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

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

Temperature Dependence of Backbone Dynamics in Human Ileal Bile Acid-Binding Protein: Implications for the Mechanism of Ligand Binding

Human ileal bile acid-binding protein (I-BABP), a member of the family of intracellular lipid binding proteins plays a key role in the cellular trafficking and metabolic regulation of bile salts. The protein has two internal and, according to a recent study, an additional superficial binding site and binds di- and trihydroxy bile salts with positive cooperativity and a high degree of site-selectivity. Previously, in the apo form, we have identified an extensive network of conformational fluctuations on the millisecond time scale, which cease upon ligation. Additionally, ligand binding at room temperature was found to be accompanied by a slight rigidification of picosecond-nanosecond (ps-ns) backbone flexibility. In the current study, temperature-dependent N-15 NMR spin relaxation measurements were used to gain more insight into the role of dynamics in human I-BABP-bile salt recognition. According to our analysis, residues sensing a conformational exchange in the apo state can be grouped into two clusters with slightly different exchange rates. The entropy-enthalpy compensation observed for both clusters suggests a disorder-order transition between a ground and a sparsely populated higher energy state in the absence of ligands. Analysis of the faster, ps-ns motion of N-15-H-1 bond vectors indicates an unusual nonlinear temperature-dependence for both ligation states. Intriguingly, while bile salt binding results in a more uniform response to temperature change throughout the protein, the temperature derivative of the generalized order parameter shows different responses to temperature increase for the two forms of the protein in the investigated temperature range. Analysis of both slow and fast motions in human I-BABP indicates largely different energy landscapes for the apo and halo states suggesting that optimization of binding interactions might be achieved by altering the dynamic behavior of specific segments in the protein

Structural and Dynamic Determinants of Molecular Recognition in Bile Acid-Binding Proteins

Disorders in bile acid transport and metabolism have been related to a number of metabolic disease states, atherosclerosis, type-II diabetes, and cancer. Bile acid-binding proteins (BABPs), a subfamily of intracellular lipid-binding proteins (iLBPs), have a key role in the cellular trafficking and metabolic targeting of bile salts. Within the family of iLBPs, BABPs exhibit unique binding properties including positive binding cooperativity and site-selectivity, which in different tissues and organisms appears to be tailored to the local bile salt pool. Structural and biophysical studies of the past two decades have shed light on the mechanism of bile salt binding at the atomic level, providing us with a mechanistic picture of ligand entry and release, and the communication between the binding sites. In this review, we discuss the emerging view of bile salt recognition in intestinal- and liver-BABPs, with examples from both mammalian and non-mammalian species. The structural and dynamic determinants of the BABP-bile–salt interaction reviewed herein set the basis for the design and development of drug candidates targeting the transcellular traffic of bile salts in enterocytes and hepatocytes

Peptide antibiotics in action: Investigation of polypeptide chains in insoluble environments by rotational-echo double resonance

AbstractRotational-echo double resonance (REDOR) is a solid-state NMR technique that has the capability of providing intra- and intermolecular distance and orientational restraints in non-crystallizable, poorly soluble heterogeneous molecular systems such as cell membranes and cell walls. In this review, we will present two applications of REDOR: the investigation of a magainin-related antimicrobial peptide in lipid bilayers and the study of a vancomycin-like glycopeptide in the cell walls of Staphylococcus aureus

Temperature Dependence of Backbone Dynamics in Human Ileal Bile Acid-Binding Protein: Implications for the Mechanism of Ligand Binding

Human ileal bile acid-binding protein (I-BABP), a member of the family of intracellular lipid binding proteins plays a key role in the cellular trafficking and metabolic regulation of bile salts. The protein has two internal and, according to a recent study, an additional superficial binding site and binds di- and trihydroxy bile salts with positive cooperativity and a high degree of site-selectivity. Previously, in the <i>apo</i> form, we have identified an extensive network of conformational fluctuations on the millisecond time scale, which cease upon ligation. Additionally, ligand binding at room temperature was found to be accompanied by a slight rigidification of picosecond–nanosecond (ps–ns) backbone flexibility. In the current study, temperature-dependent ¹⁵N NMR spin relaxation measurements were used to gain more insight into the role of dynamics in human I-BABP-bile salt recognition. According to our analysis, residues sensing a conformational exchange in the <i>apo</i> state can be grouped into two clusters with slightly different exchange rates. The entropy–enthalpy compensation observed for both clusters suggests a disorder–order transition between a ground and a sparsely populated higher energy state in the absence of ligands. Analysis of the faster, ps–ns motion of ¹⁵N–¹H bond vectors indicates an unusual nonlinear temperature-dependence for both ligation states. Intriguingly, while bile salt binding results in a more uniform response to temperature change throughout the protein, the temperature derivative of the generalized order parameter shows different responses to temperature increase for the two forms of the protein in the investigated temperature range. Analysis of both slow and fast motions in human I-BABP indicates largely different energy landscapes for the <i>apo</i> and <i>holo</i> states suggesting that optimization of binding interactions might be achieved by altering the dynamic behavior of specific segments in the protein

