Christopher M. Barbieri

Universally applicable methods for monitoring response regulator aspartate phosphorylation both in vitro and in vivo using Phos-tag™ based reagents

Recent development of the phosphate chelator, Phos-tag, together with Phos-tag pendant reagents, has provided new methods for detection of phosphorylated serine, threonine, tyrosine, and histidine residues in phosphoproteins. We have investigated the use of Phos-tag for detection and quantification of phospho-aspartate in response regulator proteins that function within two-component signaling systems. Alternative methods are especially important, because the labile nature of the acylphosphate bond in response regulator proteins has restricted the application of many traditional methods of phosphoprotein analysis. We demonstrate that Phos-tag gel stain can be used to detect phospho-Asp in response regulators and that Phos-tag acrylamide gel electrophoresis can be used to separate phosphorylated and unphosphorylated forms of response regulator proteins. The latter method, coupled to Western blot analysis, enables detection of specific phosphorylated proteins in complex mixtures such as cell lysates. Standards of phosphorylated proteins can be used to correct for hydrolysis of the labile phospho-Asp bond that invariably occurs during analysis. We have employed Phos-tag methods to characterize the phosphorylation state of the Escherichia coli response regulator PhoB both in vitro, using purified protein, and in vivo, by analyzing lysates of cells grown under different conditions of induction of the PhoR/PhoB phosphate assimilation pathway.

Structure of the Staphylococcus aureus AgrA LytTR Domain Bound to DNA Reveals a Beta Fold with an Unusual Mode of Binding

The LytTR domain is a DNA-binding motif found within the AlgR/AgrA/LytR family of transcription factors that regulate virulence factor and toxin gene expression in pathogenic bacteria. This previously uncharacterized domain lacks sequence similarity with proteins of known structure. The crystal structure of the DNA-binding domain of Staphylococcus aureus AgrA complexed with a DNA pentadecamer duplex has been determined at 1.6 A resolution. The structure establishes a 10-stranded beta fold for the LytTR domain and reveals its mode of interaction with DNA. Residues within loop regions of AgrA contact two successive major grooves and the intervening minor groove on one face of the oligonucleotide duplex, inducing a substantial bend in the DNA. Loss of DNA binding upon substitution of key interacting residues in AgrA supports the observed binding mode. This mode of protein-DNA interaction provides a potential target for future antimicrobial drug design.

Defining the mode, energetics and specificity with which a macrocyclic hexaoxazole binds to human telomeric G-quadruplex DNA

Oxazole-containing macrocycles represent a promising class of anticancer agents that target G-quadruplex DNA. We report the results of spectroscopic studies aimed at defining the mode, energetics and specificity with which a hexaoxazole-containing macrocycle (HXDV) binds to the intramolecular quadruplex formed by the human telomeric DNA model oligonucleotide d(T2AG3)4 in the presence of potassium ions. HXDV binds solely to the quadruplex nucleic acid form, but not to the duplex or triplex form. HXDV binds d(T2AG3)4 with a stoichiometry of two drug molecules per quadruplex, with these binding reactions being coupled to the destacking of adenine residues from the terminal G-tetrads. HXDV binding to d(T2AG3)4 does not alter the length of the quadruplex. These collective observations are indicative of a nonintercalative ‘terminal capping’ mode of interaction in which one HXDV molecule binds to each end of the quadruplex. The binding of HXDV to d(T2AG3)4 is entropy driven, with this entropic driving force reflecting contributions from favorable drug-induced alterations in the configurational entropy of the host quadruplex as well as in net hydration. The ‘terminal capping’ mode of binding revealed by our studies may prove to be a general feature of the interactions between oxazole-containing macrocyclic ligands (including telomestatin) and intramolecular DNA quadruplexes.

