Structural Similarity of YbeD Protein from
            <i>Escherichia coli</i>
            to Allosteric Regulatory Domains

Kozlov, Guennadi; Elias, Demetra; Semesi, A.; Yee, Adelinda; Cygler, Mirosław; Gehring, Kalle

doi:10.1128/jb.186.23.8083-8088.2004

Cited by 12 publications

(13 citation statements)

References 36 publications

(26 reference statements)

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“…Notably the extent of the acidification of the medium correlated positively with increased levels of pyruvate dehydrogenase and acetate kinase; YidC-GFP overexpression resulted in the most pronounced effect followed by LepI-GFP and YedZ-GFP. Also the level of YbeD, involved in the biosynthesis of lipoic acid, which is required as the prosthetic group of pyruvate dehydrogenase, was increased 16-fold (53). Overexpression of the soluble protein GST-GFP did not result in the aforementioned changes and consistently did not affect the pH of the culture medium (Fig.…”

Section: Overexpressed Proteins and Culture Conditions-thementioning

confidence: 97%

Consequences of Membrane Protein Overexpression in Escherichia coli

Wagner

Baars

Ytterberg

et al. 2007

Molecular & Cellular Proteomics

320

217

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Overexpression of membrane proteins is often essential for structural and functional studies, but yields are frequently too low. An understanding of the physiological response to overexpression is needed to improve such yields. Therefore, we analyzed the consequences of overexpression of three different membrane proteins (YidC, YedZ, and LepI) fused to green fluorescent protein (GFP) in the bacterium Escherichia coli and compared this with overexpression of a soluble protein, GST-GFP. Proteomes of total lysates, purified aggregates, and cytoplasmic membranes were analyzed by one-and two-dimensional gel electrophoresis and mass spectrometry complemented with flow cytometry, microscopy, Western blotting, and pulse labeling experiments. Composition and accumulation levels of protein complexes in the cytoplasmic membrane were analyzed with improved two-dimensional blue native PAGE. Overexpression of the three membrane proteins, but not soluble GST-GFP, resulted in accumulation of cytoplasmic aggregates containing the overexpressed proteins, chaperones (DnaK/J and GroEL/ S), and soluble proteases (HslUV and ClpXP) as well as many precursors of periplasmic and outer membrane proteins. This was consistent with lowered accumulation levels of secreted proteins in the three membrane protein overexpressors and is likely to be a direct consequence of saturation of the cytoplasmic membrane protein translocation machinery. Importantly accumulation levels of respiratory chain complexes in the cytoplasmic membrane were strongly reduced. Induction of the acetate-phosphotransacetylase pathway for ATP production and a downregulated tricarboxylic acid cycle indicated the activation of the Arc two-component system, which mediates adaptive responses to changing respiratory states. This study provides a basis for designing rational strategies to improve yields of membrane protein overexpression in E. coli.

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Section: Overexpressed Proteins and Culture Conditions-thementioning

confidence: 97%

Consequences of Membrane Protein Overexpression in Escherichia coli

Wagner

Baars

Ytterberg

et al. 2007

Molecular & Cellular Proteomics

320

217

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“…The structures range from stand-alone ACT domains, as seen for the YbeD protein from E. coli (14) (Fig. 3K), to large multimeric proteins with ACT domains either isolated or associating into groups of 2, 3, or 4 (6, 7, 10, 11, 14 -19, 21, The sequences are aligned with regard to residue type and secondary structure.…”

Section: Act Domain Structuresmentioning

confidence: 99%

The ACT Domain: A Small Molecule Binding Domain and Its Role as a Common Regulatory Element

Grant

2006

Journal of Biological Chemistry

190

209

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The ACT domain is a structural motif in proteins of 70 -80 amino acids that is one of a growing number of different intracellular small molecule binding domains that function in the control of metabolism, solute transport, and signal transduction (1-5). The first structure of an ACT domain was determined in 1995 with the crystal structure of Escherichia coli D-3-phosphoglycerate dehydrogenase (6), a tetrameric protein containing one ACT domain per subunit. This structure represents the archetypical ACT domain and is composed of four ␤ strands and two ␣ helices arranged in a ␤␣␤␤␣␤ fold as shown in Fig. 1. However, it was not recognized as a recurring motif until 1999 when Aravind and Koonin (2) proposed this based on a PSI-Blast (position-specific iterating-Blast) sequence data base search using the small subunit (IlvN) of acetolactate synthase. This search identified a diverse group of proteins that were noted to be involved in some way in amino acid and purine metabolism and were regulated by specific amino acids. They named this proposed domain the ACT domain after the first letters of three of the proteins, aspartate kinase-chorismate mutase-tyrA (prephenate dehydrogenase).In 2001, the structure of the Lrp-like transcriptional regulator from Pyrococcus furiosis was published (7). This was the first structure of a transcription factor that contained an ACT domain. A PSI-Blast analysis of its sequence by Ettema et al. (3) revealed an additional group of proteins, including both enzymes and transcription regulators, that they proposed contained a novel type of ACT domain, which they named the RAM domain for regulator of amino acid metabolism. The Lrplike transcriptional regulator contains the ACT domain fold (␤␣␤␤␣␤), but the sequence alignment resulting from the PSIBlast search appeared to reveal a somewhat different pattern of conservation of residue type (3) (Fig. 2). Mutagenesis data also suggested that the ligand binding sites of the Lrp-like protein were located differently than in the ACT domains described previously. Although the ACT domain from E. coli D-3-phosphoglycerate dehydrogenase and the Lrp-like transcription factor superimpose very well (1.8-Å root mean square deviation), the dimer interfaces of each are significantly different. The ACT domain dimers of phosphoglycerate dehydrogenase form a side-by-side structure producing an extended 8-stranded ␤ sheet (Fig. 3A). On the other hand, the ␤ sheets of the ACT domain dimers of the Lrp transcription factor assume a more face-to-face configuration (Fig. 3J). These observations formed the basis for the proposed division into ACT and RAM domains. As will be seen later in this review, recently determined structures demonstrate that the ACT domain shows an increasing diversity in tertiary and quaternary architecture as well as ligand binding interactions.A novel type of ACT domain-containing protein family whose members contain ACT domain repeats has also been identified by sequence analysis in Arabadopsis (8). These proteins were termed ACR proteins....

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“…Indeed, the only conserved cysteine is buried, and the solvent-accessible glutamates are dispensable (30). In addition, domains with a ␤␣␤␤␣␤ fold, including ACT domains, have been involved in dimerization, and some modules appear to evolve in the protein interaction domain (1,20). Our biochemical analysis and bacterial two-hy- brid study supported a monomeric nature of the recombinant C-terminal domain, which contrasts with canonical ACT domains.…”

Section: Discussionmentioning

confidence: 64%