Tài liệu Báo cáo khoa học: Accessibility changes within diphtheria toxin T domain when in the

Accessibility changes within diphtheria toxin T domain

when in the functional molten globule state, as determined

using hydrogen⁄deuterium exchange measurements

Petr Man1,2,*, Caroline Montagner3,*, Heidi Vitrac3

, Daniel Kavan2

, Sylvain Pichard4

, Daniel Gillet4

,

Eric Forest1 and Vincent Forge3

1 Laboratoire de Spectrome´trie de Masse des Prote´ines, Institut de Biologie Structurale (CEA, CNRS, UJF, UMR 5075), Grenoble, France

2 Laboratory of Molecular Structure Characterization, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vı´denˇ ska´ 1083,

Prague 4, Czech Republic

3 CEA; DSV; iRTSV; Laboratoire de Chimie et Biologie des Me´taux (UMR 5249); CEA-Grenoble, Grenoble, France

4 Commissariat a` l’Energie Atomique (CEA), Institut de Biologie et Technologies de Saclay (iBiTecS), Service d’Inge´nierie Mole´ culaire des

Prote´ines (SIMOPRO), F-91191 Gif sur Yvette, France

Keywords

diphtheria toxin; hydrogen ⁄ deuterium

exchanges; mass spectrometry; protein ⁄

membrane interactions; translocation domain

Correspondence

D. Gillet, Commissariat a` l’Energie Atomique

(CEA), Institut de Biologie et Technologies

de Saclay (iBiTecS), Service d’Inge´nierie

Mole´ culaire des Prote´ines (SIMOPRO),

F-91191 Gif sur Yvette, France

Fax: +33 1 69 08 94 30

Tel: +33 1 69 08 76 46

E-mail: [email protected]

E. Forest, Laboratoire de Spectrome´trie de

Masse des Prote´ines, Institut de Biologie

Structurale (CEA-CNRS-UJF), 41 rue Jules

Horowitz, 38027 Grenoble, France

Fax: +33 4 38 78 54 94

Tel: +33 4 38 78 34 03

E-mail: [email protected]

V. Forge, CEA; DSV; iRTSV; Laboratoire de

Chimie et Biologie des Me´taux (UMR 5249);

CEA-Grenoble, 17 rue des martyrs, 38054

Grenoble, France

Fax: +33 4 38 78 54 87

Tel: +33 4 38 78 94 05

E-mail: [email protected]

*These authors contributed equally to this work

(Received 7 August 2009, revised 6

November2009, accepted23November2009)

doi:10.1111/j.1742-4658.2009.07511.x

The translocation domain (T domain) of diphtheria toxin adopts a partially

folded state, the so-called molten globule state, to become functional at

acidic pH. We compared, using hydrogen ⁄ deuterium exchange experiments

associated with MS, the structures of the T domain in its soluble folded

state at neutral pH and in its functional molten globule state at acidic pH.

In the native state, the a-helices TH5 and TH8 are identified as the core of

the domain. Based on the high-resolution structure of the T domain, we

propose that TH8 is highly protected because it is buried within the native

structure. According to the same structure, TH5 is partly accessible at the

surface of the T domain. We propose that its high protection is caused by

the formation of dimers. Within the molten globule state, high protection

is still observed within the helical hairpin TH8–TH9, which is responsible

for the insertion of the T domain into the membrane. In the absence of the

lipid bilayer, this hydrophobic part of the domain self-assembles, leading

to the formation of oligomers. Overall, hydrogen ⁄ deuterium-exchange mea￾surements allow the analysis of interaction contacts within small oligomers

made of partially folded proteins. Such information, together with crystal

structure data, are particularly valuable for using to analyze the self￾assembly of proteins.

Structured digital abstract

l MINT-7298382, MINT-7298394: diphtheria toxin (uniprotkb:Q6NK15) and diphtheria toxin

(uniprotkb:Q6NK15) bind (MI:0407) by molecular sieving (MI:0071)

Abbreviations

C domain, catalytic domain; ESI-TOF, electrospray ionization-time of flight; H ⁄ D, hydrogen ⁄ deuterium; MG domain, molten globule domain;

N, native; T domain, translocation domain.

FEBS Journal 277 (2010) 653–662 ª 2009 The Authors Journal compilation ª 2009 FEBS 653