Structural Basis for Dimerization and DNA Binding of Transcription Factor FLI1

FLI1 (Friend leukemia integration 1) is a metazoan transcription factor that is upregulated in a number of cancers. In addition, rearrangements of the <i>fli1</i> gene cause sarcomas, leukemias, and lymphomas. These rearrangements encode oncogenic transcription factors, in which the DNA binding domain (DBD or ETS domain) of FLI1 on the C-terminal side is fused to a part of an another protein on the N-terminal side. Such abnormal cancer cell-specific fusions retain the DNA binding properties of FLI1 and acquire non-native protein–protein or protein–nucleic acid interactions of the substituted region. As a result, these fusions trigger oncogenic transcriptional reprogramming of the host cell. Interactions of FLI1 fusions with other proteins and with itself play a critical role in the oncogenic regulatory functions, and they are currently under intense scrutiny, mechanistically and as potential novel anticancer drug targets. We report elusive crystal structures of the FLI1 DBD, alone and in complex with cognate DNA containing a GGAA recognition sequence. Both structures reveal a previously unrecognized dimer of this domain, consistent with its dimerization in solution. The homodimerization interface is helix-swapped and dominated by hydrophobic interactions, including those between two interlocking Phe362 residues. A mutation of Phe362 to an alanine disrupted the propensity of this domain to dimerize without perturbing its structure or the DNA binding function, consistent with the structural observations. We propose that FLI1 DBD dimerization plays a role in transcriptional activation and repression by FLI1 and its fusions at promoters containing multiple FLI1 binding sites

Steady-state kinetics of NEO acetylation by <i>Mt</i>Eis and their analysis.

<p><b>A.</b> Representative dependences of the steady-state rate of acetylation of NEO on the concentration of NEO at different concentrations of AcCoA, as specified. <b>B.</b> Representative dependences of the steady-state rate of acetylation of NEO on the concentration of AcCoA at different concentrations of NEO, as specified. <b>C.</b> Dependence of the apparent rate constant (<i>k</i>_cat,AG), as obtained from data shown in panel <b>A</b>, on the concentration of AcCoA. <b>D.</b> Dependence of the apparent <i>K</i>_m,AG, as obtained from data shown in panel <b>A</b>, on the concentration of AcCoA. The theoretical curve in <b>D</b> is the best simultaneous fit of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092370#pone.0092370.e003" target="_blank">eq. (3)</a> to these values and those for acetylation of KAN as described in the text.</p

Deciphering Nature’s Intricate Way of <i>N</i>,<i>S</i>-Dimethylating l‑Cysteine: Sequential Action of Two Bifunctional Adenylation Domains

Dimethylation of amino acids consists of an interesting and puzzling series of events that could be achieved, during nonribosomal peptide biosynthesis, either by a single adenylation (A) domain interrupted by a methyltransferase (M) domain or by the sequential action of two of such independent enzymes. Herein, to establish the method by which Nature <i>N</i>,<i>S</i>-dimethylates l-Cys, we studied its formation during thiochondrilline A biosynthesis by evaluating TioS­(A_3aM_3SA_3bT₃) and TioN­(A_aM_NA_b). This study not only led to identification of the exact pathway followed in Nature by these two enzymes for <i>N</i>,<i>S</i>-dimethylation of l-Cys, but also revealed that a single interrupted A domain can <i>N</i>,<i>N</i>-dimethylate amino acids, a novel phenomenon in the nonribosomal peptide field. These findings offer important and useful insights for the development and engineering of novel interrupted A domain enzymes to serve, in the future, as tools for combinatorial biosynthesis

Aminoglycoside Multiacetylating Activity of the Enhanced Intracellular Survival Protein from <i>Mycobacterium smegmatis</i> and Its Inhibition

The enhanced intracellular survival (Eis) protein improves the survival of <i>Mycobacterium smegmatis</i> (<i>Msm</i>) in macrophages and functions as the acetyltransferase responsible for kanamycin A resistance, a hallmark of extensively drug-resistant (XDR) tuberculosis, in a large number of <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) clinical isolates. We recently demonstrated that Eis from <i>Mtb</i> (Eis_<i>Mtb</i>) efficiently multiacetylates a variety of aminoglycoside (AG) antibiotics. Here, to gain insight into the origin of substrate selectivity of AG multiacetylation by Eis, we analyzed AG acetylation by Eis_<i>Msm</i>, investigated its inhibition, and compared these functions to those of Eis_<i>Mtb</i>. Even though for several AGs the multiacetylation properties of Eis_<i>Msm</i> and Eis_<i>Mtb</i> are similar, there are three major differences. (i) Eis_<i>Msm</i> diacetylates apramycin, a conformationally constrained AG, which Eis_<i>Mtb</i> cannot modify. (ii) Eis_<i>Msm</i> triacetylates paromomycin, which can be only diacetylated by Eis_<i>Mtb</i>. (iii) Eis_<i>Msm</i> only monoacetylates hygromycin, a structurally unique AG that is diacetylated by Eis_<i>Mtb</i>. Several nonconserved amino acid residues lining the AG-binding pocket of Eis are likely responsible for these differences between the two Eis homologues. Specifically, we propose that because the AG-binding pocket of Eis_<i>Msm</i> is more open than that of Eis_<i>Mtb</i>, it accommodates apramycin for acetylation in Eis_<i>Msm</i>, but not in Eis_<i>Mtb</i>. We also demonstrate that inhibitors of Eis_<i>Mtb</i> that we recently discovered can inhibit Eis_<i>Msm</i> activity. These observations help define the structural origins of substrate preference among Eis homologues and suggest that Eis_<i>Mtb</i> inhibitors may be applied against all pathogenic mycobacteria to overcome AG resistance caused by Eis upregulation

List of <i>M. tuberculosis</i> isolates used in this study.

