The influence of ligand valency on aggregation mechanisms for inhibiting bacterial toxins.

Divalent and tetravalent analogues of ganglioside GM1 are potent inhibitors of cholera toxin and Escherichia coli heat-labile toxin. However, they show little increase in inherent affinity when compared to the corresponding monovalent carbohydrate ligand. Analytical ultracentrifugation and dynamic light scattering have been used to demonstrate that the multivalent inhibitors induce protein aggregation and the formation of space-filling networks. This aggregation process appears to arise when using ligands that do not match the valency of the protein receptor. While it is generally accepted that multivalency is an effective strategy for increasing the activity of inhibitors, here we show that the valency of the inhibitor also has a dramatic effect on the kinetics of aggregation and the stability of intermediate protein complexes. Structural studies employing atomic force microscopy have revealed that a divalent inhibitor induces head-to-head dimerization of the protein toxin en route to higher aggregates.

A beta-sheet interaction interface directs the tetramerisation of the Miz-1 POZ domain.

The POZ/BTB domain is an evolutionarily conserved motif found in approximately 40 zinc-finger transcription factors (POZ-ZF factors). Several POZ-ZF factors are implicated in human cancer, and POZ domain interaction interfaces represent an attractive target for therapeutic intervention. Miz-1 (Myc-interacting zinc-finger protein) is a POZ-ZF factor that regulates DNA-damage-induced cell cycle arrest and plays an important role in human cancer by virtue of its interaction with the c-Myc and BCL6 oncogene products. The Miz-1 POZ domain mediates both self-association and the recruitment of non-POZ partners. POZ-ZF factors generally function as homodimers, although higher-order associations and heteromeric interactions are known to be physiologically important; crucially, the interaction interfaces in such large complexes have not been characterised. We report here the crystal structure of the Miz-1 POZ domain up to 2.1 A resolution. The tetrameric organisation of Miz-1 POZ reveals two types of interaction interface between subunits; an interface of alpha-helices resembles the dimerisation interface of reported POZ domain structures, whereas a novel beta-sheet interface directs the association of two POZ domain dimers. We show that the beta-sheet interface directs the tetramerisation of the Miz-1 POZ domain in solution and therefore represents a newly described candidate interface for the higher-order homo- and hetero-oligomerisation of POZ-ZF proteins in vivo.

Scanning conformational space with a library of stereo- and regiochemically diverse aminoglycoside derivatives: the discovery of new ligands for RNA hairpin sequences.

A library of stereo- and regiochemically diverse aminoglycoside derivatives was screened at 1 microM using surface plasmon resonance (SPR) against RNA hairpin models of the bacterial A-site, and the HIV viral TAR and RRE sequences. In order to double the stereochemical diversity of the library, the compounds were screened against both enantiomers of each of these sequences. Remarkably, this initial screen suggested that the same four aminoglycoside derivatives bound most tightly to all three of the RNAs, suggesting that these compounds were good RNA binders which, nonetheless, discriminated poorly between the RNA sequences. The interactions between selected isomeric aminoglycoside derivatives and the RNA hairpins were then studied in more detail using an SPR assay. Three isomeric tight-binding aminoglycoside derivatives, which had been identified from the initial screen, were found to bind more tightly to the RNA hairpins (with K(D) values in the range 0.23 to 4.7 microM) than a fourth isomeric derivative (which had K(D) values in the range 6.0 to 30 microM). The magnitude of the tightest RNA-aminoglycoside interactions stemmed, in large part, from remarkably slow dissociation of the aminoglycosides from the RNA targets. The three tight-binding aminoglycoside derivatives were found, however, to discriminate rather poorly between alternative RNA sequences with, at best, around a twenty-fold difference in affinity for alternative RNA hairpin sequences. Within the aminoglycoside derivative library studied, high affinity for an RNA target was not accompanied by good discrimination between alternative RNA sequences.

Affinity of molecular interactions in the bacteriophage phi29 DNA packaging motor.

DNA packaging in the bacteriophage phi29 involves a molecular motor with protein and RNA components, including interactions between the viral connector protein and molecules of pRNA, both of which form multimeric complexes. Data are presented to demonstrate the higher order assembly of pRNA together with the affinity of pRNA:pRNA and pRNA:connector interactions, which are used to propose a model for motor function. In solution, pRNA can form dimeric and trimeric multimers in a magnesium-dependent manner, with dissociation constants for multimerization in the micromolar range. pRNA:connector binding is also facilitated by the presence of magnesium ions, with a nanomolar apparent dissociation constant for the interaction. From studies with a mutant pRNA, it appears that multimerization of pRNA is not essential for connector binding and it is likely that connector protein is involved in the stabilization of higher order RNA multimers. It is proposed that magnesium ions may promote conformational change that facilitate pRNA:connector interactions, essential for motor function.

