The structural overlap between <i>Vp</i>YeaZ (green), <i>Tm</i>YeaZ (purple), <i>Ec</i>YeaZ (red) and <i>St</i>YeaZ (black).

<p>The side chains of the conserved and semi-conserved residues likely to be implicated in nucleotide binding are shown for <i>Vp</i>YeaZ.</p

A: Comparison of form 1 and form 2 dimers observed in the crystal structures of <i>Vp</i>YeaZ, <i>St</i>YeaZ, <i>Ec</i>YeaZ and <i>Tm</i>YeaZ.

<p>B: Location of residues L40, L47, I74, I78, L82 (shown in red) at the interface of <i>Vp</i>YeaZ dimer (left) and positions of the equivalent residues in <i>Tm</i>YeaZ form 2 dimer (right). The residue positions are shown for one half of the dimer, with the other half represented by its molecular surface. C: A stereo diagram showing modeled positions of a nucleotide molecule in the interdomain cleft of the form 1 (left) and the form 2 dimers. The nucleotide (ATP) molecule has been positioned based on structural similarity between domains I in YeaZ and Kae1. The modelled nucleotide is shown in ball representation. In the model of the form 2 dimer complex one of the ATP molecules is shown in light grey for clarity of illustration.</p

A sequence alignment of YeaZ homologues in <i>V. parahaemolyticus</i> (V. par), <i>S. typhimurium</i> (S. typ), <i>E. coli</i> (E. col), <i>T. maritima</i> (T. mar), <i>Pasteurella multocida</i> (P. mul), <i>Xylella fastidiosa</i> (X. fas), <i>Sinorhizobi

<p>The elements of the secondary structure and the sequence numbering for <i>Vp</i>YeaZ are shown above the alignment. The fully conserved residues are shown by a black box with reverse type. The position of the conserved and semi-conserved residues which can be identified in the nucleotide-binding site of proteins belonging to the ASKHA family, are highlighted by black and gray boxes respectively, and marked with “n” underneath the aligned sequences. The residues forming a hydrophobic surface patch which becomes exposed upon dimer re-organisation are enclosed by a box and marked with “<i>h</i>” underneath the alignment. Alignment was carried out using the ClustalW server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023245#pone.0023245-Thompson1" target="_blank">[16]</a>. The figure was created using ESPript <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023245#pone.0023245-Gouet1" target="_blank">[17]</a>.</p

A: Stereo diagram of the structure of the <i>Vp</i>YeaZ monomer.

<p>Each element of the secondary structure is labeled. Domains I and II and the putative nucleotide-binding cleft are identified. The figure was prepared using PyMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023245#pone.0023245-Delano1" target="_blank">[11]</a>. B: The topology of the secondary structure elements. Residue numbers are indicated at the start and end of each secondary structure element. C: SDS-PAGE showing the time-course of VpYeaZ digestion by Glu-C protease. The positions of molecular mass markers are shown to the left. The arrow indicates a relatively stable C-terminally truncated fragment. D: Amino acid sequence of VpYeaZ with Glu-C-sensitive site identified in this study shown by the arrow.</p

CD spectra of the recombinant CagA fragments.

<p>Ellipticity in the far-UV range (200–260 nm) is plotted for (a) CagA-N at 0.075 mg/ml, (b) CagA-M at 0.05 mg/ml, (c) CagA-M_c at 0.1 mg/ml and (d) Cag-R at 0.15 mg/ml.</p

Thermal unfolding and refolding transitions of CagA domains monitored by far-UV CD.

<p>The unfolding data is shown with black dots for CagA-N (a), CagA-M (b), CagA-M_c (c) and CagA-R (d). Unfolding was reversible for CagA-M, CagA-M_c and CagA-R; the refolding data is shown with open circles. The insets show the corresponding CD spectra for CagA-M (b), CagA-M_c (c) and CagA-R (d) for the native (solid line), unfolded (85 °C, dashed line) and refolded (dotted line) states. The low signal-to-noise ratio for the CagA-<i>R </i><i>spectrum</i> reflects the fact that the scan for this fragment was performed at lower wavelengths (205 nm rather than 222 nm), where the absorbance is inherently higher and thus the data is collected at a higher dynode voltage.</p

Crystal structure of the CagA region comprising the N-terminal (Domain I) and the middle (Domain II + Domain III) domains [19].

<p>Crystal structure of the CagA region comprising the N-terminal (Domain I) and the middle (Domain II + Domain III) domains [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079367#B19" target="_blank">19</a>].</p

SEC and molecular weight (MW) and hydrodynamic radius determination of CagA-N (a), CagA-M_c (b) and CagA-R (c).

<p>Green dots superimposed on the peak indicate the MW as shown on the left-hand y-axis. Red dots represent the hydrodynamic radius calculated over the central portion of the elution peak (shown by UV trace in blue). The hydrodynamic radius values are shown on the right-hand y-axis.</p

SDS-PAGE showing the time-course of digestion of CagA-M by trypsin.

<p>The arrow indicates a relatively stable core fragment CagA-M_c.</p

Comparative schematic representation of the bacterial flagellar export apparatus and F_O/F₁ ATPase.

<p>The bacterial cytoplasmic membrane (<i>CM</i>) is shown in dark gray blocks. <i>Peri</i> stands for periplasmic face and <i>Cyto</i> for cytoplasmic face. The names components that are functionally and/or structurally related between the two complexes (as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052179#s4" target="_blank">Discussion</a>) are outlined in black and share similar colours and/or shapes.</p