Song, J, Park, YH, Nemeria, NS, Kale, S, Kakalis, L and Jordan, F (2010) Nuclear magnetic resonance evidence for the role of the flexible regions of the E1 component of the pyruvate dehydrogenase complex from gram-negative bacteria. J. Biol. Chem. 285:4680-94
Most bacterial pyruvate dehydrogenase complexes from either gram-positive or gram-negative bacteria have E1 components with an alpha(2) homodimeric quaternary structure. In a sequel to our previous publications, we present the first NMR study on the flexible regions of the E1 component from Escherichia coli and its biological relevance. We report sequence-specific NMR assignments for 6 residues in the N-terminal 1-55 region and for a glycine in each of the two mobile active center loops of the E1 component, a 200-kDa homodimer. This was accomplished by using site-specific substitutions and appropriate labeling patterns along with a peptide with the sequence corresponding to the N-terminal 1-35 amino acids of the E1 component. To study the functions of these mobile regions, we also examined the spectra in the presence of (a) a reaction intermediate analog known to affect the mobility of the active center loops, (b) an E2 component construct consisting of a lipoyl domain and peripheral subunit binding domain, and (c) a peptide corresponding to the amino acid sequence of the E2 peripheral subunit binding domain. Deductions from the NMR studies are in excellent agreement with our functional finding, providing a clear indication that the N-terminal region of the E1 interacts with the E2 peripheral subunit binding domain and that this interaction precedes reductive acetylation. The results provide the first structural support to the notion that the N-terminal region of the E1 component of this entire class of bacterial pyruvate dehydrogenase complexes is responsible for binding the E2 component.
Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Chromatography, Gel; Gram-Negative Bacteria/enzymology; Magnetic Resonance Spectroscopy/methods; Protein Binding/genetics; Protein Binding/physiology; Protein Structure, Tertiary/genetics; Protein Structure, Tertiary/physiology; Pyruvate Dehydrogenase Complex/chemistry; Pyruvate Dehydrogenase Complex/genetics; Pyruvate Dehydrogenase Complex/metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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