The effect of ligand affinity to the contact dynamics of the ligand binding domain of thyroid hormone receptor - retinoid X receptor.

Ligand-based allostery has been gaining attention for its importance in protein regulation and implication in drug design. One of the interesting cases of protein allostery is the thyroid hormone receptor - retinoid x receptor (TR:RXR), which regulates the gene expression of important physiological processes, such as development and metabolism. It is regulated by the TR native ligand triiodothyronine (T3), which displays anticooperative behavior to the RXR ligand 9-cis retinoic acid (9C). In contrast to this anticooperative behavior, 9C has been shown to increase the activity of TR:RXR. Here we probed the influence of the affinity and the interactions of the TR ligand to the allostery of the TR:RXR through contact dynamics and residue networks. The TR ligand analogs were designed to have higher (G2) and lower (N1) binding energies than T3 when docked to the TR:RXR(9C) complex. The aqueous TR(N1/T3/G2):RXR(9C) complexes were subjected to 30 ns all-atom simulations using theNAMD. The program CAMERRA was used to capture the subtle perturbations of TR:RXR by mapping the residue contact dynamics. Various parts of the TR ligands; including the hydrophilic head, the iodine substituents, and the ligand tail; have been probed for their significance in ligand affinity. The results on the T3 and G2 complexes suggest that ligand affinity can be utilized as a predictor for anticooperative systems on which ligand is more likely to dissociate or remain bound. All 3 complexes also display distinct contact networks for cross-dimer signalling and ligand communication. Understanding ligand-based allostery could potentially unveil secrets of ligand-regulated protein dynamics, a foundation for the design of better and more efficient allosteric drugs.

Crystal structure of a catalytically active, non-toxic endopeptidase derivative of Clostridium botulinum toxin A.

Botulinum neurotoxins (BoNTs) modulate cholinergic nerve terminals to result in neurotransmitter blockade. BoNTs consists of catalytic (LC), translocation (Hn) and cell-binding domains (Hc). The binding function of the Hc domain is essential for BoNTs to bind the neuronal cell membrane, therefore, removal of the Hc domain results in a product that retains the endopeptidase activity of the LC but is non-toxic. Thus, a molecule consisting of LC and Hn domains of BoNTs, termed LHn, is a suitable molecule for engineering novel therapeutics. The structure of LHA at 2.6 A reported here provides an understanding of the structural implications and challenges of engineering therapeutic molecules that combine functional properties of LHn of BoNTs with specific ligand partners to target different cell types.

Effects of a hairpin polyamide on DNA melting: comparison with distamycin and Hoechst 33258.

We have used DNase I footprinting and fluorescence melting studies to study the interaction of the hairpin polyamide Im-Py-Py-Py-(R)H2Ngamma-Im-Py-Py-Py-beta-Dp with its preferred binding sites (5'-WGWWCW; W=A or T) and other sequences. DNase I footprinting confirmed that the ligand binds to the sequence AGAACA at nanomolar concentrations and that changing the terminal A to G causes a dramatic decrease in affinity, while there was no interaction with the reverse sequence WCWWGW. Fluorescence melting studies with 11-mer duplexes showed that the polyamide had very different effects on the forward (TGWWCT) and reverse (TCTAGT) sequences. At low concentrations, the polyamide produced biphasic melting curves with TGATCT, TGTACT and TGAACT, suggesting a strong interaction. In contrast, the melting profiles with TCTAGT were always monophasic and showed much smaller concentration dependent changes in Tm. The polyamide also showed weak binding to the sequence TGATCT when one of the central AT pairs was replaced with an AC mismatch. These melting profiles were compared with those produced by the AT-selective minor groove binding agents distamycin and Hoechst 33258 at the same sites and at similar sequences containing A5 and (AT)3, which are expected to bind distamycin in the 1:1 and 2:1 modes, respectively. These ligands produced simple monophasic melting curves in which the Tm steadily increased as the ligand concentration was raised.

DNA sequence recognition by an isopropyl substituted thiazole polyamide.

We have used DNA footprinting and fluorescence melting experiments to study the sequence-specific binding of a novel minor groove binding ligand (thiazotropsin A), containing an isopropyl substituted thiazole polyamide, to DNA. In one fragment, which contains every tetranucleotide sequence, sub-micromolar concentrations of the ligand generate a single footprint at the sequence ACTAGT. This sequence preference is confirmed in melting experiments with fluorescently labelled oligonucleotides. Experiments with DNA fragments that contain variants of this sequence suggest that the ligand also binds, with slightly lower affinity, to sequences of the type XCYRGZ, where X is any base except C, and Z is any base except G.

