Assembly and stoichiometry of the core structure of the bacterial flagellar type III export gate complex.

The bacterial flagellar type III export apparatus, which is required for flagellar assembly beyond the cell membranes, consists of a transmembrane export gate complex and a cytoplasmic ATPase complex. FlhA, FlhB, FliP, FliQ, and FliR form the gate complex inside the basal body MS ring, although FliO is required for efficient export gate formation in Salmonella enterica. However, it remains unknown how they form the gate complex. Here we report that FliP forms a homohexameric ring with a diameter of 10 nm. Alanine substitutions of conserved Phe-137, Phe-150, and Glu-178 residues in the periplasmic domain of FliP (FliPP) inhibited FliP6 ring formation, suppressing flagellar protein export. FliO formed a 5-nm ring structure with 3 clamp-like structures that bind to the FliP6 ring. The crystal structure of FliPP derived from Thermotoga maritia, and structure-based photo-crosslinking experiments revealed that Phe-150 and Ser-156 of FliPP are involved in the FliP-FliP interactions and that Phe-150, Arg-152, Ser-156, and Pro-158 are responsible for the FliP-FliO interactions. Overexpression of FliP restored motility of a ∆fliO mutant to the wild-type level, suggesting that the FliP6 ring is a functional unit in the export gate complex and that FliO is not part of the final gate structure. Copurification assays revealed that FlhA, FlhB, FliQ, and FliR are associated with the FliO/FliP complex. We propose that the assembly of the export gate complex begins with FliP6 ring formation with the help of the FliO scaffold, followed by FliQ, FliR, and FlhB and finally FlhA during MS ring formation.

Structure, Function, Folding, and Aggregation of a Neuroferritinopathy-Related Ferritin Variant.

Neuroferritinopathy is a rare, adult-onset, dominantly inherited movement disorder caused by mutations in the ferritin gene. A ferritin light-chain variant related to neuroferritinopathy, in which alanine 96 is replaced with threonine (A96T), was expressed in Escherichia coli, purified, and characterized. The circular dichroism, analytical ultracentrifugation, and small-angle X-ray scattering studies have shown that both the subunit structure and the assembly of A96T are the same as those of wild-type human ferritin light chain (HuFTL). The iron-incorporation ability was also comparable to that of HuFTL. Although the structural stability against heat, acid, and denaturant was reduced, the structure was sufficiently stable under physiological conditions. The most remarkable defects observed for A96T were a lower refolding efficiency and a stronger propensity to aggregate. The possible relationship between folding deficiency and disease is discussed.

Structural stability of flagellin subunit affects the rate of flagellin export in the absence of FliS chaperone.

FliS chaperone binds to flagellin FliC in the cytoplasm and transfers FliC to a sorting platform of the flagellar type III export apparatus through the interaction between FliS and FlhA for rapid and efficient protein export during flagellar filament assembly. FliS also suppresses the secretion of an anti-σ factor, FlgM. Loss of FliS results in a short filament phenotype although the expression levels of FliC are increased considerably due to an increase in the secretion level of FlgM. Here to clarify the rate limiting step of FliC export in the absence of FliS, we isolated bypass mutants from a Salmonella ΔfliS mutant. All the bypass mutations were identified in FliC. These bypass mutations increased the export rate of FliC by ca. twofold, allowing the bypass mutant cells to produce longer filaments than the parental ΔfliS cells. Both far-UV CD measurements and limited proteolysis revealed that the bypass mutations significantly destabilize the folded structure of FliC monomer. These results suggest that an unfolding step of FliC limits the export rate of FliC in the ΔfliS mutant, thereby producing short filaments. We propose that FliS promotes FliC docking at the FlhA platform to facilitate subsequent unfolding of FliC.

1.8 Å resolution structure of ß-galactosidase with a 200†kV CRYO ARM electron microscope.

We report the determination of the structure of Escherichia coli ß-galactosidase at a resolution of ∼1.8 Šusing data collected on a 200 kV CRYO ARM microscope equipped with a K3 direct electron detector. The data were collected in a single 24 h session by recording images from an array of 7 × 7 holes at each stage position using the automated data collection program SerialEM. In addition to the expected features such as holes in the densities of aromatic residues, the map also shows density bumps corresponding to the locations of hydrogen atoms. The hydrogen densities are useful in assigning absolute orientations for residues such as glutamine or asparagine by removing the uncertainty in the fitting of the amide groups, and are likely to be especially relevant in the context of structure-guided drug design. These findings validate the use of electron microscopes operating at 200 kV for imaging protein complexes at atomic resolution using cryo-EM.

Understanding hydrogen-bonding structures of molecular crystals via electron and NMR nanocrystallography.

Understanding hydrogen-bonding networks in nanocrystals and microcrystals that are too small for X-ray diffractometry is a challenge. Although electron diffraction (ED) or electron 3D crystallography are applicable to determining the structures of such nanocrystals owing to their strong scattering power, these techniques still lead to ambiguities in the hydrogen atom positions and misassignments of atoms with similar atomic numbers such as carbon, nitrogen, and oxygen. Here, we propose a technique combining ED, solid-state NMR (SSNMR), and first-principles quantum calculations to overcome these limitations. The rotational ED method is first used to determine the positions of the non-hydrogen atoms, and SSNMR is then applied to ascertain the hydrogen atom positions and assign the carbon, nitrogen, and oxygen atoms via the NMR signals for 1H, 13C, 14N, and 15N with the aid of quantum computations. This approach elucidates the hydrogen-bonding networks in L-histidine and cimetidine form B whose structure was previously unknown.

Crystallization and preliminary X-ray analysis of the periplasmic domain of FliP, an integral membrane component of the bacterial flagellar type III protein-export apparatus.

The bacterial flagellar proteins are transported via a specific export apparatus to the distal end of the growing structure for their self-assembly. FliP is an essential membrane component of the export apparatus. FliP has an N-terminal signal peptide and is predicted to have four transmembrane (TM) helices and a periplasmic domain (FliPP) between TM-2 and TM-3. In this study, FliPP from Thermotoga maritima (TmFliPP) and its selenomethionine derivative (SeMet-TmFliPP) were purified and crystallized. TmFliPP formed a homotetramer in solution. Crystals of TmFliPP and SeMet-TmFliPP were obtained by the hanging-drop vapour-diffusion technique with 2-methyl-2,4-pentanediol as a precipitant. These two crystals grew in the hexagonal space group P6222 or P6422, with unit-cell parameters a = b = 114.9, c = 193.8 Å. X-ray diffraction data were collected from crystals of TmFliPP and SeMet-TmFliPP to 2.4 and 2.8 Šresolution, respectively.