Probing the molecular mechanism of action of co-repressor in the<i>E.coli</i>methionine repressor-operator complex using surface plasmon resonance (SPR)

Parsons, Isobel D.; Persson, Björn; Mekhalfia, Abdelaziz; Blackburn, G. Michael; Stockley, Peter G.

doi:10.1093/nar/23.2.211

Cited by 61 publications

(50 citation statements)

References 13 publications

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“…Unfortunately, we were not able to determine the pH-dependent binding of AzaAdoMet to aLSMT due to the aggregation of the enzyme at pH values less than 7.0. Nonetheless, our data are consistent with surface plasmon resonance studies of the Escherichia coli methionine repressor MetJ and AzaAdoMet in which ligand binding was only observed under acidic conditions, eliciting co-repression comparable with AdoMet (47). In summary, these results demonstrate that the strength of the CH⅐⅐⅐O interactions are strongly dependent on the degree of polarization of the heteroatom to which the methyl group is covalently bonded.…”

Section: Resultssupporting

confidence: 89%

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Catalytic Roles for Carbon-Oxygen Hydrogen Bonding in SET Domain Lysine Methyltransferases

Couture

Hauk

Thompson

et al. 2006

Journal of Biological Chemistry

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SET domain enzymes represent a distinct family of protein lysine methyltransferases in eukaryotes. Recent studies have yielded significant insights into the structural basis of substrate recognition and the product specificities of these enzymes. However, the mechanism by which SET domain methyltransferases catalyze the transfer of the methyl group from S-adenosyl-L-methionine to the lysine ⑀-amine has remained unresolved. To elucidate this mechanism, we have determined the structures of the plant SET domain enzyme, pea ribulose-1,5 bisphosphate carboxylase/oxygenase large subunit methyltransferase, bound to S-adenosyl-L-methionine, and its non-reactive analogs Aza-adenosyl-L-methionine and Sinefungin, and characterized the binding of these ligands to a homolog of the enzyme. The structural and biochemical data collectively reveal that S-adenosyl-L-methionine is selectively recognized through carbon-oxygen hydrogen bonds between the cofactor's methyl group and an array of structurally conserved oxygens that comprise the methyl transfer pore in the active site. Furthermore, the structure of the enzyme co-crystallized with the product ⑀-N-trimethyllysine reveals a trigonal array of carbon-oxygen interactions between the ⑀-ammonium methyl groups and the oxygens in the pore. Taken together, these results establish a central role for carbon-oxygen hydrogen bonding in aligning the cofactor's methyl group for transfer to the lysine ⑀-amine and in coordinating the methyl groups after transfer to facilitate multiple rounds of lysine methylation.Protein lysine methylation has emerged as a prominent post-translational modification in gene regulatory and intracellular signaling pathways. In the nucleus, site-specific methylation of lysines within histones, transcription factors, and mitotic proteins governs a diverse array of processes within the nucleus including gene expression, DNA damage checkpoint control, cell cycle progression, and mitosis (1). In 2000, a breakthrough in our understanding of this modification occurred with the discovery of the first histone-specific protein lysine methyltransferases (PKMTs) 4 (2). These enzymes possess a conserved catalytic SET domain, a 120-residue motif that was named for three Drosophila gene regulatory factors, SU(VAR)3-9, E(Z), and TRX (3). This domain shares no apparent sequence or structural homology with other S-adenosyl-L-methionine (AdoMet)-dependent enzymes, establishing the SET domain family as a novel class of methyltransferases. This seminal discovery heralded the identification of a multitude of SET domain PKMTs, which methylate histone and non-histone substrates (1).Since their identification, crystal structures of several SET domain PKMTs in complex with various substrates and products have been reported, yielding insights into the catalytic mechanism and protein substrate specificity of this methyltransferase family. These structures include the histone methyltransferases SET7/9 (4 -8), SET8 (also known as PR-SET7) (9, 10), and Neurospora DIM-5 (11) as well as pea R...

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Section: Resultssupporting

confidence: 89%

“…In this analog, the polarization of the methyl group is dependent on the protonation state of its tertiary ␥-amine (pK a ϳ7.1), which mimics the AdoMet sulfonium cation (40,47). Under basic conditions, the methyl group is relatively unpolarized due to the amine's deprotonation.…”

Section: Resultsmentioning

confidence: 99%

Catalytic Roles for Carbon-Oxygen Hydrogen Bonding in SET Domain Lysine Methyltransferases

Couture

Hauk

Thompson

et al. 2006

Journal of Biological Chemistry

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“…Sensorgrams for the aptamers binding to the fibrillar targets could not be fitted to this model due to the increasing extent of re-binding in the dissociation phase. To derive apparent affinities in these cases, we initially determined the apparent dissociation rates over a 30-s period, conditions where pseudo-first order behavior occurs (66), and then used these values in the instrument software to derive the apparent association rates and hence the apparent equilibrium constants, assuming a 1:1 binding model. The resultant 2 values (supplemental Table S1) suggest that the data were good fits to this model, which yielded apparent K D values of ϳ4 and ϳ7 nM for binding of M2 to the WL and LS fibril targets, respectively, with 2 values ϳ1.…”

Section: Selection Of Anti-␤ 2 M Amyloid Fibrilmentioning

confidence: 99%

Production and Characterization of RNA Aptamers Specific for Amyloid Fibril Epitopes

