered active site. All three minimal hairpin ribozymes bound Co(NH(3))(6)(3+) at G21/A40, the E-loop/S-turn boundary. The native structure also showed that U37 of the S-turn adopts both sequestered and exposed conformations that differ by a maximum displacement of 13 A. In the sequestered form, the U37 base packs against G36, and its 2'-hydroxyl group forms a water mediated hydrogen bond to O4' of G+1. These interactions were not observed in previous four-way-junction hairpin ribozyme structures due to crystal contacts with the U1A splicing protein. Interestingly, the U39C and U39(C3) mutations shifted the equilibrium conformation of U37 into the sequestered form through formation of new hydrogen bonds in the S-turn, proximal to the essential nucleotide A38. A comparison of all three new structures has implications for the catalytically relevant conformation of the S-turn and suggests a rationale for the distinctive metal dependence of each mutant.

Crystallization↗
PubMed2005

Crystallographic analysis of the NNA7 Fab and proposal for the mode of human blood-group recognition.

The NNA7 Fab antibody fragment recognizes the human N-type blood-group antigen comprised of the N-terminal glycopeptide of glycophorin A (GPA). A mutant form of this Fab fragment, NNA7-G91S, exhibits markedly reduced antigen binding. To provide insight into how these Fab fragments recognize this glycopeptide antigen, the crystal structures of NNA7 and NNA7-G91S were solved and refined to 1.83 and 1.97 A resolution, respectively. Both molecules are composed of the same heavy (H) chain Fd fragment, but each contains a slightly different light (L) chain owing to the G91S substitution. Specifically, the G91S mutation pushes the backbone of the neighboring H chain away from complementarity-determining region 3 (CDR3) of the L-chain variable region, allowing an additional glycerol cryoprotectant molecule to enter the antigen-combining site near the putative location of O-linked glycosylation. Each Fab fragment also possesses a well defined 2-(N-morpholino)ethanesulfonic acid (MES) molecule trapped in its antigen-combining site, as well as a crystallographic symmetry-related molecule comprising an amino-acid sequence that is virtually identical to the N-terminus of GPA. The MES molecule interacts with the H-chain CDR in a manner reminiscent of antibody-carbohydrate complexes. These results suggest a model for recognition of the glycopeptide antigen that accounts for the deleterious effect of the G91S substitution.

Alkanesulfonic Acids↗
PubMed2005

Kinetic evidence for interaction of human immunodeficiency virus type 1 reverse transcriptase with the 3'-OH of the incoming dTTP substrate.

Two previously identified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants, Q151N and V148I, are known to have reduced dNTP binding affinity but possess wild-type chemical catalysis rates. Structural modeling based on the crystal structure of the HIV-1 RT ternary complex with dTTP proposes that Q151N loses the interaction with the 3'-OH of the incoming dTTP and that V148I disrupts positioning of Q151 for this interaction. On the basis of this, we predicted that while wild-type (WT) HIV-1 RT would have decreased binding affinity to dTTP analogues lacking 3'-OH, compared to dTTP, the Q151N and V148I RT mutants should have decreased but similar affinity to both dTTP and dTTP analogues. Pre-steady-state kinetics on WT RT showed 14- and 53-fold higher K(d) values for the 3'-OH lacking ddTTP and acyTTP, compared to dTTP. In contrast, the Q151N and V148I mutants, which were predicted to have lost H-bonding interaction with the 3'-OH of dTTP, showed higher but similar K(d) values for dTTP, ddTTP, and acyTTP. Interestingly, the Q151N and V148I RTs bound to AZTTP approximately 12 and 18 times more tightly than to dTTP, respectively. Our structure modeling suggests that these RT mutants can interact with the azido moiety of AZTTP, which is 1.4 A longer than the 3'-OH of dTTP. The kinetic data presented in this report demonstrate the functional role of the Q151 residue in HIV-1 RT interaction with dTTP and its analogues containing chemical modifications at the 3'-C of the sugar moiety.

