The phosphocarrier protein IIIGlc is an integral component of the bacterial phosphotransferase (PTS) system. Unphosphorylated IIIGlc inhibits non-PTS carbohydrate transport systems by binding to diverse target proteins. The crystal structure at 2.6 A resolution of one of the targets, glycerol kinase (GK), in complex with unphosphorylated IIIGlc, glycerol, and adenosine diphosphate was determined. GK contains a region that is topologically identical to the adenosine triphosphate binding domains of hexokinase, the 70-kD heat shock cognate, and actin. IIIGlc binds far from the catalytic site of GK, indicating that long-range conformational changes mediate the inhibition of GK by IIIGlc. GK and IIIGlc are bound by hydrophobic and electrostatic interactions, with only one hydrogen bond involving an uncharged group. The phosphorylation site of IIIGlc, His90, is buried in a hydrophobic environment formed by the active site region of IIIGlc and a 3(10) helix of GK, suggesting that phosphorylation prevents IIIGlc binding to GK by directly disrupting protein-protein interactions.

The crystal structure of a proteolytically modified form ofthe Escherichia coli phosphocarrier and signal transducing protein ifC has been determined by multiple isomorphous and molecular replacement. The model has been refined to an R-factor of 0.166 for data between 6-and 2.1-A resolution with an rms deviation of 0.020 A from ideal bond lengths and 3.20 from ideal bond angles. The molecule is a 3-sheet sandwich, with six antiparallel strands on either side.Several short distorted helices line the periphery of the active site, which is a shallow extremely hydrophobic depression -18 A in diameter near the center of one face. The side chains of the active site histidine residues 75 and 90 face each other at the center of the depression, with the N3 positions exposed to solvent, separated by 3.3 A in an excellent position to form adducts with phosphate. Chloroplatinate forms a divalent adduct with both histidyl side chains, suggesting that the phosphodonor reaction might proceed through a similar transition state. The hydrophobic patch forms the primary crystal contact, suggesting a mode of association of m with other components of the phosphoenolpyruvate-dependent phosphotransferase system.The phosphoenolpyruvate:glycose phosphotransferase system (PTS) catalyzes the transport and phosphorylation of a number of simple sugars. In Escherichia coli, glucose uptake results from a sequential phosphate transfer reaction involving histidine residues offourPTS proteins: phosphoenolpyruvate enzyme I > histidine-containing protein (HPr) ITIgic II>gc glucose. The PTS also regulates chemotaxis to PTS sugars, adenylate cyclase, certain non-PTS permeases, and the transcription of some operons (for reviews, see refs. 1-6). Genetic (7) and biochemical (2) studies show that these regulatory phenomena are controlled by the crr gene product IIIlc (Mr,18,099 10382The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

In enteric bacteria, the phosphoenolpyruvate:glycose phosphotransferase system (PTS) catalyzes the uptake and phosphorylation of its sugar substrates. In addition, the PTS regulates adenylate cyclase, glycerol kinase (GK), and a number of non-PTS permeases (for reviews, see refs. 1-4). In the present report, we show that the binding of IIIic to GK generates a putative intermolecular zinc coordination site. Zn(II) not only specifically binds at this site but substantially increases the apparent affinity of the two proteins and the inhibitory potency of 1IIGIc for GK. These results have significant implications for the mechanisms of regulation of multiple proteins by IIIiGC. MATERIALS AND METHODSCrystals ofthe IIIGIc/GK complex were prepared by hangingdrop vapor diffusion as described (8) and transferred to storage buffer containing 20 mM cation chloride, 20 mM ADP, 50 mM glycerol 3-phosphate (G3P), and 0.6 M sodium acetate in 100 mM Mes buffer, pH 6.1. Normally, all solutions were prepared in ultrafiltered water (Nanopure II; Barnstead), but the MgCl2 solution described below was inadvertently prepared with reverse-osmosis water (15-fold higher conductivity than the ultrafiltered water).Diffraction data using graphite-monochromated CuKa radiation were collected on a San Diego Multiwire Systems area detector and reduced by the supplied software. Crystallographic refinement was performed with the TNT package of programs (9), and electron density maps were inspected with FRODO (10).A site-directed mutant of 111GIc, H75Q (His75 -* Gln), was prepared as reported (11). GK assays were conducted (12) with the homogeneous enzyme in the presence or absence of IJIGic and Zn(II) as indicated. The effect of Zn(II) on the coupling enzyme system was investigated. ZnADP is a substrate for pyruvate kinase, but the efficacy of the coupling system is not affected by the Zn(II) concentrations used in these experiments. The specific activity of GK is constant over the range of enzyme concentrations (0.5-2.5 pg/ml) used with 0.1 mM ZnCl2 added to the assay mixture.Abbreviations: GK, glycerol kinase; PTS, phosphotransferase system; G3P, glycerol 3-phosphate.$To whom reprint requests should be sent at the * address. 3544The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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