Structural Analysis of the Escherichia coli KdpFABC Holoenzyme and its Funktional Domains

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In the case of changes in turgor across the cell membrane of Escherichia coli, the cell induces the high-affinity K+-translocating KdpFABC complex, a P-type transport ATPase (P-ATPase) of unique subunit composition and structure. In order to study structural and functional modules of this transport system, the KdpFABC complex as well as functionally important, but soluble domains of KdpB (catalytic subunit) and KdpC should be crystallized and the structures should be solved by X-ray crystallography. The dynamics (ATP binding, phosphorylation, dephosphorylation) and the interplay of the soluble domains, which are now available in homogeneous form, should be studied in solution by NMR. From these studies important information can be obtained about the conformational changes occurring during the reaction cycle of the enzyme complex. The expression of the kdpFABC operon is regulated by the membrane-bound sensor kinase KdpD and the soluble response regulator KdpE. The communication between these two regulatory proteins occurs via phosphorylation and dephosphorylation reactions. Both proteins, as well as a functionally important, but soluble domain of KdpD, should be crystallized and their structures should be solved by X-ray crystallography. Crystals of KdpE have already been obtained. Therefore, the continuation of this part of the project will be given priority. Since the regulatory proteins involved in environmental signalling all exhibit a similar structure (modular design), the information obtained for KdpD and KdpE are pertinent for the understanding of the signal transduction mechanism at the molecular level.