A Disulfide Bridge Allows for Site‐Selective Binding in Liver Bile Acid Binding Protein Thereby Stabilising the Orientation of Key Amino Acid Side Chains

Abstract

The presence of a disulfide bridge in liver bile acid binding protein (L-BABP/S-S) allows for site-selective binding of two bile acids, glycochenodeoxycholic (GCDA) and glycocholic acid (GCA), differing only in the presence of a hydroxyl group. The protein form devoid of the disulfide bridge (L-BABP) binds both bile salts without discriminating ability. We investigate the determinants of the molecular recognition process in the formation of the heterotypic L-BABP/S-S complex with GCDA [corrected] and GCA [corrected] located in the superficial and inner protein sites, respectively. The comparison of the NMR spectroscopy structure of heterotypic holo L-BABP/S-S, the first reported for this protein family, with that of the homotypic L-BABP complex demonstrates that the introduction of a S-S link between adjacent strands changes the conformation of three key residues, which function as hot-spot mediators of molecular discrimination. The favoured χ(1) rotameric states (t, g(+) and g(-) for E99, Q100 and E109 residues, respectively) allow the onset of an extended intramolecular hydrogen-bond network and the consequent stabilisation of the side-chain orientation of a buried histidine, which is capable of anchoring a specific ligand.

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“…The chemical shift changes observed between apo (without ligand) and holo (in the presence of ligand) protein at IRG:BABP ratio of 3 are plotted in Figure A. Residues significantly affected by IRG addition, namely S61, F62, G65, T71, T72, G75, K77, C80, A85, C91, H98, V102, I111, and K123, have been mapped on the BABP structure, previously determined by NMR (Figure B). Key residues involved in binding are located in the strand regions delimiting the protein cavity, at the level of βD‐βJ strands, indicating that IRG is hosted in the protein internal cavity.…”

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“…Much work has been then devoted to unravel the mechanism of allostery, describing the structural determinants of ligand binding and evaluating the role of protein dynamics in ligand recognition [30, 31, 36]. Specifically one highly conserved buried histidine (H98) was indicated to play a central role in establishing a network of hydrogen bonds and salt bridges among buried residues, defining a sort of continuous polar ‘‘spine’’ connecting remote strands of the liver apo protein (Figure 2) [20, 32]. Indeed a mutation of H98 was shown to disrupt the energetic communication functional to efficient binding [29].…”

“…It was shown that when the human ileal protein is incubated with a mixture of the two bile salts, GCA binds nearly exclusively to one site, while GCDA binds nearly exclusively to the other site [33]. In the case of the chicken liver scaffold, the presence of a disulphide bridge was required to confer site selectivity to the protein [20]. Indeed sequence analysis of non-mammalian chicken liver BABP (access code P80226 in Swiss-Prot) reported the presence of a cysteine residue at position 80 and of a threonine residue at position 91.…”

“…Alignment of non-mammalian liver BABPs shows that a cysteine in position 91 is present in nearly all aligned sequences, with the exception of lizard and axolotl [22]. Both chicken liver proteins, with and without the disulphide bridge, were produced and their dynamics and interactions with endogenous ligands studied [20, 41]. Only the protein endowed with the disulphide bridge displayed site-selectivity, as clearly shown by the first trace of 1 H- 15 N HSQC experiments (Figure 3), whereby a sample of T91C-BABP: 15 N-GCA complex (1:2) (Figure 3, black line) after addition of unlabelled GCDA (u-GCDA) (Figure 3, grey line) showed the disappearance of the resonance corresponding to site 1, occupied by u-GCDA.…”

“…The more hydrophobic GCDA is bound to site 1, which displays a more hydrophobic character [20]. The presence of a S-S link between adjacent strands was shown to favour specific rotameric states for residues E99, Q100 and E109, allowing for the onset of an extended intramolecular hydrogen bond network and the consequent stabilization of the side-chain orientation of the buried H98, thus capable of anchoring GCA.…”