Bunka

Mantle

Morten

et al. 2007

Journal of Biological Chemistry

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One of the most fascinating features of amyloid fibrils is their generic cross-␤ architecture that can be formed from many different and completely unrelated proteins. Nonetheless, amyloid fibrils with diverse structural and phenotypic properties can form, both in vivo and in vitro, from the same protein sequence. Here, we have exploited the power of RNA selection techniques to isolate small, structured, single-stranded RNA molecules known as aptamers that were targeted specifically to amyloidlike fibrils formed in vitro from ␤ 2 -microglobulin (␤ 2 m), the amyloid fibril protein associated with dialysis-related amyloidosis. The aptamers bind with high affinity (apparent K D ϳ nM) to ␤ 2 m fibrils with diverse morphologies generated under different conditions in vitro, as well as to amyloid fibrils isolated from tissues of dialysis-related amyloidosis patients, demonstrating that they can detect conserved epitopes between different fibrillar species of ␤ 2 m. Interestingly, the aptamers also recognize some other, but not all, amyloid fibrils generated in vitro or isolated from ex vivo sources. Based on these observations, we have shown that although amyloid fibrils share many common structural properties, they also have features that are unique to individual fibril types.A number of proteins and peptides undergo specific aggregation in vivo, leading to a range of pathological disorders, collectively known as amyloidoses (1, 2). These diseases are characterized by the deposition of normally soluble proteins or peptides into insoluble fibrillar arrays with a cross-␤ architecture (1, 2). About 30 different proteins have been identified as the fibrillar component of human amyloid deposits to date, although studies have shown that amyloid-like fibrils can be generated in vitro from virtually any protein sequence under suitable experimental conditions (3, 4). Remarkably, although amyloid and amyloid-like fibrils are formed from diverse precursor proteins with unrelated primary sequences and tertiary folds, they all adopt a cross-␤ molecular architecture, identified by x-ray fiber diffraction (5, 6) and bind a number of histological dyes, such as thioflavin T (ThT) and Congo Red (7,8), the latter showing a characteristic optical anisotropy with apple-green birefringence when viewed using cross-polarized light (9). The ubiquitous ability of amyloid fibrils to bind serum amyloid P component (10, 11), glycosaminoglycans and apolipoprotein E (12), together with the finding that antibodies (e.g. WO1) raised against A␤-(1-40) amyloid fibrils are also able to recognize amyloid fibrils formed from a wide variety of proteins and peptides, unrelated in sequence and structure (13), reinforces the view that amyloid fibrils possess a common core structure. Despite the immense interest in this field (1, 14), the mechanism of how normally soluble proteins or peptides are transformed into the ordered cross-␤ structure of amyloid is largely unknown, although partial denaturation of the native protein, or folding of disordered states to...

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“…Each met-box is bound by a repressor dimer that also makes protein-protein contacts to neighbouring dimers leading to co-operative saturation of the operators [3,13,16]. Binding of two AdoMet (S-adenosylmethionine) co-repressor molecules to sites on the opposite side of the protein from the DNA-binding motif [2,16] has been postulated to create an unusual long-range electrostatic interaction with the phosphodiester backbone of DNA, raising DNA affinity at least 100-fold [17,18]. Sequence specificity arises via direct amino acid side chain hydrogen-bonding to the bases at positions 2 and 3 in the met-box, and symmetry-related positions in the larger operator, and via sequence-dependent distortions of the operator duplex at the centre of met-boxes and at the junction between them [2,3,13].…”

Section: Introductionmentioning

confidence: 99%

Transcript analysis reveals an extended regulon and the importance of protein–protein co-operativity for the Escherichia coli methionine repressor

Marincs

Manfield

Stead

et al. 2006

Biochemical Journal

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We have used DNA arrays to investigate the effects of knocking out the methionine repressor gene, metJ, on the Escherichia coli transcriptome. We assayed the effects in the knockout strain of supplying wild-type or mutant MetJ repressors from an expression plasmid, thus establishing a rapid assay for in vivo effects of mutations characterized previously in vitro. Repression is largely restricted to known genes involved in the biosynthesis and uptake of methionine. However, we identified a number of additional genes that are significantly up-regulated in the absence of repressor. Sequence analysis of the 5 promoter regions of these genes identified plausible matches to met-box sequences for three of these, and subsequent electrophoretic mobility-shift assay analysis showed that for two such loci their repressor affinity is higher than or comparable with the known metB operator, suggesting that they are directly regulated. This can be rationalized for one of the loci, folE, by the metabolic role of its encoded enzyme; however, the links to the other regulated loci are unclear, suggesting both an extension to the known met regulon and additional complexity to the role of the repressor. The plasmid gene replacement system has been used to examine the importance of protein-protein co-operativity in operator saturation using the structurally characterized mutant repressor, Q44K. In vivo, there are detectable reductions in the levels of regulation observed, demonstrating the importance of balancing protein-protein and protein-DNA affinity.

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Probing the molecular mechanism of action of co-repressor in theE.colimethionine repressor-operator complex using surface plasmon resonance (SPR)

Cited by 61 publications

References 13 publications

Catalytic Roles for Carbon-Oxygen Hydrogen Bonding in SET Domain Lysine Methyltransferases

Catalytic Roles for Carbon-Oxygen Hydrogen Bonding in SET Domain Lysine Methyltransferases

Production and Characterization of RNA Aptamers Specific for Amyloid Fibril Epitopes

Transcript analysis reveals an extended regulon and the importance of protein–protein co-operativity for the Escherichia coli methionine repressor

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