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

The solution structure of the final phosphoryl transfer complex in the glucose-specific arm of the Escherichia coli phosphotransferase system, between enzyme IIA (IIA) and the cytoplasmic B domain (IIB) of the glucose transporter IICB, has been solved by NMR. The interface ( 1200-Å buried surface) is formed by the interaction of a concave depression on IIA with a convex protrusion on IIB. The phosphoryl donor and acceptor residues, His-90 of IIA and Cys-35 of IIB (residues of IIB 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 (pKa 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 and the Arg-38/Arg-40 pair of IIB 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 and IIB. The structures of IIA IIB and the two upstream complexes of the glucose phosphotransferase system (EI HPr and IIA HPr) reveal a cascade in which highly overlapping binding sites on HPr and IIA recognize structurally diverse proteins. In bacteria, carbohydrate transport across the membrane, mediated by the phosphoenolpyruvate:sugar phosphotransferase system (PTS), involves the tight coupling of translocation and phosphorylation. The PTS is a classical example of a signal transduction pathway involving phosphoryl transfer (1), whereby a phosphoryl group originating on phosphoenolpyruvate is transferred to the translocated carbohydrate via a series of three bimolecular protein-protein complexes. The first two steps of the PTS are common to all sugars: enzyme I (EI) is autophosphorylated by phosphoenolpyruvate and subsequently donates the phosphoryl group to the histidine phosphocarrier protein HPr. The proteins downstream from HPr are sugarspecific, comprising four distinct families of IIA permeases (2–4). In the case of the glucose branch of the PTS, the phosphoryl group is transferred from HPr to IIA and thence from IIA to the C-terminal cytoplasmic domain (IIB) of the glucose transporter IICB. In addition to their function within the PTS cascade, the PTS proteins also serve to regulate other pathways (2). Thus, dephosphorylated enzyme I inhibits bacterial chemotaxis (5); dephosphorylated HPr functions as a positive regulatory subunit of glycogen phosphorylase (6); dephosphorylated IIA is a negative regulator of glycerol kinase (7), as well as a variety of non-PTS permeases (2), whereas phosphorylated IIA is a positive regulator of adenylyl cyclase (8); finally, the dephosphorylated form of IICB sequesters the global repressor Mlc, thereby initiating PTS gene transcription in response to the uptake of glucose from the extracellular environment (9–12). This multiplicity of interactions in which individual proteins specifically recognize a wide variety of structurally diverse targets serves as a paradigm for understanding protein-protein interactions and the factors determining their specificity. The glucose-specific transporter, IICB, comprises an Nterminal transmembrane domain thought to consist of eight membrane-spanning helices (residues 17–323) connected to a C-terminal cytoplasmic domain (IIB, residues 401–476) via a long flexible linker (13–15). Recently, we have solved the solution NMR structures of the initial protein-protein complex of the Escherichia coli PTS between the N-terminal domain of enzyme I (EIN) and HPr (16) and the subsequent complexes of * 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. 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/). § 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: 301-4960825; E-mail: [email protected]. 1 The abbreviations used are: PTS, phosphoenolpyruvate:sugar phosphotransferase system; EI, enzyme I; EIN, N-terminal domain of enzyme I; HPr, histidine-containing phosphocarrier protein; IIA, glucose-specific enzyme IIA; IICB, the glucose-specific transporter; IIB, cytoplasmic B domain of IICB; IIB, the cytoplasmic B domain of the sucrose-specific transporter; NOE, nuclear Overhauser effect; r.m.s., root mean square; PTP, protein-tyrosine phosphatase. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 27, Issue of July 4, pp. 25191–25206, 2003 Printed in U.S.A.