Despite this, we were able to model the initial helical structural element, corresponding to residues 274C308, of this region. core regions of the FliD molecule, and that disordered/flexible areas form the five lower leg constructions (Vonderviszt et al., 1998)?that?are known to interact with the FliC filament. FliD exhibits low sequence similarity to the flagellar hook proteins and to?FliC. However, it shares the disordered terminal areas?of these flagellar proteins,?a?common structural characteristic that is thought to control the?polymerization of flagellar proteins and to play an important role in connection with the FliC filament (Vonderviszt et al., 1998). These areas are the most conserved in FliD sequences across bacteria. Flagellum-mediated motility is vital for the virulence and pathogenicity of numerous bacteria, including (Black et al., 1988), (Allen-Vercoe and Woodward, 1999; Marchetti et al., 2004), (La Ragione et al., 2000), (Krukonis and DiRita, 2003), and?(Arora et al., 2005), as well as the major causative agent of gastric malignancy (Kim et al., 1999). To date, however, no high-resolution structure of any FliD protein exists. To better determine PHA-665752 the tasks of FliD in bacterial motility and pathogenesis, we determined the first X-ray crystal structure of FliD at 2.2?? resolution, and assessed the structural contributions of its flexible areas using a multitude of complementary biophysical and practical analyses. Results Crystal structure of the FliD protein from PAO1 To facilitate crystallization of FliD PHA-665752 from your PAO1 strain, we erased the expected coiled-coil domains on both the N- and C-termini of full size FliD, which has?474?residues (FliD1C474), to generate the truncated FliD78-405 (Number 1a, Number 1source data 1). We indicated FliD78C405 in with an N-terminal His6-tag and purified it to homogeneity by Ni2+-NTA, size exclusion and anion exchange chromatography. We improved in the beginning weakly diffracting crystals of FliD78C405 by random matrix microseed screening (Bergfors, 2003), yielding crystals that diffracted to 2.2?? resolution. In the absence of any homologous protein that may be used like a model for PHA-665752 molecular alternative, we crystallized a seleno-methionine derivative of FliD78C405 that included four leucine-to-methionine mutations (FliD78C405/L4CM4).?This crystal provided phase information sufficient to create an initial model,?which?we used subsequently for molecular replacement with the native FliD78C405 dataset (Number 1source data 1). We modeled residues 80C273 into obvious electron denseness, including all part chains, but observed denseness of progressively poor quality in?the?C-terminus beyond residue 273 (Figure 1figure product 2a). Therefore, we were able to model with confidence only a single helix in this region, related to residues 274C308, with incomplete side chain constructions. To determine whether the remaining region of the protein actually existed in the crystals and not just in the protein preparation used for crystallization, we analyzed crystals using liquid chromatography-mass spectrometry (LC-MS) and SDS-PAGE. Both analyses indicated the crystals consisted of an approximate 50:50 mixture of the FliD78C405 protein used for crystallization and a further proteolyzed Rabbit polyclonal to SP1.SP1 is a transcription factor of the Sp1 C2H2-type zinc-finger protein family.Phosphorylated and activated by MAPK. version having a molecular excess weight of about 27?kDa. The N-terminal His6-tag is still detectable by Western blot (Number 1figure product 2b). Thus, the proteolyzed form corresponds approximately to residues 78C319 of FliD. The 86 residues absent from your C-terminus inside a human population of FliD proteins are clearly not required for crystal packing, suggesting that they are highly flexible? actually inside a crystalline environment. Open in a separate window Number 1. Crystal structure of FliD reveals structural similarity to additional flagellar proteins.(a) Schematic representation of the FliD proteins used in these studies. Protein website/region boundaries are labeled and are drawn approximately to level. (b) Crystal structure of the FliD78C405 monomer subunit with spectrum coloring from your N-terminus (blue) to the C-terminus (reddish). Head website 1, head website 2 and the lower leg region are indicated. (c) Superposition of the FliD78C405 crystal structure (domain coloring as with panel (a)) and FlgK/HAP1/hook filament capping protein (cyan). (d) Superposition of the FliD78C405 crystal structure (domain coloring as with panel (a)) and flagellin/FliC (magenta). DOI: http://dx.doi.org/10.7554/eLife.18857.003 Figure 1source data 1.Crystallographic data collection, phasing and refinement statistics.DOI: http://dx.doi.org/10.7554/eLife.18857.004 Click here to view.(13K, xlsx) Number 1figure product 1. Open in a separate window Protein sequence of FliD1C474.The protein sequence of FliD from PAO1 is shown. The tertiary website structure based on the offered X-ray crystal structure and the?expected?secondary structure is indicated. DOI: http://dx.doi.org/10.7554/eLife.18857.005 Figure 1figure supplement 2. Open in a separate windowpane Electron denseness and protein degradation of FliD crystals.(a) The overall.