Ligand entry in human ileal bile acid-binding protein is mediated by histidine protonation

Gergő Horváth,Orsolya Toke,József Kardos,András Micsonai,Mihály Kovács,Orsolya Egyed,Changguo Tang

doi:10.1038/s41598-019-41180-7

Abstract

Human ileal bile acid-binding protein (hI-BABP) has a key role in the intracellular transport of bile salts. To explore the role of histidine protonation in the binding process, the pH-dependence of bile salt binding and internal dynamics in hI-BABP was investigated using NMR spectroscopy and biophysical tools. Thermodynamic and kinetic measurements show an increase in the overall binding affinity and the association rate constant of the first binding step below the pKa of the histidines, suggesting that ligand binding is favoured by the protonated state. The overlap between residues exhibiting a high sensitivity to pH in their backbone amide chemical shifts and protein regions undergoing a global ms conformational exchange indicate a connection between the two processes. According to 15N NMR relaxation dispersion analysis, the slow motion is most pronounced at and above the pKa of the histidines. In agreement with the NMR measurements, MD simulations show a stabilization of the protein by histidine protonation. Hydrogen-bonding and van der Waals interactions mediating the flow of information between the C/D- and G/H-turn regions hosting the three histidines, suggest a complex way of pH-governed allosteric regulation of ligand entry involving a transition between a closed and a more open protein state.

Highlights

Intracellular lipid binding proteins are small, 14–15 kDa polypeptide chains that are thought to function in the transcellular trafficking of fatty acids, retinoids, and bile salts[1,2]
Our results unveil an intimate relation between histidine protonation and an opening/closing motion mediating ligand entry in the apo form of human ileal bile acid-binding protein (hI-bile acid binding proteins (BABP)) raising the possibility of a pH-dependent mechanism pf bile salt uptake in the enterocytes of the distal small intestine
To quantitate the thermodynamic parameters of ligand binding, isothermal titration calorimetry (ITC) data sets were fit to the stepwise binding model shown in eq 1 by Bayesian analysis

Summary

Introduction

Intracellular lipid binding proteins (iLBPs) are small, 14–15 kDa polypeptide chains that are thought to function in the transcellular trafficking of fatty acids, retinoids, and bile salts[1,2]. Proton transfer mechanisms and tautomer equilibria near physiological pH may have a role in conformational transitions associated with ligand entry and/or a subsequent communication between the binding sites, in particular that hydrogen bond networks have been shown to be key determinants of positive binding cooperativity www.nature.com/scientificreports in human I-BABP21. Due to their multiple tautomeric and charged states, histidines play an important role in binding processes in proteins, in particular that the pKa of their imidazole ring is usually close to physiological pH allowing them to act as either acids or bases[22,23]. Our results unveil an intimate relation between histidine protonation and an opening/closing motion mediating ligand entry in the apo form of hI-BABP raising the possibility of a pH-dependent mechanism pf bile salt uptake in the enterocytes of the distal small intestine

Methods

Results

Conclusion