X-ray crystallographic analysis of three membrane proteins : The nicotinic acetylcholine receptor from Torpedo californica, Omp85 and TtoA from Thermus thermophilus HB27

Abstract

The work presented here was focused on the purification, crystallization and X-ray structure determination of three different transmembrane proteins - the nicotinic acetylcholine receptor (nAChR) from Torpedo californica and two outer membrane proteins from Thermus thermophilus HB27, TtOmp85, and one of its potential substrates, TtoA.The nAChR is a ligand-gated ion channel which belongs to the superfamily of Cys-loop receptors and plays a keyrole in synaptic transmission of neuronal signals. nAChRs consist of five homologous subunits composed as tissue-specific homo- or heteropentamers. So far, only a structure of 4.0 Å resolution derived from electron microscopy on tubular 2D-crystals is available for whole nAChR pentamers from Torpedo marmorata. Albeit two high resolution structures of prokaryotic homologues were published recently, a high-resolution structure of the nAChR is still missing. Microcrystals obtained from preparations of detergent-solubilized nAChR-enriched membranes from the electric organ of the pacific ray Torpedo california were reported already in 1988, but the properties of those crystals remained uncharacterized. In the work presented here, such crystals were examined by synchroton radiation and diffraction patterns typical for protein crystals were recorded. Diffraction of those crystals was limited to 20 Å resolution, possible spacegroup and unit-cell parameters were determined with the recorded data. Neither refinement of the crystallization conditions nor optimization of the purification protocol allowed to grow crystals of solubilized nAChR suitable for high resolution X-ray structure determination.Proteins belonging to the Omp85 family are involved in the assembly of beta-barrel outer membrane proteins or in the translocation of proteins across the outer membrane in bacteria, mitochondria and chloroplasts. The thermophilic bacterium Thermus thermophilus represents an intermediate between Gram-positive and Gram-negative bacteria with an only roughly characterized outer membrane. It encodes one Omp85-like protein, TtOmp85, which represents an ancestral type of this family. TtOmp85 was overexpressed in T. thermophilus strain HB27 and natively purified from preparations of the outer membrane. In the presence of detergent, purified TtOmp85 existed mainly as a monomer, composed of two stable protease-resistant modules. Detergent solubilized TtOmp85 was used for crystallization experiments and microcrystals appeared under crystallization conditions including PEG400 as precipitant. Crystals diffracted to 5.0 Å resolution and belonged to spacegroup C222 with unit cell dimensions of a = 96.3 Å, b = 416.1 Å, c = 94.3 Å; alpha = beta = gamma = 90°. Only a partial dataset could be collected because diffraction was anisotropic, depending on the orientation of the plate shaped microcrystals in the X-ray beam, and crystals rapidly accumulated radiation damage during proceeding datacollection. The structure of TtOmp85 could not be solved with the available data. Preparations of detergent solubilized TtOmp85 were provided for accompanying studies in collaborating working groups to further characterize TtOmp85.Only a few further outer membrane proteins from T. thermophilus are described. TtoA, a new beta-barrel outer membrane protein, was identified by bioinformatic analysis of the genome sequence of strain HB27. Results of an in vitro binding assay where TtoA bound to TtOmp85 suggest that insertion of proteins in the outer membrane in T. thermophilus might be similar to the mechanisms found in modern Gram-negative bacteria. TtoA was successfully crystallized and its structure was determined to a resolution of 2.8 Å, representing the first crystal structure of an outer membrane protein from a thermophilic bacterium. Crystals belonged to space group P3(1)21 with unit cell parameters a = b = 166.7 Å, c = 97.5 Å; alpha = beta = 90°, gamma = 120°. TtoA consists of an eight-stranded beta-barrel with a large extracellular part to which a divalent cation is bound. A five-stranded extracellular beta-sheet protrudes out of the membrane-embedded transmembrane barrel and is stabilized by a disulfide bridge. The edge of this beta-sheet forms crystal contacts with neighbouring TtoA molecules that could mimic physiologic interactions with other proteins.