Wayne F. Anderson

Nucleotide sequence of the dmsABC operon encoding the anaerobic dimethylsulphoxide reductase of Escherichia coli

The nucleotide sequence of a 6.5 kilobasepair chromosomal DNA fragment encoding the anaerobic dimethylsulphoxide (DMSO) reductase operon of Escherichia coli has been determined. The DMSO reductase structural operon was shown to consist of three open reading frames, namely dmsABC, encoding polypeptides with predicted molecular weights of 87350, 23070, and 30789 Daltons, respectively. The DMS A polypeptide displayed a high degree of amino acid sequence homology with the single‐subunit enzyme, biotin sulphoxide reductase (bisC) and with formate dehydrogenase (fdhF), suggesting that the active site and molybdopterin cofactor binding site that is common to these enzymes is located in the DMS A subunit. A comparison of the predicted N‐terminal amino acids of the dmsA gene product to those of the 82600 subunit of purified DMSO reductase indicated that post‐translational processing of a 16 amino acid peptide at the amino terminus of DMS A had occurred. The DMS B polypeptide contains 16 cysteine residues organized in four clusters, two of which are typical of 4Fe–4S binding domains. The DMS C polypeptide is composed of eight segments of hydrophobic amino acids of appropriate length to cross the cytoplasmic membrane, suggesting that this subunit functions to anchor the enzyme to the membrane.

Structural, kinetic and proteomic characterization of acetyl phosphate-dependent bacterial protein acetylation.

The emerging view of Nε-lysine acetylation in eukaryotes is of a relatively abundant post-translational modification (PTM) that has a major impact on the function, structure, stability and/or location of thousands of proteins involved in diverse cellular processes. This PTM is typically considered to arise by the donation of the acetyl group from acetyl-coenzyme A (acCoA) to the ε-amino group of a lysine residue that is reversibly catalyzed by lysine acetyltransferases and deacetylases. Here, we provide genetic, mass spectrometric, biochemical and structural evidence that Nε-lysine acetylation is an equally abundant and important PTM in bacteria. Applying a recently developed, label-free and global mass spectrometric approach to an isogenic set of mutants, we detected acetylation of thousands of lysine residues on hundreds of Escherichia coli proteins that participate in diverse and often essential cellular processes, including translation, transcription and central metabolism. Many of these acetylations were regulated in an acetyl phosphate (acP)-dependent manner, providing compelling evidence for a recently reported mechanism of bacterial Nε-lysine acetylation. These mass spectrometric data, coupled with observations made by crystallography, biochemistry, and additional mass spectrometry showed that this acP-dependent acetylation is both non-enzymatic and specific, with specificity determined by the accessibility, reactivity and three-dimensional microenvironment of the target lysine. Crystallographic evidence shows acP can bind to proteins in active sites and cofactor binding sites, but also potentially anywhere molecules with a phosphate moiety could bind. Finally, we provide evidence that acP-dependent acetylation can impact the function of critical enzymes, including glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, and RNA polymerase.

Crystal structures of YkuI and its complex with second messenger cyclic Di-GMP suggest catalytic mechanism of phosphodiester bond cleavage by EAL domains.

Cyclic di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger that is involved in the regulation of cell surface-associated traits and the persistence of infections. Omnipresent GGDEF and EAL domains, which occur in various combinations with regulatory domains, catalyze c-di-GMP synthesis and degradation, respectively. The crystal structure of full-length YkuI from Bacillus subtilis, composed of an EAL domain and a C-terminal PAS-like domain, has been determined in its native form and in complex with c-di-GMP and Ca2+. The EAL domain exhibits a triose-phosphate isomerase-barrel fold with one antiparallel β-strand. The complex with c-di-GMP-Ca2+ defines the active site of the putative phosphodiesterase located at the C-terminal end of the β-barrel. The EAL motif is part of the active site with Glu-33 of the motif being involved in cation coordination. The structure of the complex allows the proposal of a phosphodiesterase mechanism, in which the divalent cation and the general base Glu-209 activate a catalytic water molecule for nucleophilic in-line attack on the phosphorus. The C-terminal domain closely resembles the PAS-fold. Its pocket-like structure could accommodate a yet unknown ligand. YkuI forms a tight dimer via EAL-EAL and trans EAL-PAS-like domain association. The possible regulatory significance of the EAL-EAL interface and a mechanism for signal transduction between sensory and catalytic domains of c-di-GMP-specific phosphodiesterases are discussed.