Regulation of Response Regulator Autophosphorylation through Interdomain Contacts

DNA-binding response regulators (RRs) of the OmpR/PhoB subfamily alternate between inactive and active conformational states, with the latter having enhanced DNA-binding affinity. Phosphorylation of an aspartate residue in the receiver domain, usually via phosphotransfer from a cognate histidine kinase, stabilizes the active conformation. Many of the available structures of inactive OmpR/PhoB family proteins exhibit extensive interfaces between the N-terminal receiver and C-terminal DNA-binding domains. These interfaces invariably involve the α4-β5-α5 face of the receiver domain, the locus of the largest differences between inactive and active conformations and the surface that mediates dimerization of receiver domains in the active state. Structures of receiver domain dimers of DrrB, DrrD, and MtrA have been determined, and phosphorylation kinetics were analyzed. Analysis of phosphotransfer from small molecule phosphodonors has revealed large differences in autophosphorylation rates among OmpR/PhoB RRs. RRs with substantial domain interfaces exhibit slow rates of phosphorylation. Rates are greatly increased in isolated receiver domain constructs. Such differences are not observed between autophosphorylation rates of full-length and isolated receiver domains of a RR that lacks interdomain interfaces, and they are not observed in histidine kinase-mediated phosphotransfer. These findings suggest that domain interfaces restrict receiver domain conformational dynamics, stabilizing an inactive conformation that is catalytically incompetent for phosphotransfer from small molecule phosphodonors. Inhibition of phosphotransfer by domain interfaces provides an explanation for the observation that some RRs cannot be phosphorylated by small molecule phosphodonors in vitro and provides a potential mechanism for insulating some RRs from small molecule-mediated phosphorylation in vivo.

G-Quadruplexes Induce Apoptosis in Tumor Cells

Several G-rich oligodeoxynucleotides (ODNs), which are capable of forming G-quadruplexes, have been shown to exhibit antiproliferative activity against tumor cell lines and antitumor activity in nude mice carrying prostate and breast tumor xenografts. However, the molecular basis for their antitumor activity remains unclear. In the current study, we showed that a variety of telomeric G-tail oligodeoxynucleotides (TG-ODNs) exhibited antiproliferative activity against many tumor cells in culture. Systematic mutational analysis of the TG-ODNs suggests that the antiproliferative activity depends on the G-quadruplex conformation of these TG-ODNs. TG-ODNs were also shown to induce poly(ADP-ribose) polymerase-1 cleavage, phosphatidylserine flipping, and caspase activation, indicative of induction of apoptosis. TG-ODN-induced apoptosis was largely ataxia telangiectasia mutated (ATM) dependent. Furthermore, TG-ODN-induced apoptosis was inhibited by the c-Jun NH(2)-terminal kinase (JNK) inhibitor SP600125. Indeed, TG-ODNs were shown to activate the JNK pathway in an ATM-dependent manner as evidenced by elevated phosphorylation of JNK and c-Jun. Interestingly, a number of G-quadruplex ODNs (GQ-ODN) derived from nontelomeric sequences also induced ATM/JNK-dependent apoptosis, suggesting a possible common mechanism of tumor cell killing by GQ-ODNs.

Differential Selectivity of Natural and Synthetic Aminoglycosides towards the Eukaryotic and Prokaryotic Decoding A Sites.

The lack of absolute prokaryotic selectivity of natural antibiotics is widespread and is a significant clinical problem. The use of this disadvantage of aminoglycoside antibiotics for the possible treatment of human genetic diseases is extremely challenging. Here, we have used a combination of biochemical and structural analysis to compare and contrast the molecular mechanisms of action and the structure–activity relationships of a new synthetic aminoglycoside, NB33, and a structurally similar natural aminoglycoside apramycin. The data presented herein demonstrate the general molecular principles that determine the decreased selectivity of apramycin for the prokaryotic decoding site, and the increased selectivity of NB33 for the eukaryotic decoding site. These results are therefore extremely beneficial for further research on both the design of new aminoglycoside‐based antibiotics with diminished deleterious effects on humans, as well as the design of new aminoglycoside‐based structures that selectively target the eukaryotic ribosome.

Comprehensive Analysis of OmpR Phosphorylation, Dimerization and DNA Binding Supports a Canonical Model for Activation

