Technical Highlight - March 2013
Short description: Structure determination of a membrane protein using poorly diffracting microcrystals illustrates a powerful new application of solid-state NMR spectroscopy.
Membrane protein structure determination faces many difficulties, including formation of well-diffracting crystals from detergent-purified samples. Solution-state NMR experiments are also challenging due to the large size of protein-detergent assemblies, leading to poor data sensitivity and resolution. Solid-state NMR (ssNMR) spectroscopy has unique capabilities and can yield both structural and dynamic information from samples oriented in media, such as lipid bilayers. Compared to solution NMR, the major advantage of ssNMR is that it is not limited by molecular weight. Moreover, magic angle spinning (MAS) of samples averages anisotropic interactions between nuclei, resulting in increased resolution.
Habeck, Linke, van Rossum and colleagues have now developed a novel application of MAS ssNMR using the bacterial integral membrane protein YadA as a test case. A member of the trimeric autotransporter adhesin (TAA) family, YadA is used by the pathogen Y enterocolitica to adhere to host cells during infection. Microcrystals of the YadA membrane anchor region (YadA-M) were obtained during crystallization trials but were not of sufficient quality to solve the structure. The authors decided instead to use the microcrystals for ssNMR experiments and obtained a comprehensive data set from a single sample. Although 24 MAS experiments were recorded, 13 of those provided the necessary structural information due to the high quality of the data. The chemical shift data and distance restraints revealed that the YadA-M structure is composed of an N-terminal α-helix and four β-strands.
The actual structure determination did pose challenges, given that some ssNMR restraint types are less tightly defined than those in solution. In addition, as with other proteins that form homo-oligomers, there is considerable ambiguity associated with resolving intra- and inter-monomer interactions. The authors addressed this issue via an iterative approach, using the inferential structure determination method previously applied to sparse and hybrid data sets. The final calculations were only compatible with a trimer and the structure ensemble is well defined, with an r.m.s.d. of 0.84 ± 0.32 Å for Cα atoms.
The trimeric YadA-M structure consists of a β-barrel and a coiled-coil composed of the N-terminal α-helices that pass through the central cavity. Evolutionary analysis of TAA family members supports the observation from the NMR data that residues in the barrel lumen and an adjacent ASSA sequence present structural flexibility. The structure also allowed the authors to propose an intermediate involving the ASSA sequence during the autotransport mechanism.
S.A. Shahid et al. Membrane-protein structure determination by solid-state NMR spectroscopy of microcrystals.
Nat Methods. 9, 1212-1217 (2012). doi:10.1038/nmeth.2248
S.A. Shahid, S. Markovic, D. Linke, B.J. van Rossum. Assignment and secondary structure of the YadA membrane protein by solid-state MAS NMR.
Sci Rep. 2, 803 (2012). doi:10.1038/srep00803