Proposed α-helical super-secondary structure associated with protein-DNA recognition

Abstract Knowledge of the three-dimensional structure of the bacteriophage λ Cro repressor, combined with an analysis of amino acid sequences and DNA coding sequences for this and other proteins that recognize and bind specific base sequences of double-helical DNA, suggests that a portion of the structure of the Cro repressor that is involved in DNA binding also occurs in the Cro protein from bacteriophage 434, the cII protein from bacteriophage λ, the Salmonella phage P22 c2 repressor and the cI repressor from bacteriophage λ. This α-helical super-secondary structure may be a common structural motif in proteins that bind double-helical DNA in a base sequence-specific manner.

MEK4 Function, Genistein Treatment, and Invasion of Human Prostate Cancer Cells

BACKGROUND Dietary intake of genistein by patients with prostate cancer has been associated with decreased metastasis and mortality. Genistein blocks activation of p38 mitogen-activated protein kinase and thus inhibits matrix metalloproteinase-2 (MMP-2) expression and cell invasion in cultured cells and inhibits metastasis of human prostate cancer cells in mice. We investigated the target for genistein in prostate cancer cells. METHODS Prostate cell lines PC3-M, PC3, 1532NPTX, 1542NPTX, 1532CPTX, and 1542CPTX were used. All cell lines were transiently transfected with a constitutively active mitogen-activated protein kinase kinase 4 (MEK4) expression vector (to increase MEK4 expression), small interfering RNA against MEK4 (to decrease MEK4 expression), or corresponding control constructs. Cell invasion was assessed by a Boyden chamber assay. Gene expression was assessed by a quantitative reverse transcription-polymerase chain reaction. Protein expression was assessed by Western blot analysis. Modeller and AutoDock programs were used for modeling of the structure of MEK4 protein and ligand docking, respectively. MMP-2 transcript levels were assessed in normal prostate epithelial cells from 24 patients with prostate cancer from a phase II randomized trial comparing genistein treatment with no treatment. Statistical significance required a P value of .050 or less. All statistical tests were two-sided. RESULTS Overexpression of MEK4 increased MMP-2 expression and cell invasion in all six cell lines. Decreased MEK4 expression had the opposite effects. Modeling showed that genistein bound to the active site of MEK4. Genistein inhibited MEK4 kinase activity with a half maximal inhibitory concentration of 0.40 microM (95% confidence interval [CI] = 0.36 to 0.45 muM). The MMP-2 transcript level in normal prostate epithelial cells was statistically significantly higher in the untreated group (100%) than in the genistein-treated group (24%; difference = 76%, 95% CI = 38% to 115%; P = .045). CONCLUSIONS We identified MEK4 as a proinvasion protein in six human prostate cancer cell lines and the target for genistein. We showed, to our knowledge for the first time, that genistein treatment, compared with no treatment, was associated with decreased levels of MMP-2 transcripts in normal prostate cells from prostate cancer-containing tissue.

Many gene-regulatory proteins appear to have a similar α-helical fold that binds DNA and evolved from a common precursor

SummaryAmino acid and DNA sequence comparisons suggest that many sequence-specific DNA-binding proteins have in common and homologous region of about 22 amino acids. This region corresponds to two consecutive α-helices that occur in bot Cro and cI repressor proteins of bacteriophage λ and in catabolite gene activator protein ofEscherichia coli and are presumed to interact with DNA. The results obtained here suggest that this α-helical DNA-binding fold occurs in many proteins that regulate gene expression. It also appears that this DNA-binding unit evolved from a common evolutionary precursor.

Comparison of the structures of Cro and λ repressor proteins from bacteriophage λ

The three-dimensional structures of cro repressor protein and of the amino-terminal domain of λ repressor protein, both from bacteriophage λ, are compared. The second and third α-helices, α2 and α3, are shown to have essentially identical conformations in the two proteins, confirming the significance of the amino acid sequence homology previously noted between these and other DNA binding proteins in the region corresponding to these helices. The correspondence between the two-helical units in cro and λ repressor protein is better than the striking agreement noted previously between two-helical units in cro and catabolite gene-activator protein. Parts of the first α-helices of repressor and cro show a structural correspondence that suggests a revised sequence homology between the two proteins in their extreme amino-terminal regions. In particular, there is a short loop between the α1 and α2 helices of λ repressor that is missing from cro. This structural difference may account for the observed differences found with different cros and repressors in the pattern of phosphates whose ethylation prevents the binding of these proteins to their specific recognition sites. Although the two proteins have strikingly similar α2-α3 helical units that are presumed to bind to DNA in an essentially similar manner, stereochemical restrictions prevent the α2-α3 units of the respective proteins aligning on the DNA in exactly the same way.

