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Background: The phosphoenolpyruvate: sugar phosphotransferase system (PTS) is a bacterial and mycoplasma system responsible for the uptake of some sugars, concomitant with their phosphorylation. The sugar-specific component of the system, enzyme II (EII), consists of three domains, EIIA, EIIB and EIIC. EIIA and EIIB are cytoplasmic and EIIC is an integral membrane protein that contains the sugar-binding site. Phosphoenolpyruvate (PEP) provides the source of the phosphoryl group, which is transferred via several phosphoprotein intermediates, eventually being transferred to the internalized sugar. Along the pathway, EIIA accepts a phosphoryl group from the phosphocarrier protein HPr and transfers it to EIIB. The structure of the glucose-specific EIIA (EIIA glc ) from Mycoplasma capricolum reported here facilitates understanding of the nature of the interactions between this protein and its partners. Results: The crystal structure of EIIA glc from M. capricolum has been determined at 2.5 a resolution. Two neighboring EIIA glc molecules associate with one another in a front-to-back fashion, such that Glu 149 of one molecule forms electrostatic interactions with the active-site histidine residues, His90 and His75, of the other. Glu 149 is therefore considered to mimic the interaction that a phosphorylated histidine of a partner protein makes with EIIA. Another interaction, an ion pair between the active-site Asp94 and Lys 168 of a neighboring molecule, may be analogous to the interaction between Asp94 of EIIA glc and Arg 17 of HPr. Analysis of molecular packing in this crystal, and in the crystals of two other homologous proteins from Escherichia coli and Bacillus subtilis , reveals that in all cases active-site hydrophobic residues are involved in crystal contacts, but in each case a different region of the neighboring molecule is involved. The transition-state complexes of M. capricolum EIIA glc with HPr and EIIB glc have been modeled; in each case, different structural units are shown to interact with EIIA glc . Many of the interactions are hydrophobic with no sequence specificity. The only specific interaction, other than that formed by the phosphoryl group, involves ion pairs between two invariant aspartate residues of EIIA glc and arginine/lysine residues of HPr or EIIB glc . Conclusions: The non-discriminating nature of the hydrophobic interactions that EIIA glc forms with a variety of partners may be a consequence of the requirement for interaction with a variety of proteins that show no sequence or structural similarity. Nevertheless, specificity is provided by an ion-pair interaction that is enhanced by the apolar nature of the interface.

Background: The three-dimensional structures of histidine-containing phosphocarrier protein (HPr), a member of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), have been determined from Gram-negative and Gram-positive bacteria. The structure of HPr reported here for Mycoplasma capricolum is the first protein structure to be determined for this class of organism. Comparative structural studies with the bacterial proteins highlight sequence–structure correlations relevant to proposals about the evolutionary origin of mycoplasmas. Results The crystal structure of HPr from M. capricolum has been determined and refined at 1.8 a resolution, revealing the same overall fold as that of other HPrs of known structure. However, mycoplasma HPr resembles HPrs from Gram-positive bacteria more closely than those from Gram-negative bacteria. As in HPrs from Bacillus subtilis and Escherichia coli, the phosphoryl group carrier (His15) forms the N-terminal cap of a helix, but in contrast to the other crystal structures, the side chain of the adjacent Arg17 is conformationally disordered. A sulfate ion interacts with Ser46, a residue known to be phosphorylated in a regulatory manner. Conclusion The greater degree of structural similarity of the M. capricolum HPr to HPrs from Gram-positive rather than Gram-negative bacteria is consistent with the proposal that mycoplasma evolved from Gram-positive bacteria. The proposal that no major conformational transition is required for phosphorylation of the activesite histidine is reinforced by comparing the crystal structures with and without an anion in the active site. The conformational disorder of the Arg17 side chain suggests that its guanidinium group does not have to form specific interactions with other protein groups before phosphorylation at His15. The association of a sulfate ion with Ser46 serves as a model for HPr(Ser46∼P). As there is no evidence of a conformational change accompanying Ser46 phosphorylation, the inhibitory effect of this event may be attributable to altered surface electrostatics.