Abstract
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.
Highlights
To study the functions of these mobile regions, we 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
Summary and Conclusions—These NMR results provide the first structural support to the notion that the entire N-terminal region of the E1ec is responsible for binding the E2ec component
The N-terminal region of E1ec, not seen in the x-ray structures, gives well resolved resonances and permitted sequence specific assignment for six resonances (Trp-16, Gln-18, Gly-28, Gln-33, Gln-38, and Gly-47). These assignments complement the biochemical findings on this region; substitution of several residues in this region dramatically reduced the activity of the reconstituted complex and affected intercomponent assembly
Summary
The Wizard Plus Miniprep DNA purification system was used for purification of DNA (Promega, Madison, WI). The QuikChange site-directed mutagenesis kit was used for singlesite substitution (Stratagene, La Jolla, CA). DNA sequencing was done at the Molecular Resource Facility of the New Jersey Medical School (Newark, NJ). E. coli BL21(DE3) pLys cells were from Novagen (Novagen, EMD Chemicals, Gibbstown, NJ). The synthetic peptide corresponding to the N-terminal 1–35 amino acids of the E1ec was from SynPep (Dublin, CA). The synthetic peptide with sequence H2N-ATPLIRRLAREFGVNLAKVKGTGRKGRILREDVQAYVKEAI-OH corresponding to the E2ec PSBD was from CHI Scientific (Maynard, MA). RbCa TXN SALTS used to make super-competent cells was from BIO101, Inc. The 18 unlabeled L-amino acids used in the minimal medium were from Sigma, with the exception of L-Lys from EMD Chemicals, Inc.
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