Metabolic features of chronic fatigue syndrome

Significance Chronic fatigue syndrome is a multisystem disease that causes long-term pain and disability. It is difficult to diagnose because of its protean symptoms and the lack of a diagnostic laboratory test. We report that targeted, broad-spectrum metabolomics of plasma not only revealed a characteristic chemical signature but also revealed an unexpected underlying biology. Metabolomics showed that chronic fatigue syndrome is a highly concerted hypometabolic response to environmental stress that traces to mitochondria and was similar to the classically studied developmental state of dauer. This discovery opens a fresh path for the rational development of new therapeutics and identifies metabolomics as a powerful tool to identify the chemical differences that contribute to health and disease. More than 2 million people in the United States have myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). We performed targeted, broad-spectrum metabolomics to gain insights into the biology of CFS. We studied a total of 84 subjects using these methods. Forty-five subjects (n = 22 men and 23 women) met diagnostic criteria for ME/CFS by Institute of Medicine, Canadian, and Fukuda criteria. Thirty-nine subjects (n = 18 men and 21 women) were age- and sex-matched normal controls. Males with CFS were 53 (±2.8) y old (mean ± SEM; range, 21–67 y). Females were 52 (±2.5) y old (range, 20–67 y). The Karnofsky performance scores were 62 (±3.2) for males and 54 (±3.3) for females. We targeted 612 metabolites in plasma from 63 biochemical pathways by hydrophilic interaction liquid chromatography, electrospray ionization, and tandem mass spectrometry in a single-injection method. Patients with CFS showed abnormalities in 20 metabolic pathways. Eighty percent of the diagnostic metabolites were decreased, consistent with a hypometabolic syndrome. Pathway abnormalities included sphingolipid, phospholipid, purine, cholesterol, microbiome, pyrroline-5-carboxylate, riboflavin, branch chain amino acid, peroxisomal, and mitochondrial metabolism. Area under the receiver operator characteristic curve analysis showed diagnostic accuracies of 94% [95% confidence interval (CI), 84–100%] in males using eight metabolites and 96% (95% CI, 86–100%) in females using 13 metabolites. Our data show that despite the heterogeneity of factors leading to CFS, the cellular metabolic response in patients was homogeneous, statistically robust, and chemically similar to the evolutionarily conserved persistence response to environmental stress known as dauer.

STRUCTURE-FUNCTION CORRELATES OF AUTOANTIBODIES TO NUCLEIC ACIDS. LESSONS FROM IMMUNOCHEMICAL, GENETIC AND STRUCTURAL STUDIES

Nucleic acid binding autoantibodies are the hallmark of the human autoimmune disease, systemic lupus erythematosus (SLE) and are also prevalent in mouse models of this disease. The immunologic stimuli for the production of these antibodies as well as their pathogenic mechanisms are not well understood. However, extensive immunochemical and genetic studies, together with initial crystallographic analysis and computer modeling, have suggested several structure-function correlates which will form the basis for future research. The anti-DNA and anti-RNA autoantibodies comprise a continuous spectrum of specificities in which a delicate balance exists between the binding to the sugar-phosphate backbone and the interactions with the heterocyclic bases of the nucleic acid. Prominent in these interactions are the products of specific V-region immunoglobulin genes, some of which appear to be uniquely suitable for nucleic acid binding. Other structural elements encoded by D minigenes, N sequences and somatic mutations, help to increase the affinity of the binding interaction, and may also increase the repertoire of nucleic acid binding antibodies by combining with a relatively large number of additional V-gene products. Initial crystallographic analyses of anti-DNA antibodies indicate some fundamental differences in the structure and shape of ssDNA and dsDNA antibody combining sites. However, they also suggest a considerable degree of flexibility of both antibody and antigen, which is induced by their binding interaction.

Structural and Molecular Mechanism for Autoprocessing of MARTX Toxin of Vibrio cholerae at Multiple Sites

The multifunctional autoprocessing repeats-in-toxin (MARTX) toxin of Vibrio cholerae causes destruction of the actin cytoskeleton by covalent cross-linking of actin and inactivation of Rho GTPases. The effector domains responsible for these activities are here shown to be independent proteins released from the large toxin by autoproteolysis catalyzed by an embedded cysteine protease domain (CPD). The CPD is activated upon binding inositol hexakisphosphate (InsP6). In this study, we demonstrated that InsP6 is not simply an allosteric cofactor, but rather binding of InsP6 stabilized the CPD structure, facilitating formation of the enzyme-substrate complex. The 1.95-Å crystal structure of this InsP6-bound unprocessed form of CPD was determined and revealed the scissile bond Leu3428–Ala3429 captured in the catalytic site. Upon processing at this site, CPD was converted to a form with 500-fold reduced affinity for InsP6, but was reactivated for high affinity binding of InsP6 by cooperative binding of both a new substrate and InsP6. Reactivation of CPD allowed cleavage of the MARTX toxin at other sites, specifically at leucine residues between the effector domains. Processed CPD also cleaved other proteins in trans, including the leucine-rich protein YopM, demonstrating that it is a promiscuous leucine-specific protease.