Tài liệu Báo cáo khoa học: Disulfide bridge regulates ligand-binding site selectivity in liver bile

Disulfide bridge regulates ligand-binding site selectivity in

liver bile acid-binding proteins

Clelia Cogliati1

, Simona Tomaselli1

, Michael Assfalg2

, Massimo Pedo` 2

, Pasquale Ferranti3

,

Lucia Zetta1

, Henriette Molinari2 and Laura Ragona1

1 Laboratorio NMR, Istituto per lo Studio delle Macromolecole, CNR, Milan, Italy

2 Dipartimento di Biotecnologie, Universita` di Verona Strada le Grazie, Verona, Italy

3 Dipartimento di Scienza degli Alimenti, Universita` di Napoli Federico II, Portici, Italy

Introduction

Bile acids (BAs) are vital components of many biologi￾cal processes and play an important role in the patho￾genesis of numerous common diseases [1], but

the specific mechanisms coupling intracellular BAs to

biological targets are not well understood. BAs circu￾late between the liver and intestine through a mecha￾nism known as ‘enterohepatic circulation’, which is a

tightly regulated process, particularly by BAs them￾selves. BA-binding proteins (BABPs), belonging to the

intracellular lipid-binding protein (iLBP) family, play a

vital role in the enterohepatic circulation as cytoplas￾matic transporters of BAs. Understanding the mecha￾Keywords

backbone dynamics; disulfide bridge;

intracellular lipid-binding protein; molecular

recognition; NMR

Correspondence

L. Ragona, Lab. NMR, Istituto per lo Studio

delle Macromolecole, CNR, Via Bassini, 15,

20133, Milano, Italy

Fax: +39 02 23699620

Tel: +39 02 23699619

E-mail: [email protected]

H. Molinari, Dipartimento di Biotecnologie,

Universita` degli Studi di Verona, Strada le

Grazie, 15, 37134 Verona, Italy

Fax: +39 0458027929

Tel: +39 0458027901

E-mail: [email protected]

(Received 3 July 2009, revised 17 August

2009, accepted 18 August 2009)

doi:10.1111/j.1742-4658.2009.07309.x

Bile acid-binding proteins (BABPs) are cytosolic lipid chaperones that play

central roles in driving bile flow, as well as in the adaptation to various

pathological conditions, contributing to the maintenance of bile acid

homeostasis and functional distribution within the cell. Understanding the

mode of binding of bile acids with their cytoplasmic transporters is a key

issue in providing a model for the mechanism of their transfer from the

cytoplasm to the nucleus, for delivery to nuclear receptors. A number of

factors have been shown to modulate bile salt selectivity, stoichiometry,

and affinity of binding to BABPs, e.g. chemistry of the ligand, protein plas￾ticity and, possibly, the formation of disulfide bridges. Here, the effects of

the presence of a naturally occurring disulfide bridge on liver BABP

ligand-binding properties and backbone dynamics have been investigated

by NMR. Interestingly, the disulfide bridge does not modify the protein￾binding stoichiometry, but has a key role in modulating recognition at both

sites, inducing site selectivity for glycocholic and glycochenodeoxycholic

acid. Protein conformational changes following the introduction of a disul￾fide bridge are small and located around the inner binding site, whereas

significant changes in backbone motions are observed for several residues

distributed over the entire protein, both in the apo form and in the holo

form. Site selectivity appears, therefore, to be dependent on protein mobil￾ity rather than being governed by steric factors. The detected properties

further establish a parallelism with the behaviour of human ileal BABP,

substantiating the proposal that BABPs have parallel functions in hepato￾cytes and enterocytes.

Abbreviations

BA, bile acid; BABP, bile acid-binding protein; CA, cholate; CDA, chenodeoxycholate; CSP, chemical shift perturbation; GCA, glycocholic acid;

GCDA, glycochenodeoxycholic acid; I-BABP, human ileal bile acid-binding protein; iLBP, intracellular lipid-binding protein; L-BABP, chicken

liver bile acid-binding protein.

FEBS Journal 276 (2009) 6011–6023 ª 2009 The Authors Journal compilation ª 2009 FEBS 6011