Local and Global Dynamics in Klebsiella pneumoniae Outer Membrane Protein a in Lipid Bilayers Probed at Atomic Resolution

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• 作者：Olivier Saurel ; Iordan Iordanov ; Guillaume Nars ; Pascal Demange ; Tanguy Le Marchand ; Loren B. Andreas ; Guido Pintacuda ; Alain Milon
• 刊名：Journal of the American Chemical Society
• 出版年：2017
• 出版时间：February 1, 2017
• 年：2017
• 卷：139
• 期：4
• 页码：1590-1597
• 全文大小：515K
• ISSN：1520-5126

文摘

The role of membrane proteins in cellular mechanism strongly depends on their dynamics, and solid-state magic-angle spinning (MAS) nuclear magnetic resonance (NMR) is a unique method to exhaustively characterize motions of proteins in a lipid environment. Herein, we make use of advances in ¹H-detected MAS NMR to describe the dynamics of the membrane domain of the Outer membrane protein A of Klebsiella pneumoniae (KpOmpA). By measuring ¹H–¹⁵N dipolar-coupling as well as ¹⁵N R₁ and R_1ρ relaxation rates at fast (60 kHz) MAS and high magnetic field (1 GHz), we were able to describe the motions of the residues of the β-barrel as a collective rocking of low amplitude and of hundreds of nanoseconds time scale. Residual local motions at the edges of the strands, underscored by enhanced ¹⁵N R_1ρ relaxation rates, report on the mobility of the connected loops. In agreement with MAS NMR data, proteolysis experiments performed on the full length KpOmpA as well as on its membrane domain, reconstituted in liposomes or in detergent micelles, revealed in all cases the existence of a unique trypsin cleavage site within the membrane domain (out of 16 potential Lys and Arg sites). This site is located in the extracellular loop L3, showing that it is highly accessible to protein–protein interactions. KpOmpA is involved in cell–cell recognition, for adhesion and immune response mechanisms. The L3 region may therefore play a key role in pathogenicity.