DNA sequence specificity of triplex-binding ligands.

We have examined the ability of naphthylquinoline, a 2,7-disubstituted anthraquinone and BePI, a benzo[e]pyridoindole derivative, to stabilize parallel DNA triplexes of different base composition. Fluorescence melting studies, with both inter- and intramolecular triplexes, show that all three ligands stabilize triplexes that contain blocks of TAT triplets. Naphthylquinoline has no effect on triplexes formed with third strands composed of (TC)n or (CCT)n, but stabilizes triplexes that contain (TTC)n. In contrast, BePI slightly destabilizes the triplexes that are formed at (TC)n (CCT)n and (TTC)n. 2,7-Anthraquinone stabilizes (TC)n (CCT)n and (TTC)n, although it has the greatest effect on the latter. DNase I footprinting studies confirm that triplexes formed with (CCT)n are stabilized by the 2,7-disubstituted amidoanthraquinone but not by naphthylquinoline. Both ligands stabilize the triplex formed with (CCTT)n and neither affects the complex with (CT)n. We suggest that BePI and naphthylquinoline can only bind between adjacent TAT triplets, while the anthraquinone has a broader sequence of selectivity. These differences may be attributed to the presence (naphthylquinoline and BePI) or absence (anthraquinone) of a positive charge on the aromatic portion of the ligand, which prevents intercalation adjacent to C+GC triplets. The most stable structures are formed when the stacked rings (bases or ligand) alternate between charged and uncharged species. Triplexes containing alternating C+GC and TAT triplets are not stabilized by ligands as they would interrupt the alternating pattern of charged and uncharged residues.

Thermodynamic and kinetic stability of intermolecular triple helices containing different proportions of C+*GC and T*AT triplets.

We have used oligonucleotides containing appropriately placed fluorophores and quenchers to measure the stability of 15mer intermolecular triplexes with third strands consisting of repeats of TTT, TTC, TCC and TCTC. In the presence of 200 mM sodium (pH 5.0) triplexes that contain only T.AT triplets are unstable and melt below 30 degrees C. In contrast, triplets with repeats of TTC, TCC and CTCT melt at 67, 72 and 76 degrees C, respectively. The most stable complex is generated by the sequence containing alternating C+*GC and T*AT triplets. All four triplexes are stabilised by increasing the ionic strength or by the addition of magnesium, although triplexes with a higher proportion of C+*GC triplets are much less sensitive to changes in the ionic conditions. The enthalpies of formation of these triplexes were estimated by examining the concentration dependence of the melting profiles and show that, in the presence of 200 mM sodium at pH 5.0, each C+*GC triplet contributes about 30 kJ x mol(-1), while each T*AT contributes only 11 kJ x mol(-1). Kinetic experiments with these oligonucleotides show that in 200 mM sodium (pH 5.0) repeats of TCC and TTC have half-lives of approximately 20 min, while the triplex with alternating C+*GC and T.AT triplets has a half-life of approximately 3 days. In contrast, the dissociation kinetics of the triplex containing only T*AT are too fast to measure.

The metal ion in the active site of the membrane glucose dehydrogenase of Escherichia coli.

All pyrroloquinoline quinone (PQQ)-containing dehydrogenases whose structures are known contain Ca(2+) bonded to the PQQ at the active site. However, membrane glucose dehydrogenase (GDH) requires reconstitution with PQQ and Mg(2+) ions (but not Ca(2+)) for activity. To address the question of whether the Mg(2+) replaces the usual active site Ca(2+) in this enzyme, mutant GDHs were produced in which residues proposed to be involved in binding metal ion were modified (D354N-GDH and N355D-GDH and D354N-GDH/N355D-GDH). The most remarkable observation was that reconstitution with PQQ of the mutant enzymes was not supported by Mg(2+) ions as in the wild-type GDH, but it could be supported by Ca(2+), Sr(2+) or Ba(2+) ions. This was competitively inhibited by Mg(2+). This result, together with studies on the kinetics of the modified enzymes have led to the conclusion that, although a Ca(2+) ion is able to form part of the active site of the genetically modified GDH, as in all other PQQ-containing quinoproteins, a Mg(2+) ion surprisingly replaces Ca(2+) in the active site of the wild-type GDH.