The OmpR/PhoB family of response regulators (RRs) is the largest class of two-component system signal transduction proteins. Extensive biochemical and structural characterization of these transcription factors has provided insights into their activation and DNA-binding mechanisms. For the most part, OmpR/PhoB family proteins are thought to become activated through phosphorylation from their cognate histidine kinase partners, which in turn facilitates an allosteric change in the RR, enabling homodimerization and subsequently enhanced DNA binding. Incongruently, it has been suggested that OmpR, the eponymous member of this RR family, becomes activated via different mechanisms, whereby DNA binding plays a central role in facilitating dimerization and phosphorylation. Characterization of the rate and extent of the phosphorylation of OmpR and OmpR DNA-binding mutants following activation of the EnvZ/OmpR two-component system shows that DNA binding is not essential for phosphorylation of OmpR in vivo. In addition, detailed analyses of the energetics of DNA binding and dimerization of OmpR in both its unphosphorylated and phosphorylated state indicate that phosphorylation enhances OmpR dimerization and that this dimerization enhancement is the energetic driving force for phosphorylation-mediated regulation of OmpR-DNA binding. These findings suggest that OmpR phosphorylation-mediated activation follows the same paradigm as the other members of the OmpR/PhoB family of RRs in contrast to previously proposed models of OmpR activation.

A hybrid NMR/SAXS-based approach for discriminating oligomeric protein interfaces using Rosetta

Oligomeric proteins are important targets for structure determination in solution. While in most cases the fold of individual subunits can be determined experimentally, or predicted by homology‐based methods, protein–protein interfaces are challenging to determine de novo using conventional NMR structure determination protocols. Here we focus on a member of the bet‐V1 superfamily, Aha1 from Colwellia psychrerythraea. This family displays a broad range of crystallographic interfaces none of which can be reconciled with the NMR and SAXS data collected for Aha1. Unlike conventional methods relying on a dense network of experimental restraints, the sparse data are used to limit conformational search during optimization of a physically realistic energy function. This work highlights a new approach for studying minor conformational changes due to structural plasticity within a single dimeric interface in solution. Proteins 2015; 83:309–317.

Defining the Molecular Forces That Determine the Impact of Neomycin on Bacterial Protein Synthesis: Importance of the 2′-Amino Functionality

ABSTRACT 2-Deoxystreptamine (2-DOS) aminoglycosides exert their antibiotic actions by binding to the A site of the 16S rRNA and interfering with bacterial protein synthesis. However, the molecular forces that govern the antitranslational activities of aminoglycosides are poorly understood. Here, we describe studies aimed at elucidating these molecular forces. In this connection, we compare the bactericidal, antitranslational, and rRNA binding properties of the 4,5-disubstituted 2-DOS aminoglycoside neomycin (Neo) and a conformationally restricted analog of Neo (CR-Neo) in which the 2′-nitrogen atom is covalently conjugated to the 5′′-carbon atom. The bactericidal potency of Neo exceeds that of CR-Neo, with this enhanced antibacterial activity reflecting a correspondingly enhanced antitranslational potency. Time-resolved fluorescence anisotropy studies suggest that the enhanced antitranslational potency of Neo relative to that of CR-Neo is due to a greater extent of drug-induced reduction in the mobilities of the nucleotides at positions 1492 and 1493 of the rRNA A site. Buffer- and salt-dependent binding studies, coupled with high-resolution structural information, point to electrostatic contacts between the 2′-amino functionality of Neo and the host rRNA as being an important modulator of 1492 and 1493 base mobilities and therefore antitranslational activities.

Structures of apo- and ssDNA-bound YdbC from Lactococcus lactis uncover the function of protein domain family DUF2128 and expand the single-stranded DNA-binding domain proteome

Single-stranded DNA (ssDNA) binding proteins are important in basal metabolic pathways for gene transcription, recombination, DNA repair and replication in all domains of life. Their main cellular role is to stabilize melted duplex DNA and protect genomic DNA from degradation. We have uncovered the molecular function of protein domain family domain of unknown function DUF2128 (PF09901) as a novel ssDNA binding domain. This bacterial domain strongly associates into a dimer and presents a highly positively charged surface that is consistent with its function in non-specific ssDNA binding. Lactococcus lactis YdbC is a representative of DUF2128. The solution NMR structures of the 20 kDa apo-YdbC dimer and YdbC:dT19G1 complex were determined. The ssDNA-binding energetics to YdbC were characterized by isothermal titration calorimetry. YdbC shows comparable nanomolar affinities for pyrimidine and mixed oligonucleotides, and the affinity is sufficiently strong to disrupt duplex DNA. In addition, YdbC binds with lower affinity to ssRNA, making it a versatile nucleic acid-binding domain. The DUF2128 family is related to the eukaryotic nuclear protein positive cofactor 4 (PC4) family and to the PUR family both by fold similarity and molecular function.