kpOmpA is a membrane protein belonging to the outer Membrane Protein A family. Its transmembrane domain (216 aa) presents a significant homology with E. coli OmpA (80 %), whose three dimensional structure has been determined by X-ray crystallography and by NMR (1). The difference is mostly concentrated in the extracellular loops which are larger in the case of kpOmpA. This protein was shown to activate macrophages and dendritic cells through the TLR2 dependent pathway, and these larger loops are supposed to play a specific role in the interactions with the immune system (2,3).
The present work is focused on structural and dynamical studies of kpOmpA transmembrane domain by high resolution liquid state NMR. This domain, in fusion with a C-terminal His-tag, was overexpressed in E. Coli as inclusion bodies, and subsequently purified and refolded in 3-14 zwittergent. CD and gel electrophoresis demonstrated the formation of a beta barrel type protein. It was also produced in fully 13C, 15N, 2H labelled form for NMR studies. Preliminary experiments in a variety of detergents demonstrated that DHPC is the detergent providing the highest quality spectra. D2O exchange experiments revealed that in the NMR sample conditions a number of resonances were non exchangeable for weeks, confirming the presence of a stable fold. This sample was subjected to a whole set of 3D experiments in their TROSY version: HSQC, HNCACB, HN(CO)CACB, HNCO, HN(CA)CO, (HNC)CCCNH-TOCSY at 700 MHz (Toulouse), 600, 800 and 900 MHz with cryoprobes (Frankfort EU-NMR facility). This set of experiments has allowed the assignment of 85% of 1H and 15N resonances 90% of C’, 88% C and Cβ, and 81% of other aliphatic side-chain 13C resonances. A 4D HN-HN NOESY experiment has revealed a ensemble of H-H proximities characteristic of a β barrel, also confirmed by CSIs. A sample of methyl protonated otherwise perdeuterated protein was used to measure CH3-NH and CH3-CH3 constraints. An ensemble of NMR constraints have been determined, including 528 distance restraints, 128 H-bond restraints and 264 torsion angle restraints. The structure calculation was accomplished within CNS providing a set of 20 best structures with a rmsd of 0.564 Å within the  barrel. The dynamical analysis has been performed using 15N relaxation for ns loops dynamics and time dependent TROSY for interfacial slow ms time scale motions (4).
The protein has also been reconstituted in DMPC lipid bilayers (5), and preliminary solid state NMR MAS 2D 13C spin diffusion spectra have been acquired in order to compare the structure and dynamics in detergent and bilayer environment. As part of a European network on “structural biology of membrane proteins”, in collaboration with A. Engel (Basel), and D. Muller (Dresden), we are currently analysing kpOmpA in lipid bilayer by electron microscopy, atomic force microscopy and single molecule force microscopy.

(1) A. Pautsch et al. J Mol Biol. 2000, A. Arora et al., Nat. Struct. Biol., 2001 (2) P. Jeannin et al., Nat. immunol., 2000; Immunity 2005 (3) PhD thesis, M. Sugawara, UPS Toulouse, France, 2003 (4) M. renault et al. J. Mol. Biol., 385, 117 (2009) (5) M. Renault et al., C.R. Chimie, 2006
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