Determinants of site-selectivity in human ileal bile acid-binding protein by NMR dynamic analysis of a functionally-impaired mutant

Abstract

Human ileal bile acid-binding protein (hI-BABP), a member of the family of intracellular lipid-binding proteins, has a key role in the enterohepatic circulation of bile salts. The two internal binding sites of hI-BABP exhibit positive cooperativity accompanied by a site preference of glycocholate (GCA) and glycochenodeoxycholate (GCDA), the two most abundant bile salts in the human body. Previous study of Q51A hI-BABP in its apo state, a mutant with lost site-selectivity, suggests that disruption of the hydrogen-bonding network in the vicinity of the C/D-turn has long-range dynamic effects. To improve our understanding of the determinants of site-selectivity in hI-BABP, a comparative NMR chemical shift and spin relaxation analysis of homo- and heterotypic bile salt complexes of wild-type and Q51A hI-BABP was carried out. The wild-type GCDA-complex shows a striking similarity with the thermodynamically most stable hI-BABP:GCDA:GCA complex in terms of both structure and dynamic behaviour, suggesting that the bound GCDA at site 1 has a decisive role in conveying key stabilizing interactions in the physiologically most abundant heterotypic complex. Destabilization of hI-BABP-GCDA by the functionally impairing mutation Q51A is indicated by both the increase of ms-timescale motions in key segments of the protein as well as by increased ps-ns local fluctuations superimposed on slow motions. Our study suggests that binding interactions in hI-BABP might be modulated by altering the dynamic behaviour of specific segments in the protein with implications for targeting the intracellular trafficking of bile salts and bile salt-induced stimulation of nuclear receptors.