Membrane proteins are largely underrepresented among available atomic-resolution structures. The use of detergents in protein purification procedures hinders the formation of well-ordered crystals for X-ray crystallography and leads to slower molecular tumbling, impeding the application of solution-state NMR. Solid-state magic-angle spinning NMR spectroscopy is an emerging method for membrane-protein structural biology that can overcome these technical problems. Here we present the solid-state NMR structure of the transmembrane domain of the Yersinia enterocolitica adhesin A (YadA). The sample was derived from crystallization trials that yielded only poorly diffracting microcrystals. We solved the structure using a single, uniformly (13)C- and (15)N-labeled sample. In addition, solid-state NMR allowed us to acquire information on the flexibility and mobility of parts of the structure, which, in combination with evolutionary conservation information, presents new insights into the autotransport mechanism of YadA.

MAS-NMR was used to study the structure and dynamics at ambient temperatures of the membrane-anchor domain of YadA (YadA-M) in a pellet of the outer membrane of E. coli in which it was expressed. YadA is an adhesin from the pathogen Yersinia enterocolitica that is involved in interactions with the host cell, and it is a model protein for studying the autotransport process. Existing assignments were sucessfully transferred to a large part of the YadA-M protein in the E. coli lipid environment by using (13) C-(13) C DARR and PDSD spectra at different mixing times. The chemical shifts in most regions of YadA-M are unchanged relative to those in microcrystalline YadA-M preparations from which a structure has previously been solved, including the ASSA region that is proposed to be involved in transition-state hairpin formation for transport of the soluble domain. Comparisons of the dynamics between the microcrystalline and membrane-embedded samples indicate greater flexibility of the ASSA region in the outer-membrane preparation at physiological temperatures. This study will pave the way towards MAS-NMR structure determination of membrane proteins, and a better understanding of functionally important dynamic residues in native membrane environments.

We report the complete solid-state MAS NMR resonance assignment of a medium-sized, trimeric membrane protein, YadA-M. The protein YadA (Yersinia adhesin A) is an important virulence factor of enteropathogenic Yersinia species (such as Yersinia enterocolitica and Yersinia pseudotuberculosis). YadA is localized on the bacterial cell surface and is involved in adhesion to host cells and tissues. It is anchored in the outer membrane by a transmembrane anchor domain (YadA-M). This domain hosts the so-called autotransporter function of YadA: it transports its own N-terminal domain through the outer membrane. The assignment is based on a dataset that consisted of several MAS NMR correlation spectra, recorded on a single, uniformly 13C, 15N- labelled microcrystalline preparation. Except for the single C-terminal residue and the mobile strep tag, we were able to completely assign YadA-M. From this, secondary structure elements were predicted, which, combined with several long-range interstrand restraints, yielded the architecture of the β-sheet.