Tài liệu Báo cáo khoa học: Basis of recognition between the NarJ chaperone and the N-terminus of the

Basis of recognition between the NarJ chaperone and the

N-terminus of the NarG subunit from Escherichia coli

nitrate reductase

Silva Zakian1

, Daniel Lafitte2

, Alexandra Vergnes1

, Cyril Pimentel3

, Corinne Sebban-Kreuzer3

,

Rene´ Toci1

, Jean-Baptiste Claude4

, Franc¸oise Guerlesquin3 and Axel Magalon1

1 Laboratoire de Chimie Bacte´rienne, Institut de Microbiologie de la Me´diterrane´e, Centre National de la Recherche Scientifique, Marseille,

France

2 MaP site Timone, UMR INSERM 911, Universite´ d’Aix-Marseille II, France

3 Interactions et Modulateurs de Re´ponses, Institut de Microbiologie de la Me´diterrane´e, Centre National de la Recherche Scientifique,

Marseille, France

4 Information Ge´nomique et Structurale, Marseille, France

Introduction

A new family of molecular chaperones, conserved in

most prokaryotes, performs essential roles in the

biogenesis of both exported and nonexported metallo￾proteins [1,2]. They share a common fold composed

entirely of a-helices and several flexible regions [1,2].

A particular feature of these chaperones is their ability

to interact with twin-arginine signal sequences of

exported metalloenzymes or N-terminal sequences of

nonexported ones [2,3]. The mechanisms governing

such interactions are of paramount importance in the

context of metalloprotein biogenesis.

These interactions are well illustrated by the non￾exported membrane-bound nitrate reductase complex

(NarGHI) of Escherichia coli, harbouring no fewer

than eight metal centres in three distinct subunits

[4–6], and the NarJ chaperone. Dynamic interactions

Keywords

chaperone; metalloproteins; nitrate

reductase; NMR; translocation

Correspondence

A. Magalon, Laboratoire de Chimie

Bacte´rienne, Institut de Microbiologie de la

Me´diterrane´e, Centre National de la

Recherche Scientifique, 31, chemin Joseph

Aiguier 13402 Marseille Cedex 09, France

Fax: +33 491 718 914

Tel: +33 491 164 668

E-mail: [email protected]

(Received 8 December 2009, revised 25

January 2010, accepted 4 February 2010)

doi:10.1111/j.1742-4658.2010.07611.x

A novel class of molecular chaperones co-ordinates the assembly and

targeting of complex metalloproteins by binding to an amino-terminal

peptide of the cognate substrate. We have previously shown that the NarJ

chaperone interacts with the N-terminus of the NarG subunit coming from

the nitrate reductase complex, NarGHI. In the present study, NMR

structural analysis revealed that the NarG(1–15) peptide adopts an a-helical

conformation in solution. Moreover, NarJ recognizes and binds the helical

NarG(1–15) peptide mostly via hydrophobic interactions as deduced from

isothermal titration calorimetry analysis. NMR and differential scanning

calorimetry analysis revealed a modification of NarJ conformation during

complex formation with the NarG(1–15) peptide. Isothermal titration calo￾rimetry and BIAcore experiments support a model whereby the protonated

state of the chaperone controls the time dependence of peptide interaction.

Structured digital abstract

l MINT-7557484: NarJT (uniprotkb:P0AF26) and NarG (uniprotkb:P09152) bind (MI:0407) by

isothermal titration calorimetry (MI:0065)

l MINT-7557456: NarJT (uniprotkb:P0AF26) and NarG (uniprotkb:P09152) bind (MI:0407) by

nuclear magnetic resonance (MI:0077)

Abbreviations

DSC, differential scanning calorimetry; HSQC, heteronuclear single quantum coherence; ITC, isothermal titration calorimetry; koff, off rate

constant; kon, on rate constant; TorA, trimethylamine N-oxide reductase.

1886 FEBS Journal 277 (2010) 1886–1895 ª 2010 The Authors Journal compilation ª 2010 FEBS