Tài liệu Báo cáo khoa học: Allosteric modulation of myristate and Mn(III)heme binding to human serum

Allosteric modulation of myristate and Mn(III)heme

binding to human serum albumin

Optical and NMR spectroscopy characterization

Gabriella Fanali1

, Riccardo Fesce1

, Cristina Agrati1

, Paolo Ascenzi2,3 and Mauro Fasano1

1 Dipartimento di Biologia Strutturale e Funzionale, and Centro di Neuroscienze, Universita` dell’Insubria, Busto Arsizio (VA), Italy

2 Dipartimento di Biologia, and Laboratorio Interdisciplinare di Microscopia Elettronica, Universita` ‘Roma Tre’, Italy

3 Istituto Nazionale per le Malattie Infettive I.R.C.C.S. ‘Lazzaro Spallanzani’, Roma, Italy

Human serum albumin (HSA) is the most prominent

protein in plasma, but it is also found in tissues and

secretions throughout the body. HSA abundance (its

concentration being 45 mgÆmL)1 in the serum of

human adults) contributes significantly to colloid￾osmotic blood pressure. HSA, best known for its

extraordinary ligand binding capacity, is constituted

by a single nonglycosylated all-a chain of 65 kDa con￾taining three homologous domains (labelled I, II, and

III), each composed of two (A and B) subdomains.

The three domains have different binding capacity

for a broad variety of ligands such as aminoacids

(Trp and Cys), hormones, metal ions, and bilirubin.

Moreover, HSA has a high affinity for heme and is

Keywords

allostery; fatty acid binding; heme binding;

human serum albumin; NMR relaxation

Correspondence

M. Fasano, Dipartimento di Biologia

Strutturale e Funzionale, Universita`

dell’Insubria, Via Alberto da Giussano 12,

I-21052 Busto Arsizio (VA), Italy

Fax: +39 0331 339459

Tel: +39 0331 339450

E-mail: [email protected]

Website: http://fisio.dipbsf.uninsubria.it/cns/

fasano

(Received 21 April 2005, revised 25 July

2005, accepted 26 July 2005)

doi:10.1111/j.1742-4658.2005.04883.x

Human serum albumin (HSA) is best known for its extraordinary ligand

binding capacity. HSA has a high affinity for heme and is responsible for

the transport of medium and long chain fatty acids. Here, we report myri￾state binding to the N and B conformational states of Mn(III)heme–HSA

(i.e. at pH 7.0 and 10.0, respectively) as investigated by optical absorbance

and NMR spectroscopy. At pH 7.0, Mn(III)heme binds to HSA with lower

affinity than Fe(III)heme, and displays a water molecule coordinated to the

metal. Myristate binding to a secondary site FAx, allosterically coupled to

the heme site, not only increases optical absorbance of Mn(III)heme-bound

HSA by a factor of approximately three, but also increases the Mn(III)-

heme affinity for the fatty acid binding site FA1 by 10–500-fold. Cooper￾ative binding appears to occur at FAx and accessory myristate binding

sites. The conformational changes of the Mn(III)heme–HSA tertiary struc￾ture allosterically induced by myristate are associated with a noticeable

change in both optical absorbance and NMR spectroscopic properties of

Mn(III)heme–HSA, allowing the Mn(III)-coordinated water molecule to

exchange with the solvent bulk. At pH ¼ 10.0 both myristate affinity for

FAx and allosteric modulation of FA1 are reduced, whereas cooperation

of accessory sites and FAx is almost unaffected. Moreover, Mn(III)heme

binds to HSA with higher affinity than at pH 7.0 even in the absence of

myristate, and the metal-coordinated water molecule is displaced. As a

whole, these results suggest that FA binding promotes conformational

changes reminiscent of N to B state HSA transition, and appear of general

significance for a deeper understanding of the allosteric modulation of

ligand binding properties of HSA.

Abbreviations

FA, fatty acid; HSA, human serum albumin; MSE, mean square error; NMRD, nuclear magnetic relaxation dispersion.

4672 FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS