Absorptive enterocytes in the small intestine contain four homologous lipid-binding proteins which represent over 5% of the total soluble cellular protein. These proteins are termed ileal lipid-binding protein, intestinal fatty acid-binding protein, """"""""liver"""""""" fatty acid-binding protein, and cellular retinol-binding protein II. They are proposed to serve distinct functional roles in absorption, transport and metabolism of different classes of dietary and biliary lipids. Because of their different specificities, these proteins may ultimately represent excellent targets for the rational design of agents to selectively reduce the intestinal absorption of saturated and unsaturated fatty acids, bile salts, and cholesterol. However, the factors that control molecular recognition in this family of proteins has not yet been established. The main objective of this research is to identify the structural, dynamic and energetic determinants of ligand-binding affinity and specificity for the four distinct lipid-binding proteins from the small intestine.
The specific aims of the proposed research are: (i) to characterize the binding pro mutant and chimeric lipid- binding proteins, (ii) to establish sequence-specific 1H, 13C, and 15N NMR assignments and determine the solution structures for several mutant proteins with altered ligand specificities, and (iii) to design and synthesize novel ligands that bind with high affinity to act as inhibitors. The feasibility of the proposed experiments is firmly established by preliminary results, including the high-yield expression and purification of natural abundance and isotope-enriched proteins, the development of biophysical assays for probing lipid-protein interactions, the generation of mutants with altered specificity, and the collection and assignment of triple-resonance 3-D NMR data with excellent sensitivity and resolution. A high intake of dietary fat has been associated with an increased risk of atherosclerosis and colon cancer. A detailed knowledge of the molecular factors governing intestinal fat absorption and homeostasis will ultimately lead to more precise dietary recommendations and novel pharmacologic strategies to prevent these prevalent and life-threatening diseases. In particular, a detailed study of the molecular determinants of lipid-binding specificity, stoichiometry and affinity in the lipid-binding proteins from intestine will contribute fundamentally important information for this purpose.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK048046-01
Application #
2148080
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1994-05-01
Project End
1998-04-30
Budget Start
1994-05-01
Budget End
1995-04-30
Support Year
1
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Washington University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Horváth, Gerg?; Bencsura, Ákos; Simon, Ágnes et al. (2016) Structural determinants of ligand binding in the ternary complex of human ileal bile acid binding protein with glycocholate and glycochenodeoxycholate obtained from solution NMR. FEBS J 283:541-55
Beck, Moriah R; Dekoster, Gregory T; Cistola, David P et al. (2009) NMR structure of a fungal virulence factor reveals structural homology with mammalian saposin B. Mol Microbiol 72:344-53
Beck, Moriah R; DeKoster, Gregory T; Hambly, David M et al. (2008) Structural features responsible for the biological stability of Histoplasma's virulence factor CBP. Biochemistry 47:4427-38
Toke, Orsolya; Monsey, John D; Cistola, David P (2007) Kinetic mechanism of ligand binding in human ileal bile acid binding protein as determined by stopped-flow fluorescence analysis. Biochemistry 46:5427-36
Toke, Orsolya; Monsey, John D; DeKoster, Gregory T et al. (2006) Determinants of cooperativity and site selectivity in human ileal bile acid binding protein. Biochemistry 45:727-37
Tochtrop, Gregory P; DeKoster, Gregory T; Covey, Douglas F et al. (2004) A single hydroxyl group governs ligand site selectivity in human ileal bile acid binding protein. J Am Chem Soc 126:11024-9
Ogbay, Benhur; Dekoster, Gregory T; Cistola, David P (2004) The NMR structure of a stable and compact all-beta-sheet variant of intestinal fatty acid-binding protein. Protein Sci 13:1227-37
Tochtrop, Gregory P; Bruns, Jamie L; Tang, Changguo et al. (2003) Steroid ring hydroxylation patterns govern cooperativity in human bile acid binding protein. Biochemistry 42:11561-7
Tochtrop, Gregory P; DeKoster, Gregory T; Cistola, David P et al. (2002) A simple efficient synthesis of [23,24]-(13)C(2)-labeled bile salts as NMR probes of protein-ligand interactions. Bioorg Med Chem Lett 12:433-5
Tochtrop, Gregory P; Richter, Klaus; Tang, Changguo et al. (2002) Energetics by NMR: site-specific binding in a positively cooperative system. Proc Natl Acad Sci U S A 99:1847-52

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