Kinetic mechanism of ligand binding in human ileal bile acid binding protein as determined by stopped-flow fluorescence analysis.
Cooperative ligand binding to human ileal bile acid binding protein (I-BABP) was studied using the stopped-flow fluorescence technique. The kinetic data obtained for wild-type protein are in agreement with a four-step mechanism where after a fast conformational change on the millisecond time scale, the ligands bind in a sequential manner, followed by another, slow conformational change on the time scale of seconds. This last step is more pronounced in the case of glycocholate (GCA), the bile salt that binds with high positive cooperativity and is absent in mutant I-BABP proteins that lack positive cooperativity in their bile salt binding. These results suggest that positive cooperativity in human I-BABP is related to a slow conformational change of the protein, which occurs after the second binding step. Analogous to that in the intestinal fatty acid binding protein (I-FABP), we hypothesize that ligand binding in I-BABP is linked to a disorder-order transition between an open and a closed form of the protein.
Toke
Orsolya
O
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA. toke@chemres.hu
Monsey
John D
JD
Cistola
David P
DP
D001647:Bile Acids and Salts / Q000737:chemistry* / Q000493:pharmacokinetics*; D001665:Binding Sites / Q000235:genetics; D005999:Glycochenodeoxycholic Acid / Q000737:chemistry / Q000493:pharmacokinetics; D006000:Glycocholic Acid / Q000737:chemistry / Q000493:pharmacokinetics; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000493:pharmacokinetics*; D007082:Ileum / Q000737:chemistry* / Q000378:metabolism*; D008024:Ligands; D008954:Models, Biological; D015233:Models, Statistical; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)
D001647:Bile Acids and Salts; D005999:Glycochenodeoxycholic Acid; D006000:Glycocholic Acid; D006913:Hydroxysteroid Dehydrogenases; D008024:Ligands; C435496:AKR1C2 protein, human; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)