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Research Themes Membrane proteins

Microbiome: Solid-State NMR, Crystallized

SBKB [doi:10.1038/sbkb.2012.161]
Technical Highlight - September 2013
Short description: The structure of a bacterial membrane protein was determined using solid-state NMR methods on protein microcrystals.

Structure ensemble of
YadA-M, determined by solid-state MAS NMR. 1

Obtaining high-resolution structures of membrane proteins is difficult. Detergents used to solubilize membrane proteins can hamper the growth of well-ordered crystals needed for X-ray crystallographic methods and also lead to slower molecular tumbling, which prevents the use of solution-state NMR techniques. Solid-state NMR (ssNMR) spectroscopy has been used to study the dynamics and structures of proteins, and is emerging as a useful tool in high-resolution structural studies of membrane proteins.

Recently, Habeck, van Rossum, Linke and colleagues have used solid-state magic-angle spinning (MAS) NMR to determine the structure of the membrane anchor (or translocator) domain of YadA, a trimeric autotransporter adhesin (TAA) from Yersinia enterocolitica. Many TAAs are adhesion molecules with important pathogenic roles. Of interest is the authors' use of YadA membrane anchor (YadA-M) microcrystals, obtained from attempts to isolate large, well-ordered crystals of detergent-solubilized trimeric YadA-M for structure determination by X-ray crystallography.

A single uniformly 13C- and 15N-labeled protein sample was used to obtain 1,192 nonredundant distance restraints from homo- and heteronuclear MAS NMR correlation experiments. An iterative approach was then employed to remove ambiguity between intra- and interprotomer contacts, a complication that arises for some ssNMR distance restraints. The authors solved the structure of the YadA-M trimer using inferential structure determination, a method that helps calculate structural information when sparse or hybrid structural data are used. An approximate structure of a YadA-M protomer was built using an error-tolerant restraint potential, which allowed calculation of the protomer structure when restraints only satisfied by the trimer were present. The refined protomer was then assembled into a trimer, using rotational symmetry restraints and symmetry axis determined from the NMR data.

The final ssNMR structure of YadA-M (PDB 2LME) is a trimeric β-barrel with an N-terminal helical domain passing through it. The protomers each contribute a four-stranded β-sheet to form the barrel and an N-terminal helix to form the coiled-coil helical domain. Overall, the structure is quite similar to the crystal structure of Hia from Haemophilus influenzae, revealing features that are conserved across TAA family members. The structure also suggests a mechanism of autotransport similar to that proposed for monomeric autotransporters.

Membrane-protein crystallization trials frequently result in microcrystalline samples not suitable for X-ray crystallography. The authors show that solid-state MAS NMR is an alternative in these cases; no changes to the protein preparation protocols were made and a complex labeling strategy is unnecessary.

Michelle Montoya

References

  1. S.A. Shahid et al. Membrane-protein structure determination by solid-state NMR spectroscopy of microcrystals.
    Nat. Meth. 9, 1212-1217 (2012). doi:10.1038/nmeth.2248

  2. 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

Structural Biology Knowledgebase ISSN: 1758-1338
Funded by a grant from the National Institute of General Medical Sciences of the National Institutes of Health