Tài liệu Báo cáo khoa học: Resolving the native conformation ofEscherichia coli OmpA docx

Resolving the native conformation of Escherichia coli

OmpA

Alexander Negoda, Elena Negoda and Rosetta N. Reusch

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA

Introduction

Outer membrane protein A (OmpA), a major outer

membrane protein of Escherichia coli, is a highly con￾served and multifunctional integral membrane protein

that has served as a model system for studies of outer

membrane targeting and protein folding [1]. However,

despite intense study for several decades, the native

structure of the protein has not yet been resolved.

A number of genetic and biochemical studies have

provided evidence for a two-domain structure of

OmpA, in which the N-terminal domain (residues

1–170) crosses the membrane eight times in antiparallel

b-strands, and the 155-residue C-terminal domain

resides in the periplasm, where it may interact with

peptidoglycan [2–6]. Additional evidence for a two￾domain structure comes from Raman spectroscopy [7]

CD and fluorescence studies [8–16]. The crystal

structure of the N-terminal 171 residues of OmpA,

determined by Pautsch and Schulz [17,18], shows an

eight-stranded amphipathic b-barrel with no continu￾ous water channel. High-resolution NMR [19,20] and

Keywords

cOHB-modification; disulfide bond; outer

membrane protein; protein folding; protein

targeting

Correspondence

R. N. Reusch, Department of Microbiology

and Molecular Genetics, Michigan State

University, East Lansing, MI 48824, USA

Fax: +1 517 353 8957

Tel: +1 517 884 5388

E-mail: [email protected]

(Received 7 July 2010, revised 17 August

2010, accepted 20 August 2010)

doi:10.1111/j.1742-4658.2010.07823.x

The native conformation of the 325-residue outer membrane protein A

(OmpA) of Escherichia coli has been a matter of contention. A narrow￾pore, two-domain structure has vied with a large-pore, single-domain struc￾ture. Our recent studies show that Ser163 and Ser167 of the N-terminal

domain (1–170) are modified in the cytoplasm by covalent attachment of

oligo-(R)-3-hydroxybutyrates (cOHBs), and further show that these modifi￾cations are essential for the N-terminal domain to be incorporated into

planar lipid bilayers as narrow pores ( 80 pS, 1 m KCl, 22 C). Here, we

examined the potential effect(s) of periplasmic modifications on pore struc￾ture by comparing OmpA isolated from outer membranes (M-OmpA) with

OmpA isolated from cytoplasmic inclusion bodies (I-OmpA). Chemical and

western blot analysis and 1

H-NMR showed that segment 264–325 in

M-OmpA, but not in I-OmpA, is modified by cOHBs. Moreover, a disul￾fide bond is formed between Cys290 and Cys302 by the periplasmic enzyme

DsbA. Planar lipid bilayer studies indicated that narrow pores formed by

M-OmpA undergo a temperature-induced transition into stable large pores

( 450 pS, 1 m KCl, 22 C) [energy of activation (Ea) = 33.2 kcalÆmol)1

],

but this transition does not occur with I-OmpA or with M-OmpA that has

been exposed to disulfide bond-reducing agents. The results suggest that

the narrow pore is a folding intermediate, and demonstrate the decisive

roles of cOHB-modification, disulfide bond formation and temperature in

folding OmpA into its native large-pore configuration.

Abbreviations

C8E4, n-octyl tetraethylene glycol monoether; cOHBs, conjugated oligo-(R)-3-hydroxybutyrates; DPhPC, diphytanoylphosphatidylcholine; Ea,

energy of activation; I-OmpA, outer membrane protein A isolated from cytoplasmic inclusion bodies; LDS, lithium dodecylsulfate; M-OmpA,

outer membrane protein A isolated from outer membranes; OHBs, oligo-(R)-3-hydroxybutyrates; OmpA, outer membrane protein A; PVDF,

poly(vinylidene difluoride); 2-ME, 2-mercaptoethanol.

FEBS Journal 277 (2010) 4427–4437 ª 2010 The Authors Journal compilation ª 2010 FEBS 4427