NMR Analyses of the Structure and Dynamics of Klebsiella Pneumoniae OMPA Domains and Full Length Protein

Collaborations:Andreas Engel and Daniel Muller, ETH Zurich, Basel, Switzerland.G. Pintacuda, CRMN, UMR5280, Villeurbanne, France.The transmembrane domain of kpOmpA possesses four long extracellular loops which exhibit substantial sequence variability throughout OmpA homologues in Enterobacteria. These loops are responsible for the immunological properties of the protein, such as cellular and humoral recognitions. Using liquid state NMR we have determined the 3D structure of kpOmpA in DHPC micelles (M. Renault et al., J. Mol. Biol. 2009). In a micellar environment, a complex dynamical behavior has been observed: a rigid barrel core, ms motion at the micellar-water interface, and sub-ns motion within the loops. Using solid state NMR relaxation and proteolysis experiments, we have demonstrated the persistence of this complex motional behavior in E. coli polar lipid bilayers (I. Iordanov et al., Biochim. Biophys. Acta, 2012). Using single molecule force spectroscopy (with D. Muller and A. Engel) we have shown that kpOmpA is able to unfold and refold reversibly its β-barrel core (P. Bosshart et al., Structure 2012).Recent advances involve: a) characterizing the structure of its C-terminal domain and its interaction with the peptido-glycane; b) analyzing ssNMR spectra of N-terminal membrane domain in liposomes using MAS at 1 GHz and 60 kHz spinning frequency (with G. Pintacuda); c) comparing the NMR spectra of the various domains and the full length protein in solution, in liposomes and in intact cell envelopes using cellular solid state NMR as established in (M. Renault et al., PNAS 2012).