Acyclovir↗
PubMed2004

Ornithine cyclodeaminase: structure, mechanism of action, and implications for the mu-crystallin family.

Ornithine cyclodeaminase catalyzes the conversion of L-ornithine to L-proline by an NAD(+)-dependent hydride transfer reaction that culminates in ammonia elimination. Phylogenetic comparisons of amino acid sequences revealed that the enzyme belongs to the mu-crystallin protein family whose three-dimensional fold has not been reported. Here we describe the crystal structure of ornithine cyclodeaminase in complex with NADH, refined to 1.80 A resolution. The enzyme consists of a homodimeric fold whose subunits comprise two functional regions: (i) a novel substrate-binding domain whose antiparallel beta-strands form a 14-stranded barrel at the oligomeric interface and (ii) a canonical Rossmann fold that interacts with a single dinucleotide positioned for re hydride transfer. The adenosyl moiety of the cofactor resides in a solvent-exposed crevice on the protein surface and makes contact with a "domain-swapped"-like coil-helix module originating from the dyad-related molecule. Diffraction data were also collected to 1.60 A resolution on crystals grown in the presence of l-ornithine. The structure revealed that the substrate carboxyl group interacts with the side chains of Arg45, Lys69, and Arg112. In addition, the ammonia leaving group hydrogen bonds to the side chain of Asp228 and the site of hydride transfer is 3.8 A from C4 of the nicotinamide. The absence of an appropriately positioned water suggested that a previously proposed mechanism that calls for hydrolytic elimination of the imino intermediate must be reconsidered. A more parsimonious description of the chemical mechanism is proposed and discussed in relation to the structure and function of mu-crystallins.

Amino Acid Motifs↗
PubMed2004

The structure of a yeast RNA-editing deaminase provides insight into the fold and function of activation-induced deaminase and APOBEC-1.

Activation-induced deaminase (AID) uses base deamination for class-switch recombination and somatic hypermutation and is related to the mammalian RNA-editing enzyme apolipoprotein B editing catalytic subunit 1 (APOBEC-1). CDD1 is a yeast ortholog of APOBEC-1 that exhibits cytidine deaminase and RNA-editing activity. Here, we present the crystal structure of CDD1 at 2.0-A resolution and its use in comparative modeling of APOBEC-1 and AID. The models explain dimerization and the need for trans-acting loops that contribute to active site formation. Substrate selectivity appears to be regulated by a central active site "flap" whose size and flexibility accommodate large substrates in contrast to deaminases of pyrimidine metabolism that bind only small nucleosides or free bases. Most importantly, the results suggested both AID and APOBEC-1 are equally likely to bind single-stranded DNA or RNA, which has implications for the identification of natural AID targets.

APOBEC-1 Deaminase↗
PubMed2004

Crystallization and X-ray diffraction analysis of ornithine cyclodeaminase from Pseudomonas putida.

Ornithine cyclodeaminase (OCD) is a member of the micro-crystallin protein family, the biological activity of which is the conversion of L-ornithine to L-proline and ammonia. In order to elucidate the functional groups of this enzyme that are involved in catalysis, the crystallization of OCD from Pseudomonas putida was undertaken. Using microbatch-under-oil screening at the high-throughput crystallization laboratory (HTC) at the Hauptman-Woodward Medical Research Institute Inc. (HWI Buffalo, NY, USA), numerous crystallization conditions were rapidly identified. Several conditions could be reproduced on a larger scale as vapor-diffusion experiments in-house. The best diffraction-quality crystals were obtained from solutions of 40%(v/v) 2-methyl-2,4-pentanediol buffered at pH 6.0 with 0.1 M MES and diffracted X-rays to 1.68 A resolution. Crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 70.0, b = 78.3, c = 119.4 A. The V(M) was 2.1 A(3) Da(-1), corresponding to 42% solvent, which is consistent with two 38.5 kDa molecules per asymmetric unit. The structure determination is under way using experimental phasing methods.