<p>−, no mutation, CIP, ciprofloxacin, OFX, ofloxacin, LVX, levofloxacin, MXF, moxifloxacin. Resistance defined as; CIP (>2 µg/mL), OFX (>2 µg/mL), LVX (>1 µg/mL) and MXF (>0.5 µg/mL). Highlighted in bold font are MICs considered resistant to that specific FQ.</p

MIC of <i>gyrB</i> transductants/mutants.

<p>WT, wild type. Resistance defined as; CIP (>2 µg/mL), OFX (>2 µg/mL), LVX (>1 µg/mL) and MXF (>0.5 µg/mL). Highlighted in bold font are MICs considered resistant to that specific FQ.</p

A structural model of <i>M. tuberculosis</i> gyrase inhibition.

<p><b>A.</b> A model of <i>M. tuberculosis</i> gyrase in complex with DNA and levofloxacin. The model was built based on the crystal structure of the complex of <i>Streptococcus pneumoniae</i> (PDB ID: 3K9F) as described in Materials and Methods. The GyrA subunit is shown in yellow, GyrB is in green, DNA is in orange, the levofloxacin molecule is shown as pink sticks. <b>B.</b> A zoomed-in view of the quinolone binding site. Residues that directly interact with the quinolone and whose mutations cause resistance are shown by blue sticks. The two residues whose double, but not single, mutations cause fluoroquinolone resistance are shown by red sticks.</p

Structural Insight into MtmC, a Bifunctional Ketoreductase-Methyltransferase Involved in the Assembly of the Mithramycin Trisaccharide Chain

More and more post-PKS tailoring enzymes are recognized as being multifunctional and codependent on other tailoring enzymes. One of the recently discovered intriguing examples is MtmC, a bifunctional TDP-4-keto-d-olivose ketoreductase-methyltransferase, whichin codependence with glycosyltransferase MtmGIVis a key contributor to the biosynthesis of the critical trisaccharide chain of the antitumor antibiotic mithramycin (MTM), produced by <i>Streptomyces argillaceus</i>. We report crystal structures of three binary complexes of MtmC with its methylation cosubstrate SAM, its coproduct SAH, and a nucleotide TDP as well as crystal structures of two ternary complexes, MtmC-SAH-TDP-4-keto-d-olivose and MtmC-SAM-TDP, in the range of 2.2–2.7 Å resolution. The structures reveal general and sugar-specific recognition and catalytic structural features of MtmC. Depending on the catalytic function that is conducted by MtmC, it must bind either NADPH or SAM in the same cofactor binding pocket. A tyrosine residue (Tyr79) appears as a lid covering the sugar moiety of the substrate during the methyl transfer reaction. This residue swings out of the active site by ∼180° in the absence of the substrate. This unique conformational change likely serves to release the methylated product and, possibly, to open the active site for binding the bulkier cosubstrate NADPH prior to the reduction reaction

Amino acid substitutions in <i>M. tuberculosis gyrB</i>.

<p><b>A</b>, numbering system according to <a href="http://genolist.pasteur.fr/TubercuList/annotation" target="_blank">http://genolist.pasteur.fr/TubercuList/annotation</a>, <b>B,</b> numbering system according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039754#pone.0039754-Zhou1" target="_blank">[45] </a><b>C</b>, numbering system according to <a href="http://tuberculist.epfl.ch/index.html" target="_blank">http://tuberculist.epfl.ch/index.html</a> annotation. Highlighted in bold are the mutations we analyzed in this study.</p

Quantifying Additive Interactions of the Osmolyte Proline with Individual Functional Groups of Proteins: Comparisons with Urea and Glycine Betaine, Interpretation of <i>m</i>‑Values

To quantify interactions of the osmolyte l-proline with protein functional groups and predict their effects on protein processes, we use vapor pressure osmometry to determine chemical potential derivatives dμ₂/d<i>m</i>₃ = μ₂₃, quantifying the preferential interactions of proline (component 3) with 21 solutes (component 2) selected to display different combinations of aliphatic or aromatic C, amide, carboxylate, phosphate or hydroxyl O, and amide or cationic N surface. Solubility data yield μ₂₃ values for four less-soluble solutes. Values of μ₂₃ are dissected using an ASA-based analysis to test the hypothesis of additivity and obtain α-values (proline interaction potentials) for these eight surface types and three inorganic ions. Values of μ₂₃ predicted from these α-values agree with the experiment, demonstrating additivity. Molecular interpretation of α-values using the solute partitioning model yields partition coefficients (<i>K</i>_p) quantifying the local accumulation or exclusion of proline in the hydration water of each functional group. Interactions of proline with native protein surfaces and effects of proline on protein unfolding are predicted from α-values and ASA information and compared with experimental data, with results for glycine betaine and urea, and with predictions from transfer free energy analysis. We conclude that proline stabilizes proteins because of its unfavorable interactions with (exclusion from) amide oxygens and aliphatic hydrocarbon surfaces exposed in unfolding and that proline is an effective in vivo osmolyte because of the osmolality increase resulting from its unfavorable interactions with anionic (carboxylate and phosphate) and amide oxygens and aliphatic hydrocarbon groups on the surface of cytoplasmic proteins and nucleic acids