Abstract
Enzyme I (EI) is the phosphoenolpyruvate (PEP)-protein phosphotransferase at the entry point of the PEP-dependent sugar phosphotransferase system, which catalyzes carbohydrate uptake into bacterial cells. In the first step of this pathway EI phosphorylates the heat-stable phospho carrier protein at His-15 using PEP as a phosphoryl donor in a reaction that requires EI dimerization and autophosphorylation at His-190. The structure of the full-length protein from Staphylococcus carnosus at 2.5A reveals an extensive interaction surface between two molecules in adjacent asymmetric units. Structural comparison with related domains indicates that this surface represents the biochemically relevant contact area of dimeric EI. Each monomer has an extended configuration with the phosphohistidine and heat-stable phospho carrier protein-binding domains clearly separated from the C-terminal dimerization and PEP-binding region. The large distance of more than 35A between the active site His-190 and the PEP binding site suggests that large conformational changes must occur during the process of autophosphorylation, as has been proposed for the structurally related enzyme pyruvate phosphate dikinase. Our structure for the first time offers a framework to analyze a large amount of research in the context of the full-length model.
Highlights
Group translocation is the membrane transport mechanism by which a substrate is chemically modified to an impermeable derivative as it crosses the cell membrane
The phosphotransferase system (PTS) catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate (PEP) to a sugar while it is being transported across the membrane
It consists of two universal components, Enzyme I [4, 5], hereafter referred to as EI, and the heat-stable histidine phospho carrier protein (HPr), and in addition several membrane-associated components, which are sugar-specific and are collectively designated as Enzyme II (EII) complexes [3]
Summary
Protein Expression and Purification—EI from S. carnosus was overexpressed in Escherichia coli (strain DH5␣) harboring the plasmid pUC-ptsO2.6X [23]. A second crystallization condition was obtained using sitting drops and 30% (w/v) polyethylene glycol 4000, 0.2 sodium malonate, and 0.1 M Tris-HCl, pH 8.5, as crystallization buffer This second condition did not require additives and produced only monoclinic crystals with similar symmetry properties and diffraction power. In the later stages of model building, structure completion was continued with data collected from another crystal grown under the original conditions for the monoclinic form. This crystal was soaked in (NH4)2Os4Br6, which improved diffraction data but did not lead to a heavy atom derivative and was considered as a native crystal (Table 1). Structural visualizations were done with the programs MOLSCRIPT along with Raster3D (Figs. 1B, 2, and 3) [29, 30] and PYMOL (Figs. 1A, 1C, and 4A). The atomic coordinates have been deposited with the Protein Data Bank (www.rcsb.org), under accession code 2HRO
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