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Purification, crystallization and structure determination of native GroEL from Escherichia coli lacking bound potassium ions.

ABSTRACT: GroEL is a member of the ATP-dependent chaperonin family that promotes the proper folding of many cytosolic bacterial proteins. The structures of GroEL in a variety of different states have been determined using X-ray crystallography and cryo-electron microscopy. In this study, a 3.02 A crystal structure of the native GroEL complex from Escherichia coli is presented. The complex was purified and crystallized in the absence of potassium ions, which allowed evaluation of the structural changes that may occur in response to cognate potassium-ion binding by comparison to the previously determined wild-type GroEL structure (PDB code 1xck), in which potassium ions were observed in all 14 subunits. In general, the structure is similar to the previously determined wild-type GroEL crystal structure with some differences in regard to temperature-factor distribution.

SUBMITTER: Kiser PD

PROVIDER: S-EPMC2335072 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Purification, crystallization and structure determination of native GroEL from Escherichia coli lacking bound potassium ions.

Kiser Philip D PD Lodowski David T DT Palczewski Krzysztof K

Acta crystallographica. Section F, Structural biology and crystallization communications 20070505 Pt 6

GroEL is a member of the ATP-dependent chaperonin family that promotes the proper folding of many cytosolic bacterial proteins. The structures of GroEL in a variety of different states have been determined using X-ray crystallography and cryo-electron microscopy. In this study, a 3.02 A crystal structure of the native GroEL complex from Escherichia coli is presented. The complex was purified and crystallized in the absence of potassium ions, which allowed evaluation of the structural changes tha ...[more]

PMID: 17554162

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Project description:The absence of PtsN, the terminal phosphoacceptor of the phosphotransferase system comprising PtsP-PtsO-PtsN, in Escherichia coli confers a potassium-sensitive (K(s)) phenotype as the external K(+) concentration ([K(+)]e) is increased above 5 mM. A growth-inhibitory increase in intracellular K(+) content, resulting from hyperactivated Trk-mediated K(+) uptake, is thought to cause this K(s) We provide evidence that the K(s) of the ?ptsN mutant is associated with K(+) limitation. Accordingly, the moderate K(s) displayed by the ?ptsN mutant was exacerbated in the absence of the Trk and Kup K(+) uptake transporters and was associated with reduced cellular K(+) content. Conversely, overproduction of multiple K(+) uptake proteins suppressed the K(s) Expression of PtsN variants bearing the H73A, H73D, and H73E substitutions of the phosphorylation site histidine of PtsN complemented the K(s) Absence of the predicted inner membrane protein YcgO (also called CvrA) suppressed the K(s), which was correlated with elevated cellular K(+) content in the ?ptsN mutant, but the ?ptsN mutation did not alter YcgO levels. Heterologous overexpression of ycgO also led to K(s) that was associated with reduced cellular K(+) content, exacerbated by the absence of Trk and Kup and alleviated by overproduction of Kup. Our findings are compatible with a model that postulates that K(s) in the ?ptsN mutant occurs due to K(+) limitation resulting from activation of K(+) efflux mediated by YcgO, which may be additionally stimulated by [K(+)]e, implicating a role for PtsN (possibly its dephosphorylated form) as an inhibitor of YcgO activity.This study examines the physiological link between the phosphotransferase system comprising PtsP-PtsO-PtsN and K(+) ion metabolism in E. coli Studies on the physiological defect that renders an E. coli mutant lacking PtsN to be growth inhibited by external K(+) indicate that growth impairment results from cellular K(+) limitation that is mediated by YcgO, a predicted inner membrane protein. Additional observations suggest that dephospho-PtsN may inhibit and external K(+) may stimulate K(+) limitation mediated by YcgO. It is speculated that YcgO-mediated K(+) limitation may be an output of a response to certain stresses, which by modulating the phosphotransfer capacity of the PtsP-PtsO-PtsN phosphorelay leads to growth cessation and stress tolerance.

Dataset Information

Purification, crystallization and structure determination of native GroEL from Escherichia coli lacking bound potassium ions.

Publications

Purification, crystallization and structure determination of native GroEL from Escherichia coli lacking bound potassium ions.

Similar Datasets

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