Christopher A. Waddling

Intertwined structure of the DNA-binding domain of intron endonuclease I-TevI with its substrate

I‐TevI is a site‐specific, sequence‐tolerant intron endonuclease. The crystal structure of the DNA‐binding domain of I‐TevI complexed with the 20 bp primary binding region of its DNA target reveals an unusually extended structure composed of three subdomains: a Zn finger, an elongated segment containing a minor groove‐binding α‐helix, and a helix–turn–helix. The protein wraps around the DNA, mostly following the minor groove, contacting the phosphate backbone along the full length of the duplex. Surprisingly, while the minor groove‐binding helix and the helix–turn–helix subdomain make hydrophobic contacts, the few base‐specific hydrogen bonds occur in segments that lack secondary structure and flank the intron insertion site. The multiple base‐specific interactions over a long segment of the substrate are consistent with the observed high site specificity in spite of sequence tolerance, while the modular composition of the domain is pertinent to the evolution of homing endonucleases.

Crystal structure of a deletion mutant of human thymidylate synthase Delta (7-29) and its ternary complex with Tomudex and dUMP.

The crystal structures of a deletion mutant of human thymidylate synthase (TS) and its ternary complex with dUMP and Tomudex have been determined at 2.0 Å and 2.5 Å resolution, respectively. The mutant TS, which lacks 23 residues near the amino terminus, is as active as the wild‐type enzyme. The ternary complex is observed in the open conformation, similar to that of the free enzyme and to that of the ternary complex of rat TS with the same ligands. This is in contrast to Escherichia coli TS, where the ternary complex with Tomudex and dUMP is observed in the closed conformation. While the ligands interact with each other in identical fashion regardless of the enzyme conformation, they are displaced by about 1.0 Å away from the catalytic cysteine in the open conformation. As a result, the covalent bond between the catalytic cysteine sulfhydryl and the base of dUMP, which is the first step in the reaction mechanism of TS and is observed in all ternary complexes of the E. coli enzyme, is not formed. This displacement results from differences in the interactions between Tomudex and the protein that are caused by differences in the environment of the glutamyl tail of the Tomudex molecule. Despite the absence of the closed conformation, Tomudex inhibits human TS ten‐fold more strongly than E. coli TS. These results suggest that formation of a covalent bond between the catalytic cysteine and the substrate dUMP is not required for effective inhibition of human TS by cofactor analogs and could have implications for drug design by eliminating this as a condition for lead compounds.

A phage tubulin assembles dynamic filaments by an atypical mechanism to center viral DNA within the host cell.

Tubulins are essential for the reproduction of many eukaryotic viruses, but historically, bacteriophage were assumed not to require a cytoskeleton. Here, we identify a tubulin-like protein, PhuZ, from bacteriophage 201φ2-1 and show that it forms filaments in vivo and in vitro. The PhuZ structure has a conserved tubulin fold, with an unusual, extended C terminus that we demonstrate to be critical for polymerization in vitro and in vivo. Longitudinal packing in the crystal lattice mimics packing observed by EM of in-vitro-formed filaments, indicating how interactions between the C terminus and the following monomer drive polymerization. PhuZ forms a filamentous array that is required for positioning phage DNA within the bacterial cell. Correct positioning to the cell center and optimal phage reproduction only occur when the PhuZ filament is dynamic. Thus, we show that PhuZ assembles a spindle-like array that functions analogously to the microtubule-based spindles of eukaryotes.

Broad-spectrum allosteric inhibition of herpesvirus proteases.

Herpesviruses rely on a homodimeric protease for viral capsid maturation. A small molecule, DD2, previously shown to disrupt dimerization of Kaposi’s sarcoma-associated herpesvirus protease (KSHV Pr) by trapping an inactive monomeric conformation and two analogues generated through carboxylate bioisosteric replacement (compounds 2 and 3) were shown to inhibit the associated proteases of all three human herpesvirus (HHV) subfamilies (α, β, and γ). Inhibition data reveal that compound 2 has potency comparable to or better than that of DD2 against the tested proteases. Nuclear magnetic resonance spectroscopy and a new application of the kinetic analysis developed by Zhang and Poorman [Zhang, Z. Y., Poorman, R. A., et al. (1991) J. Biol. Chem. 266, 15591–15594] show DD2, compound 2, and compound 3 inhibit HHV proteases by dimer disruption. All three compounds bind the dimer interface of other HHV proteases in a manner analogous to binding of DD2 to KSHV protease. The determination and analysis of cocrystal structures of both analogues with the KSHV Pr monomer verify and elaborate on the mode of binding for this chemical scaffold, explaining a newly observed critical structure–activity relationship. These results reveal a prototypical chemical scaffold for broad-spectrum allosteric inhibition of human herpesvirus proteases and an approach for the identification of small molecules that allosterically regulate protein activity by targeting protein–protein interactions.

