Stability data of FlgD from Helicobacter pylori and structural comparison with other homologs

Flagellin component D (FlgD) from Helicobacter pylori is involved in the assembly of the hook of flagella, helical tubular structures that provide motility in non-filamentous bacteria. Data provided in this article refer to HpFlgD from strains 26695 (HpFlgD_26695) and G27 (HpFlgD_G27). Within this article, information on the secondary structure content and different type of interfaces found in the two crystal forms of HpFlgD (monoclinic, HpFlgD_m and tetragonal, HpFlgD_t) are provided, as well as the list of the hydrogen bonds between monomers that are relevant for their assembly into a tetramer. Additionally, data involving investigation of the size of HpFlgD in the solution and the crystallized HpFlgD are presented, “Crystal structure of truncated FlgD from the human pathogen Helicobacter pylori” [1]. The superposition of the different domains of HpFlgD (Fn-III and tudor domains) with the similar domains found in other species is shown, as well as the superposition of HpFlgD and modeled HpFlgE (flagellar hook protein).

found in other species is shown, as well as the superposition of HpFlgD and modeled HpFlgE (flagellar hook protein).
& 2016 Elsevier Inc.. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Subject area
Chemistry More specific subject area

Protein crystallography and biophysics
Type of data Table, text file, graph, figure How data was acquired Mass spectroscopy (quadrupole-TOF spectrometer, RP-HPLC), X-ray diffraction (Swiss Light Source, SLS) Data format Raw, analyzed Experimental factors Crystals of native HpFlgD_26695 were dissolved in the appropriate buffer, as well as a sample of HpFlgD_26695 protein solution, and were run on a SDS-PAGE. The isolated bands were in gel digested with trypsin and the extracted peptides were further analyzed with nano-electrospray ionization mass spectrometry (nano-ESI MS).

Experimental features
The full length HpFlgD_G27 monomer mass was determined by reverse phase chromatography (RP-HPLC). Mass measurements were performed with a quadrupole-TOF spectrometer and the obtained spectra was further analyzed using the MASSLYNX software.

Data source location
Padua, Italy andfor mass spectroscopy dataVilligen, Switzerland, SLS À for crystallography data Data accessibility Data is with this article.

Value of the data
Providing the data on the protein stability can benefit other researchers willing to follow the same techniques.
Interpretation of differences and similarities in the structural organization of FlgD homologs can be useful for future investigations on the role of FlgD in flagellar biogenesis.
Previously unreported data on the secondary structure composition of the full length FlgD.

Data
This article presents data on the HpFlgD stability in terms of the protein size. This investigation was done in order to understand which part of the degraded protein crystallized. The data is based on the CD and mass spectra analysis (RP-HPLC, nano-ESI). In addition, comparison of different types of interfaces found in the crystal structures of the two crystal forms of HpFlgD [1] are given, as well as the amino acid residues responsible for the quaternary structure assembly. The difference between the domain orientation in HpFlgD and the similar domains in other organisms is also shown.

Experimental design, materials and methods
Secondary structure analysis of diluted HpFlgD (2 mg mL À 1 ) was performed by circular dichroism (CD) using a spectropolarimeter (Jasco Analytical Instruments) in the far UV region (190-260 nm), Fig. 1. Afterwards, the data were deconvoluted using software CDNN [2] and are shown as contributions of the various components to the protein secondary structure (Table 1).       The level of degradation of HpFlgD_26695 and crystallized HpFlgD_26695 was monitored by the SDS-PAGE. The sample from the crystal of the tetragonal form of HpFlgD_26695 (Fig. 2b and c) was prepared by dissolving the crystal in the SDS-PAGE loading buffer. This sample together with a full length HpFlgD_26695 was checked by SDS-PAGE (Fig. 3a). The bands obtained from the crystallized sample and full length HpFlgD_26695 were isolated and in gel digested with trypsin. The fractions of the extracted peptides were dried out, dissolved in 50% acetonitrile, supplemented with 0.1% formic acid and directly injected in the nano-ESI source. Mass measurements were performed with a quadrupole-TOF spectrometer (Waters, Manchester, UK) (capillary voltage: 2800-3000 V; cone voltage: 45 V; scan time: 1 s; interscan: 0.1 s). Analysis of the spectra was performed by using the MASSLYNX software (Micromass, Wynthenshow, UK). The data obtained from the mass analysis are presented in Fig. 4.
The mass of the HpFlgD_G27 monomer was determined by mass analysis of the peaks isolated by reverse phase chromatography (C4-column, RP-HPLC), Fig. 5. Table 2 Different types of interfaces between the molecules in the crystal structure of HpFlgD. N HB and N SB refer to the number of hydrogen bonds and salt bridges, respectively. The interfaces labeled a À f with a* are shown in Fig. 6, while the interface labeled as t* refers to the interface responsible for the tetramerization (as shown in Figs. 4b and 6 [1]).

Crystal system
Interface type Table 3 Hydrogen bonds (Å) between monomers that are relevant for their assembly into a tetramer.

Monomer1
Monomer2 Presence of the His tag at the C-terminus of the full length HpFlgD_26695 and crystallized HpFlgD_26695 was evaluated with anti-His antibodies (Mouse monoclonal, 1:1000 dilution) and secondary antibodies (Goat anti-mouse HRP, 1:10,000) (Western blotting technique), Fig. 3b. Fig. 6 shows different types of interfaces present in both crystal forms of HpFlgD. In Table 2 the interface area, the number of hydrogen bonds and salt bridges involved in each interface are shown. The list of hydrogen bonds responsible for the tetramerization is presented in Table 3.
Superposition of the Fn-III domain in HpFlgD with the fibronectin domain in 1FNA [3] is presented in Fig. 7, while the superposition of the tudor domain in HpFlgD and the same domain in PaFlgD (PDB ID: 3OSV, [4]) and XcFlgD (PDB ID: 3C12, [5]) can be seen in Fig. 8. Fig. 9 presents the overlayed structures of HpFlgD and modeled HpFlgE. Modeled HpFlgE was prepared by homology using software Phyre 2 [6].

Acknowledgments
This work was supported by the University of Padua, by PRIN 2010-2011 (MIUR) "Unraveling structural and functional determinants behind Helicobacter pylori pathogenesis and persistence".