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BIOLOGICAL SCIENCES / BIOPHYSICS
Structure of phosphorylated enzyme I, the phosphoenolpyruvate:sugar phosphotransferase system sugar translocation signal protein
*Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, MD 20850; Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892; Biological, Chemical, and Physical Sciences, Illinois Institute of Technology, Chicago, IL 60616; and ¶Biochemical Science Division, Chemical Sciences and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899-0001
Communicated by Ada Yonath, Weizmann Institute of Science, Rehovot, Israel, September 7, 2006 (received for review July 24, 2006)
Bacterial transport of many sugars, coupled to their phosphorylation, is carried out by the phosphoenolpyruvate (PEP):sugar phosphotransferase system and involves five phosphoryl group transfer reactions. Sugar translocation initiates with the Mg2+-dependent phosphorylation of enzyme I (EI) by PEP. Crystals of Escherichia coli EI were obtained by mixing the protein with Mg2+ and PEP, followed by oxalate, an EI inhibitor. The crystal structure reveals a dimeric protein where each subunit comprises three domains: a domain that binds the partner PEP:sugar phosphotransferase system protein, HPr; a domain that carries the phosphorylated histidine residue, His-189; and a PEP-binding domain. The PEP-binding site is occupied by Mg2+ and oxalate, and the phosphorylated His-189 is in-line for phosphotransfer to/from the ligand. Thus, the structure represents an enzyme intermediate just after phosphotransfer from PEP and before a conformational transition that brings His-189
P in proximity to the phosphoryl group acceptor, His-15 of HPr. A model of this conformational transition is proposed whereby swiveling around an 
-helical linker disengages the His domain from the PEP-binding domain. Assuming that HPr binds to the HPr-binding domain as observed by NMR spectroscopy of an EI fragment, a rotation around two linker segments orients the His domain relative to the HPr-binding domain so that His-189P and His-15 are appropriately stationed for an in-line phosphotransfer reaction.
sugar transport | phosphorylation | x-ray crystallography
Author contributions: O.H. designed research; A.T., K.L., P.-P.Z., G.K., C.C.H.C., J.S., A.H., P.T.R., A.P., and O.H. performed research; A.T., K.L., and O.H. analyzed data; and A.T., K.L., and O.H. wrote the paper.
Present address: Centocor R&D, 145 King of Prussia Road, Radnor, PA 19087.
The authors declare no conflict of interest.
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 2HWG).
||To whom correspondence should be addressed. E-mail: osnat{at}carb.nist.gov
© 2006 by The National Academy of Sciences of the USA
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