ABC proteins or traffic ATPases are members of a large superfamily of translocators, both prokaryotic and eukaryotic. The eukaryotic members include the cystic fibrosis transmembrane regulator (CFTR), the P-glycoprotein of multidrug resistance (MDR), the gene responsible for adrenoleukodistrophy (ALD), the heterodimeric transporter associated with antigen processing (Tap1/Tap2), the transporter involved in Stargard macular dystrophy, and many others of medical importance. Among the prokaryotic members are bacterial periplasmic permeases, which have been extensively characterized. Considering the extensive homology between all of these proteins it is likely that there will also be strong similarities between eukaryotic and prokaryotic systems in their mechanism of action. Thus, prokaryotic permeases provide excellent models for achieving a general understanding of the activity and function of these translocators. The proposed work aims at understanding in detail the 3-dimensional structure of a model system: the histidine permease. We plan: 1). The spatial organization of the permease will be assessed by chemical cross-linking, NMR, and spin-labeling studies which will provide information on the three-dimensional structure by defining the nearest neighbor contact between the individual components. We will purify the individual cross-linked products, perform proteolytic digestion, HPLC separation of the peptides and mass spectrometry analysis. Comparison between patterns obtained from cross-linked products and from untreated complex will indicate the sites of contact. Experiments will also be performed on the permease in various stages of its activity cycle. 2). We will also try and obtain the crystal structure of the entire permease. If successful, this would provide the best description of the structure. 3). Crystal structures will be obtained of various mutants of the separated ATP-binding domains (HisP), which will provide information on which signals are needed to initiate ATP hydrolysis and on signaling mechanisms. Examination of these and other aspects of the histidine periplasmic permease will contribute to the understanding of general mechanistic questions for these transporters and will help unravel how their several sub-domains function. An understanding of the structure of these systems is essential for developing new approaches for investigating eukaryotic transporters. This knowledge can then be applied to help develop therapeutic tools for human diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK012121-37
Application #
6760144
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Sechi, Salvatore
Project Start
1977-01-01
Project End
2005-06-30
Budget Start
2004-07-01
Budget End
2005-06-30
Support Year
37
Fiscal Year
2004
Total Cost
$240,150
Indirect Cost
Name
Children's Hospital & Res Ctr at Oakland
Department
Type
DUNS #
076536184
City
Oakland
State
CA
Country
United States
Zip Code
94609
Ames, G F; Nikaido, K; Wang, I X et al. (2001) Purification and characterization of the membrane-bound complex of an ABC transporter, the histidine permease. J Bioenerg Biomembr 33:79-92
Kreimer, D I; Chai, K P; Ferro-Luzzi Ames, G (2000) Nonequivalence of the nucleotide-binding subunits of an ABC transporter, the histidine permease, and conformational changes in the membrane complex. Biochemistry 39:14183-95
Liu, P Q; Liu, C E; Ames, G F (1999) Modulation of ATPase activity by physical disengagement of the ATP-binding domains of an ABC transporter, the histidine permease. J Biol Chem 274:18310-8
Nikaido, K; Ames, G F (1999) One intact ATP-binding subunit is sufficient to support ATP hydrolysis and translocation in an ABC transporter, the histidine permease. J Biol Chem 274:26727-35
Liu, C E; Liu, P Q; Wolf, A et al. (1999) Both lobes of the soluble receptor of the periplasmic histidine permease, an ABC transporter (traffic ATPase), interact with the membrane-bound complex. Effect of different ligands and consequences for the mechanism of action. J Biol Chem 274:739-47
Liu, P Q; Ames, G F (1998) In vitro disassembly and reassembly of an ABC transporter, the histidine permease. Proc Natl Acad Sci U S A 95:3495-500
Trakhanov, S; Kreimer, D I; Parkin, S et al. (1998) Cadmium-induced crystallization of proteins: II. Crystallization of the Salmonella typhimurium histidine-binding protein in complex with L-histidine, L-arginine, or L-lysine. Protein Sci 7:600-4
Liu, C E; Ames, G F (1997) Characterization of transport through the periplasmic histidine permease using proteoliposomes reconstituted by dialysis. J Biol Chem 272:859-66
Nikaido, K; Liu, P Q; Ames, G F (1997) Purification and characterization of HisP, the ATP-binding subunit of a traffic ATPase (ABC transporter), the histidine permease of Salmonella typhimurium. Solubility, dimerization, and ATPase activity. J Biol Chem 272:27745-52
Liu, C E; Liu, P Q; Ames, G F (1997) Characterization of the adenosine triphosphatase activity of the periplasmic histidine permease, a traffic ATPase (ABC transporter). J Biol Chem 272:21883-91

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