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J. Biol. Chem., Vol. 278, Issue 27, 25191-25206, July 4, 2003

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Solution Structure of the Phosphoryl Transfer Complex between the Signal-transducing Protein IIA^Glucose and the Cytoplasmic Domain of the Glucose Transporter IICB^Glucose of the Escherichia coli Glucose Phosphotransferase System^*

Mengli Cai  , David C. Williams, Jr.   ¶, Guangshun Wang  ||, Byeong Ryong Lee **, Alan Peterkofsky ** and G. Marius Clore  

From the Laboratory of Chemical Physics, NIDDK, and the ^**Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

The solution structure of the final phosphoryl transfer complex in the glucose-specific arm of the Escherichia coli phosphotransferase system, between enzyme IIA^Glucose (IIA^Glc) and the cytoplasmic B domain (IIB^Glc) of the glucose transporter IICB^Glc, has been solved by NMR. The interface (1200-Ų buried surface) is formed by the interaction of a concave depression on IIA^Glc with a convex protrusion on IIB^Glc. The phosphoryl donor and acceptor residues, His-90 of IIA^Glc and Cys-35 of IIB^Glc (residues of IIB^Glc are denoted in italics) are in close proximity and buried at the center of the interface. Cys-35 is primed for nucleophilic attack on the phosphorus atom by stabilization of the thiolate anion (pK_a 6.5) through intramolecular hydrogen bonding interactions with several adjacent backbone amide groups. Hydrophobic intermolecular contacts are supplemented by peripheral electrostatic interactions involving an alternating distribution of positively and negatively charged residues on the interaction surfaces of both proteins. Salt bridges between the Asp-38/Asp-94 pair of IIA^Glc and the Arg-38/Arg-40 pair of IIB^Glc neutralize the accumulation of negative charge in the vicinity of both the S atom of Cys-35 and the phosphoryl group in the complex. A pentacoordinate phosphoryl transition state is readily accommodated without any change in backbone conformation, and the structure of the complex accounts for the preferred directionality of phosphoryl transfer between IIA^Glc and IIB^Glc. The structures of IIA^Glc·IIB^Glc and the two upstream complexes of the glucose phosphotransferase system (EI·HPr and IIA^Glc·HPr) reveal a cascade in which highly overlapping binding sites on HPr and IIA^Glc recognize structurally diverse proteins.

Received for publication, March 17, 2003

The atomic coordinates and experimental NMR restraints (code 1O2F) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers Uiversity, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by the Intramural AIDS Targeted Antiviral Program of the Office of the Director of the National Institutes of Health (to G. M. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These two authors contributed equally to this work.

^¶ Recipient of a Pharmacology Research Associate Training postdoctoral fellowship from NIGMS, National Institutes of Health.

^|| Present address: Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-6805.

To whom correspondence should be addressed: Laboratory of Chemical Physics, Bldg. 5, Rm. B1-30I, NIDDK, National Institutes of Health, Bethesda, MD 20892-0510. Tel.: 301-496-0782; Fax: 301-496-0825; E-mail: mariusc{at}intra.niddk.nih.gov.

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