Structural and regulatory studies on protein components of the E. coli sugar transport system known as the phosphoenolpyruvate:sugar phosphotransferase system (PTS) continued. The first component of the PTS (enzyme I, EI) is phosphorylated by phosphoenolpyruvate (PEP) on an active site histidine in a Mg(2+)-requiring reaction to produce pyruvate. New studies, in collaboration with the Clore laboratory (NIDDK), have elucidated the three-dimensional structure of the cytoplasmic B domain of the Escherichia coli mannitol transporter using NMR. The ordered IIBMtl domain consists of a four-stranded parallel beta-sheet flanked by two helices on one face and an additional helix on the opposite face with a characteristic Rossman fold. The active site cysteine (Cys-384) is primed for nucleophilic attack at the phosphorylated histidine (His-554) of the IIAMtl domain. The structure of IIBMtl is similar to that of protein tyrosine phosphatase. In collaboration with the Wang laboratory (Eppley Institute), structural analysis of the nitrogen regulatory arm of the PTS has been started. The X-ray structure of IIANtr has previously been reported by others. To lay the groundwork for the deduction of the solution structure of the complex of IIANtr with NPr, the chemical shift assignments for IIANtr have been made. The N-terminal domain of glucose IIA confers amphitropism to the protein, allowing it to shuttle between the membrane and cytoplasm. The structure of a synthetic peptide corresponding to the N-terminal domain was studied. The effects of phospholipids or detergent chain length on the structure and translational diffusion coefficient of the peptide were investigated by NMR. Three anionic phospholipids and four lipid-mimicking anionic detergents were evaluated. In all cases, the cationic peptide adopts an amphipathic helical structure. While the chain-length of the two-chain phospholipids has a negligible effect on the peptide conformation, the effect of chain length of single-chain detergents is more significant. Short-chain anionic phospholipids are proposed to be useful membrane-mimetic models for the structural elucidation of membrane-binding peptides. The bacterial PTS regulates a variety of physiological processes, some of which are mediated by Enzyme IIAGlc. A novel IIAGlc binding protein was discovered by ligand fishing using surface plasmon resonance. The protein, named FrsA (for fermentation/respiration switch protein), is the 47 kDa product of the yafA gene, previously denoted as ?function unknown?. FrsA forms a 1:1 complex with unphosphorylated IIAGlc with a high affinity. Disruption of frsA increased cellular respiration on several sugars, while increased FrsA expression resulted in an increased fermentation rate of some sugars. The results are interpreted to indicate that IIAGlc regulates the flux between respiration and fermentation by sensing the available sugar species via a phosphorylation state dependent interaction with FrsA.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Intramural Research (Z01)
Project #
1Z01HL000151-34
Application #
6966841
Study Section
(LCB)
Project Start
Project End
Budget Start
Budget End
Support Year
34
Fiscal Year
2004
Total Cost
Indirect Cost
Name
U.S. National Heart Lung and Blood Inst
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Wang, Guangshun; Peterkofsky, Alan; Keifer, Paul A et al. (2005) NMR characterization of the Escherichia coli nitrogen regulatory protein IIANtr in solution and interaction with its partner protein, NPr. Protein Sci 14:1082-90
Williams Jr, David C; Cai, Mengli; Suh, Jeong-Yong et al. (2005) Solution NMR structure of the 48-kDa IIAMannose-HPr complex of the Escherichia coli mannose phosphotransferase system. J Biol Chem 280:20775-84
Koo, Byoung-Mo; Yoon, Mi-Jeong; Lee, Chang-Ro et al. (2004) A novel fermentation/respiration switch protein regulated by enzyme IIAGlc in Escherichia coli. J Biol Chem 279:31613-21
Keifer, Paul A; Peterkofsky, Alan; Wang, Guangshun (2004) Effects of detergent alkyl chain length and chemical structure on the properties of a micelle-bound bacterial membrane targeting peptide. Anal Biochem 331:33-9
Legler, Patricia M; Cai, Mengli; Peterkofsky, Alan et al. (2004) Three-dimensional solution structure of the cytoplasmic B domain of the mannitol transporter IImannitol of the Escherichia coli phosphotransferase system. J Biol Chem 279:39115-21
Sondej, Melissa; Vazquez-Ibar, Jose Luis; Farshidi, Arta et al. (2003) Characterization of a lactose permease mutant that binds IIAGlc in the absence of ligand. Biochemistry 42:9153-9
Dimitrova, Mariana N; Peterkofsky, Alan; Ginsburg, Ann (2003) Opposing effects of phosphoenolpyruvate and pyruvate with Mg(2+) on the conformational stability and dimerization of phosphotransferase enzyme I from Escherichia coli. Protein Sci 12:2047-56
Wang, Guangshun; Keifer, Paul A; Peterkofsky, Alan (2003) Solution structure of the N-terminal amphitropic domain of Escherichia coli glucose-specific enzyme IIA in membrane-mimetic micelles. Protein Sci 12:1087-96
Li, Xia; Peterkofsky, Alan; Wang, Guangshun (2003) 1H, 15N, and 13C chemical shift assignments of the Escherichia coli nitrogen regulatory phosphocarrier IIA(Ntr). J Biomol NMR 27:401-2
Reddy, Prasad T; Prasad, C Rama; Reddy, P Hemalatha et al. (2003) Cloning and expression of the gene for a novel protein from Mycobacterium smegmatis with functional similarity to eukaryotic calmodulin. J Bacteriol 185:5263-8

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