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J. Biol. Chem., Vol. 283, Issue 16, 11024-11037, April 18, 2008

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Solution NMR Structures of Productive and Non-productive Complexes between the A and B Domains of the Cytoplasmic Subunit of the Mannose Transporter of the Escherichia coli Phosphotransferase System^*

From the Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

Solution structures of complexes between the isolated A (IIA^Man) and B (IIB^Man) domains of the cytoplasmic component of the mannose transporter of Escherichia coli have been solved by NMR. The complex of wild-type IIA^Man and IIB^Man is a mixture of two species comprising a productive, phosphoryl transfer competent complex and a non-productive complex with the two active site histidines, His-10 of IIA^Man and His-175 of IIB^Man, separated by 25Å_. Mutation of the active site histidine, His-10, of IIA^Man to a glutamate, to mimic phosphorylation, results in the formation of a single productive complex. The apparent equilibrium dissociation constants for the binding of both wild-type and H10E IIA^Man to IIB^Man are approximately the same (K_D 0.5 mM). The productive complex can readily accommodate a transition state involving a pentacoordinate phosphoryl group with trigonal bipyramidal geometry bonded to the N2 atom of His-10 of IIA^Man and the N1 atom of His-175 of IIB^Man with negligible (<0.2Å₎ local backbone conformational changes in the immediate vicinity of the active site. The non-productive complex is related to the productive one by a 90° rotation and 37Å translation of IIB^Man relative to IIA^Man, leaving the active site His-175 of IIB^Man fully exposed to solvent in the non-productive complex. The interaction surface on IIA^Man for the non-productive complex comprises a subset of residues used in the productive complex and in both cases involves both subunits of IIA^Man. The selection of the productive complex by IIA^Man(H10E) can be attributed to neutralization of the positively charged Arg-172 of IIB^Man at the center of the interface. The non-productive IIA^Man-IIB^Man complex may possibly be relevant to subsequent phosphoryl transfer from His-175 of IIB^Man to the incoming sugar located on the transmembrane IIC^Man-IID^Man complex.

Received for publication, January 11, 2008

The atomic coordinates and experimental NMR restraints (codes 2JZH, 2JZN, 2JZO, and 1VSQ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by the intramural program of NIDDK, National Institutes of Health (NIH), and the Intramural AIDS Targeted Antiviral Program of the Office of the Director of the NIH (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.

¹ Both authors contributed equally to this work.

² Present address: Dept. of Pathology, Virginia Commonwealth University, Richmond, VA 23298-0662.

³ Present address: NICHD, NIH, Bethesda, MD 20892.

⁴ To whom correspondence should be addressed: Laboratory of Chemical Physics, Bldg. 5, Rm. B1-30I, NIDDK, NIH, Bethesda, MD 20892-0520. Tel.: 301-496-0782; Fax: 301-496-0825; E-mail: mariusc{at}intra.niddk.nih.gov.

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