Cytochrome P450 125A4, the Third Cholesterol C-26 Hydroxylase from Mycobacterium smegmatis

Mycobacterium tuberculosis (Mtb) and Mycobacterium smegmatis (Msmeg) can grow on cholesterol as the sole carbon source. In Mtb the utilization of cholesterol can be initiated by CYP125A1 or CYP142A1 and in Msmeg by the orthologous CYP125A3 and CYP142A2. Double knockout of the two enzymes in Mtb prevents its growth on cholesterol, but the double knockout of Msmeg is still able to grow, albeit at a slower rate. We report here that Msmeg has a third enzyme, CYP125A4, that also oxidizes cholesterol, although it has a much higher activity for the oxidation of 7α-hydroxycholesterol. The ability of Msmeg CYP125A4 (and Mtb CYP125A1) to oxidize 7α-hydroxycholesterol is due, at least in part, to the presence of a smaller amino acid side chain facing C-7 of the sterol substrate than in CYP125A3. The ability to oxidize 7-substituted steroids broadens the range of sterol carbon sources for growth, but even more importantly in Mtb, additional biological effects are possible due to the potent immunomodulatory activity of 7α,26-dihydroxycholesterol.

Crystal structures of the nitrate salt of 24-pyrimidinium crown 6 {5,12,19,26,33,40-hexaamino-3,10,17,24,31,38-hexamethyl[1.6 ]- (1,5)pyrimidiniophane} and its degradation product in water–methanol mixtures

Reflux of thiamin [3-(4-amino-2-methylpyrimidin-5-ylmethyl)-5-(2-hydroxyethyl)-4-methylth iazolium] mononitrate in methanol for 2 h, followed by recrystallization from methanol, produced single crystals of [L][NO 3 ] 6 ·16H 2 O {L = 5,12,19,26,33,40-hexaamino-3,10,17,24,31,38-hexamethyl[1. 6 ](1,5)pyrimidiniophane, or 24-pyrimidinium crown 6}. The compound crystallizes in the rhombohedral space group R, a = b = c = 14.131(11) A, α = β = γ = 115.26(5)°, Z = 1, R = 0.0469, 1352 independent reflections with F > 2.0σ(F). Reflux of [L][NO 3 ] 6 ·16H 2 O in methanol–water (3∶2) for 182 h produced single crystals of the nitric acid salt of 4-amino-5-methoxymethyl-2-methylpyrimidine. The compound crystallizes in the triclinic space group P, a = 9.116(6), b = 9.452(5), c = 12.742(8) A, α = 101.14(5), β = 107.13(5), γ = 93.27(5)°, Z = 4, R = 0.0644, 2690 independent reflections with F > 2.0σ(F). The nitric acid salt of 4-amino-2-methylpyrimidine-5-methanol has been isolated and identified by 1 H NMR spectroscopy from the thermal decomposition reaction of [L][NO 3 ] 6 ·16H 2 O in water. The alcohol has also been observed by 1 H NMR spectroscopy in the decomposition of [L] 6+ in aqueous solutions at room temperature.

Ring-Opening Polymerization as a Route to New Inorganic Macromolecules

Abstract Cyclic inorganic compounds are potential precursors to new inorganic polymer systems via ring-opening polymerization. In this paper we review some of our recent work aimed at preparing inorganic polymers containing main group elements. For example, the ring-opening polymerization of the cyclic thionylphosphazenes NSOX(NPCl2)2 (X = Cl or F) is discussed. This provides a route to poly(thionylphosphazenes), a new class of stable inorganic polymers with backbones of phosphorus, nitrogen, and sulfur(V1) atoms. We also describe some of our preliminary work aimed at the investigation of the synthesis, structure, and polymerization behaviour of boracyclosiloxanes (RBO)(R2SiO)n.

Crystal structures of the CO and NOBound DosS GAF-A domain and implications for DosS signaling in Mycobacterium tuberculosis.

DosS is a sensor in Mycobacterium tuberculosis that differentially responds to O2, NO, and CO, as well as to changes in the redox state of the prosthetic heme iron atom. The ferrous protein and its Fe(II)NO and Fe(II)CO complexes undergo autophosphorylation and subsequently transfer the phosphate group to DosR, a nuclear factor, to activate it. In contrast, autophosphorylation is negligible with the ferric protein and the Fe(II)O2 complex. To clarify the basis for this differential response to gases, we have determined the crystal structures of the NO and COcomplexes of the DosS GAF-A domain, which contains the heme to which the gases bind. Comparison of these crystal structures with those reported for the phosphorylation-inactive ferric GAF-A domain suggest that the GAF-A domain is in a dynamic equilibrium between active and inactive states, and that the position of Glu87 in the heme cavity, which depends on the which gas is bound, acts as a modulator of the equilibrium, and therefore of catalytic activity.

Catalytic elimination of organics over noble metal catalysts deposited on metal structures of low mass

Abstract A Carberry reactor operated with the basket spinning at a speed of 3000 rpm was used to obtain catalytic rates in the absence of external mass transfer resistance for isobutane and styrene oxidations. Catalyst position in the spinning basket was found to be critical to obtain reproducible results. Alumina- and Silicalite-supported platinum catalysts deposited on low-mass stainless steel screens and rolled into jelly-rolls gave conversion efficiencies up to 100% for styrene oxidation. For isobutane oxidation, the catalytic rate increased over a period (about 15 h) before reaching a steady value, confirming a previously reported activation effect. Such an effect was not observed for styrene oxidation.