Multiple Timescale Dynamic Analysis of Functionally-Impairing Mutations in Human Ileal Bile Acid-Binding Protein

Human ileal bile acid-binding protein (hI-BABP) has a key role in the enterohepatic circulation of bile salts. Its two internal binding sites exhibit positive cooperativity accompanied by a site-selectivity of glycocholate (GCA) and glycochenodeoxycholate (GCDA), the two most abundant bile salts in humans. To improve our understanding of the role of dynamics in ligand binding, we introduced functionally impairing single-residue mutations at two key regions of the protein and subjected the mutants to NMR relaxation analysis and MD simulations. According to our results, mutation in both the vicinity of the C/D (Q51A) and the G/H (Q99A) turns results in a redistribution of motional freedom in apo hI-BABP. Mutation Q51A, deteriorating the site-selectivity of GCA and GCDA, results in the channeling of ms fluctuations into faster motions in the binding pocket hampering the realization of key side chain interactions. Mutation Q99A, abolishing positive binding cooperativity for GCDA, leaves ms motions in the C-terminal half unchanged but by decoupling &beta;D from a dynamic cluster of the N-terminal half displays an increased flexibility in the vicinity of site 1. MD simulations of the variants indicate structural differences in the portal region and mutation-induced changes in dynamics, which depend on the protonation state of histidines. A dynamic coupling between the EFGH portal, the C/D-region, and the helical cap is evidenced highlighting the interplay of structural and dynamic effects in bile salt recognition in hI-BABP

Secondary Structure and Lipid Contact of a Peptide Antibiotic in Phospholipid Bilayers by REDOR

The chemical shifts of specific (13)C and (15)N labels distributed throughout KIAGKIA-KIAGKIA-KIAGKIA (K3), an amphiphilic 21-residue antimicrobial peptide, prove that the peptide is in an all α-helical conformation in the bilayers of multilamellar vesicles (MLVs) containing dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol (1:1). Rotational-echo double-resonance (REDOR) (13)C{(31)P} and (15)N{(31)P} experiments on the same labeled MLVs show that on partitioning into the bilayer, the peptide chains remain in contact with lipid headgroups. The amphipathic lysine side chains of K3 in particular appear to play a key role in the electrostatic interactions with the acidic lipid headgroups. In addition to the extensive peptide-headgroup contact, (13)C{(19)F} REDOR experiments on MLVs containing specifically (19)F-labeled lipid tails suggest that a portion of the peptide is surrounded by a large number of lipid acyl chains. Complementary (31)P{(19)F} REDOR experiments on these MLVs show an enhanced headgroup-lipid tail contact resulting from the presence of K3. Despite these distortions, static (31)P NMR lineshapes indicate that the lamellar structure of the membrane is preserved

Internal Motions and Exchange Processes in Human Ileal Bile Acid Binding Protein As Studied by Backbone ¹⁵N Nuclear Magnetic Resonance Spectroscopy

Human ileal bile acid binding protein (I-BABP), a member of the family of intracellular lipid binding proteins, is thought to play a role in the enterohepatic circulation of bile salts. Previously, we have shown by stopped-flow fluorescence analysis that positive binding cooperativity exhibited by I-BABP in its interactions with glycocholate (GCA) and glycochenodeoxycholate (GCDA), the two primary bile salts in humans, is related to a slow conformational change in the protein. In this study, we used backbone ¹⁵N relaxation nuclear magnetic resonance (NMR) techniques to obtain residue-specific information about the internal dynamics of apo I-BABP and the doubly ligated I-BABP:GCA:GCDA complex on various time scales. According to our NMR data, bile salt binding is accompanied by a slight rigidification of the ¹⁵N–¹H bond vectors on the picosecond to nanosecond time scale, with most pronounced changes occurring in the C–D region. In contrast to the minor effects of ligation on fast motions, relaxation dispersion NMR experiments indicate a marked difference between the two protein states on the microsecond to millisecond time scale. In the apo form, an extensive network of conformational fluctuations is detected throughout segments of the EFGHIJ β-strands and the C–D loop, which cease upon complexation. Our NMR data are in agreement with a conformational selection model we proposed earlier for I-BABP and support the hypothesis of an allosteric mechanism of ligand binding. According to the NMR measurements, the helical cap region may have a less crucial role in mediating ligand entry and release than what has been indicated for fatty acid binding proteins