Tài liệu Báo cáo khoa học: Crystal structure of importin-a bound to a peptide bearing the nuclear

Crystal structure of importin-a bound to a peptide bearing

the nuclear localisation signal from chloride intracellular

channel protein 4

Andrew V. Mynott1

, Stephen J. Harrop1

, Louise J. Brown2

, Samuel N. Breit3

, Bostjan Kobe4,5 and

Paul M. G. Curmi1,3

1 School of Physics, University of New South Wales, Sydney, NSW, Australia

2 Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia

3 St Vincent’s Centre for Applied Medical Research, St Vincent’s Hospital and University of New South Wales, Sydney, NSW, Australia

4 School of Chemistry and Molecular Biosciences and Centre for Infectious Disease Research, University of Queensland, Brisbane, Qld,

Australia

5 Institute for Molecular Bioscience, University of Queensland, Brisbane, Qld, Australia

Introduction

The importin-a:b nuclear import pathway is one of the

best understood nuclear trafficking systems in the cell

[1]. The pathway operates via the importin-a receptor,

an armadillo (ARM) repeat protein, that recognizes and

binds directly to cargo protein in the cytoplasm. The im￾portin-a:importin-b:cargo complex travels through the

nuclear pore, with importin-b primarily responsible for

negotiating passage through the nuclear pore complex.

This transport process is dependent on the ability of im￾portin-a to recognize specific nuclear localization signals

(NLSs) presented by the cargo protein. The acidic envi￾ronment of the importin-a binding sites confers a high

Keywords

chloride intracellular channel protein; CLIC4;

importin-a; nuclear localization signal (NLS);

nucleocytoplasmic transport

Correspondence

P. Curmi, School of Physics, University of

New South Wales, Sydney, NSW 2052,

Australia

Fax: +61 2 9385 6060

Tel: +61 2 9385 4552

E-mail: [email protected]

(Received 17 November 2010, revised 31

January 2011, accepted 23 February 2011)

doi:10.1111/j.1742-4658.2011.08086.x

It has been reported that a human chloride intracellular channel (CLIC)

protein, CLIC4, translocates to the nucleus in response to cellular stress,

facilitated by a putative CLIC4 nuclear localization signal (NLS). The

CLIC4 NLS adopts an a-helical structure in the native CLIC4 fold. It is

proposed that CLIC4 is transported to the nucleus via the classical nuclear

import pathway after binding the import receptor, importin-a. In this

study, we have determined the X-ray crystal structure of a truncated form

of importin-a lacking the importin-b binding domain, bound to a CLIC4

NLS peptide. The NLS peptide binds to the major binding site in an

extended conformation similar to that observed for the classical simian

virus 40 large T-antigen NLS. A Tyr residue within the CLIC4 NLS makes

surprisingly favourable interactions by forming side-chain hydrogen bonds

to the importin-a backbone. This structural evidence supports the hypothe￾sis that CLIC4 translocation to the nucleus is governed by the importin-a

nuclear import pathway, provided that CLIC4 can undergo a conforma￾tional rearrangement that exposes the NLS in an extended conformation.

Database

Structural data are available in the protein Data Bank under the accession number 3OQS.

Structured digital abstract

l CLIC4 and importin alpha bind by x-ray crystallography (View interaction)

Abbreviations

ARM, armadillo; CLIC, chloride intracellular channel; NLS, nuclear localization signal; RSCC, real space correlation coefficient; TAg, simian

virus 40 (SV40) large T-antigen.

1662 FEBS Journal 278 (2011) 1662–1675 ª 2011 The Authors Journal compilation ª 2011 FEBS