Structural basis of RNA binding discrimination between bacteriophages Qbeta and MS2.

Sequence-specific interactions between RNA stem-loops and coat protein (CP) subunits play vital roles in the life cycles of the RNA bacteriophages, e.g., by allowing translational repression of their replicase cistrons and tagging their own RNA genomes for encapsidation. The CPs of bacteriophages Qbeta and MS2 each discriminate in favor of their cognate translational operators, even in the presence of closely related operators from other phages in vivo. Discrete mutations within the MS2 CP have been shown to relax this discrimination in vitro. We have determined the structures of eight complexes between such mutants and both MS2 and Qbeta stem-loops with X-ray crystallography. In conjunction with previously determined in vivo repression data, the structures enable us to propose the molecular basis for the discrimination mechanism.

Structural analysis of Bacillus subtilis SPP1 phage helicase loader protein G39P.

The Bacillus subtilis SPP1 phage-encoded protein G39P is a loader and inhibitor of the phage G40P replicative helicase involved in the initiation of DNA replication. We have carried out a full x-ray crystallographic and preliminary NMR analysis of G39P and functional studies of the protein, including assays for helicase binding by a number of truncated mutant forms, in an effort to improve our understanding of how it both interacts with the helicase and with the phage replisome organizer, G38P. Our structural analyses reveal that G39P has a completely unexpected bipartite structure comprising a folded N-terminal domain and an essentially unfolded C-terminal domain. Although G39P has been shown to bind its G40P target with a 6:6 stoichiometry, our crystal structure and other biophysical characterization data reveal that the protein probably exists predominantly as a monomer in solution. The G39P protein is proteolytically sensitive, and our binding assays show that the C-terminal domain is essential for helicase interaction and that removal of just the 14 C-terminal residues abolishes interaction with the helicase in vitro. We propose a number of possible scenarios in which the flexibility of the C-terminal domain of G39P and its proteolytic sensitivity may have important roles for the function of G39P in vivo that are consistent with other data on SPP1 phage DNA replication.

Kinetic Studies on the Redox Interconversion of GOase(semi) and GOase(ox) Forms of Galactose Oxidase with Inorganic Complexes as Redox Partners.

Redox interconversions between the GOase(semi) (Cu(II), Tyr) and tyrosyl radical containing GOase(ox) (Cu(II), Tyr(*)) oxidation states of the Cu-containing enzyme galactose oxidase (GOase) from Fusarium NRRL 2903 have been studied. The inorganic complexes [Fe(CN)(6)](3)(-) (410 mV), [Co(phen)(3)](3+) (370 mV), [W(CN)(8)](3)(-) (530 mV), and [Co(dipic)(2)](-) (362 mV) (E degrees ' values vs NHE; dipic = 2,6-dicarboxylatopyridine) were used as oxidants for GOase(semi), and [Fe(CN)(6)](4)(-) and [Co(phen)(3)](2+) as reductants for GOase(ox). On oxidation of GOase(semi) a radical is generated at the coordinated phenolate of Tyr-272 to give GOase(ox). The one-electron reduction potential E degrees ' (25 degrees C) for the GOase(ox)/GOase(semi) couple varies with pH and is 400 mV vs NHE at pH 7.5, the smallest value so far observed for a tyrosyl radical. The reactions are very sensitive to pH, or more precisely to pK(a) values of GOase(semi) and GOase(ox), and the charge on the inorganic reagent. For example, with [Fe(CN)(6)](3)(-) as oxidant, the rate constant (25 degrees C)/M(-)(1) s(-)(1) of 0.16 x 10(3) (pH approximately 9.5) increases to 4.3 x 10(3) (pH approximately 5.5), while for [Co(phen)(3)](3+) a value of 4.9 x 10(3) (pH approximately 9.5) decreases to 0.04 x 10(3) (pH approximately 5.5), I = 0.100 M (NaCl). From the kinetics a single GOase(semi) acid dissociation process, pK(a) = 8.0 (average), has been confirmed by UV-vis spectrophotometric studies (7.9). The corresponding value for GOase(ox) is 6.7. No comparable kinetic or spectrophotometric pH dependences are observed with the Tyr495Phe variant, indicating the axial Tyr-495 as the site of protonation. Neutral CH(3)CO(2)H and HN(3) species bind at the substrate binding site of GOase(semi), thus mimicking the behavior of primary alcohols RCH(2)OH, the natural substrate of GOase. On coordination, loss of a proton occurs, and inhibition of the oxidation with [Fe(CN)(6)](3)(-) is observed.