Ammonia-Lyases↗
PubMed2004

Purification, crystallization and X-ray diffraction analysis of a recombinant Fab that recognizes a human blood-group antigen.

The NNA7 Fab fragment recognizes the human glycopeptide N blood-group antigen and has a high affinity for N-type glycophorin A (GPA). To provide insight into how antibodies recognize glycopeptide antigens, soluble Fab fragments were expressed in Escherichia coli, purified and crystallized using the hanging-drop vapor-diffusion method at 293 K. The best crystals were obtained from solutions of PEG monomethyl ether 5000 containing 4-8 mM yttrium chloride (YCl3). This rare-earth ion, which could be substituted with various lanthanides, changed the habit of crystals from multinucleated rods with a diffraction limit of 4.25 A resolution to a diamond-shaped morphology that grew as single crystals and diffracted X-rays to 1.75 A resolution. Data were collected that indicated that the crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 57.9, b = 77.1, c = 118.1 A and one Fab fragment per asymmetric unit. A molecular-replacement solution has been obtained and 86% of the molecule was fitted by use of an automated refinement procedure (ARP).

Antigen-Antibody Complex↗
PubMed2004

Activation induced deaminase: the importance of being specific.

Activation-induced deaminase (AID) is required for class switch recombination and somatic hypermutation in immunoglobulin genes. Although the preponderance of evidence suggests that AID functions by deaminating deoxycytidine in DNA, the question remains whether it can also deaminate cytidine in mRNA, as originally proposed based on its homology to RNA-editing enzymes. Recently, the biological relevance of assaying mammalian enzymes for DNA deaminase activity using Escherichia coli DNA as a reporter has been questioned, representing another round in the ongoing debate.

APOBEC-1 Deaminase↗
PubMed2003

Crystal structure of the leadzyme at 1.8 A resolution: metal ion binding and the implications for catalytic mechanism and allo site ion regulation.

The leadzyme is a small ribozyme, derived from in vitro selection, which catalyzes site specific, Pb(2+)-dependent RNA cleavage. Pb(2+) is required for activity; Mg(2+) inhibits activity, while many divalent and trivalent ions enhance it. The leadzyme structure consists of an RNA duplex interrupted by a trinucleotide bulge. Here, crystal structures determined to 1.8 A resolution, both with Mg(2+) as the sole divalent counterion and with Mg(2+) and Sr(2+) (which mimics Pb(2+) with respect to binding but not catalysis), reveal the metal ion interactions with both the ground state and precatalytic conformations of the leadzyme. Mg(H(2)O)(6)(2+) ions bridge complementary strands of the duplex at multiple locations by binding tandem purines of one RNA strand in the major groove. At one site, Mg(H(2)O)(6)(2+) ligates the phosphodiester backbone of the trinucleotide bulge in the ground state conformation, but not in the precatalytic conformation, suggesting (a) Mg(2+) may inhibit leadzyme activity by stabilizing the ground state and (b) metal ions which displace Mg(2+) from this site may activate the leadzyme. Binding of Sr(2+) to the presumed catalytic Pb(2+) site in the precatalytic leadzyme induces local structural changes in a manner that would facilitate alignment of the catalytic ribose 2'-hydroxyl with the scissile bond for cleavage. These data support a model wherein binding of a catalytic ion to a precatalytic conformation of the leadzyme, in conjunction with the flexibility of the trinucleotide bulge, may facilitate structural rearrangements around the scissle phosphodiester bond favoring configurations that allow bond cleavage.

Allosteric Site↗
PubMed2003

Messenger RNA editing in mammals: new members of the APOBEC family seeking roles in the family business.

Alteration of mRNA sequence through base modification mRNA editing frequently generates protein diversity. Several proteins have been identified as being similar to C-to-U mRNA editing enzymes based on their structural domains and the occurrence of a catalytic domain characteristic of cytidine deaminases. In light of the hypothesis that these proteins might represent novel mRNA editing systems that could affect proteome diversity, we consider their structure, expression and relevance to biomedically significant processes or pathologies.

APOBEC-1 Deaminase↗