Mapping of the hormone-sensitive lipase binding site on the adipocyte fatty acid-binding protein (AFABP). Identification of the charge quartet on the AFABP/aP2 helix-turn-helix domain.
The hormone-sensitive lipase (HSL) and adipocyte fatty acid-binding protein (AFABP/aP2) form a physical complex that affects basal and hormone-stimulated adipocyte fatty acid efflux. Previous work has established that AFABP/aP2-HSL complex formation requires that HSL be in its activated, phosphorylated form and AFABP/aP2 have a bound fatty acid. To identify the HSL binding site of AFABP/aP2 a combination of alanine-scanning mutagenesis and fluorescence resonance energy transfer was used. Mutation of Asp17, Asp18, Lys21, or Arg30 (but not other amino acids in the helix-turn-helix region) to alanine inhibited interaction with HSL without affecting fatty acid binding. The cluster of residues on the helical domain of AFABP/aP2 form two ion pairs (Asp17-Arg30 and Asp18-Lys21) and identifies the region we have termed the charge quartet as the HSL interaction site. To demonstrate direct association, the non-interacting AFABP/aP2-D18K mutant was rescued by complementary mutation of HSL (K196E). The charge quartet is conserved on other FABPs that interact with HSL such as the heart and epithelial FABPs but not on non-interacting proteins from the liver or intestine and may be a general protein interaction domain utilized by fatty acid-binding proteins in regulatory control of lipid metabolism.
Smith
Anne J
AJ
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota-Twin Cities, Minneapolis, Minnesota 55455, USA.
Sanders
Mark A
MA
Juhlmann
Brittany E
BE
Hertzel
Ann V
AV
Bernlohr
David A
DA
D017667:Adipocytes / Q000201:enzymology*; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000502:physiology; D002460:Cell Line; D050556:Fatty Acid-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005227:Fatty Acids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D010449:Peptide Mapping / Q000379:methods; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D002787:Sterol Esterase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D046912:Multiprotein Complexes; D002787:Sterol Esterase