A Disordered Region in the EvpP Protein from the Type VI Secretion System of <i>Edwardsiella tarda</i> is Essential for EvpC Binding

<div><p>The type VI secretion system (T6SS) of pathogenic bacteria plays important roles in both virulence and inter-bacterial competitions. The effectors of T6SS are presumed to be transported either by attaching to the tip protein or by interacting with HcpI (haemolysin corregulated protein 1). In <i>Edwardsiella tarda</i> PPD130/91, the T6SS secreted protein EvpP (<i><u>E</u>. tarda</i><u>v</u>irulent <u>p</u>rotein P) is found to be essential for virulence and directly interacts with EvpC (Hcp-like), suggesting that it could be a potential effector. Using limited protease digestion, nuclear magnetic resonance heteronuclear Nuclear Overhauser Effects, and hydrogen-deuterium exchange mass spectrometry, we confirmed that the dimeric EvpP (40 kDa) contains a substantial proportion (40%) of disordered regions but still maintains an ordered and folded core domain. We show that an N-terminal, 10-kDa, protease-resistant fragment in EvpP connects to a shorter, 4-kDa protease-resistant fragment through a highly flexible region, which is followed by another disordered region at the C-terminus. Within this C-terminal disordered region, residues Pro143 to Ile168 are essential for its interaction with EvpC. Unlike the highly unfolded T3SS effector, which has a lower molecular weight and is maintained in an unfolded conformation with a dedicated chaperone, the T6SS effector seems to be relatively larger, folded but partially disordered and uses HcpI as a chaperone.</p></div

EvpP is a dimeric protein in solution.

<p>(A) The recombinant wild type EvpP purified as a dimeric protein with an elution volume of 72 ml from the Superdex 75 gel filtration column (HiLoad 16/60, GE Healthcare). (B) Dynamic light scattering data showing that the hydrodynamic radius of EvpP ranged from 2.92 nm to 3.09 nm, with a mean value of 3.01 nm. The estimated molecular weight ranged from 41.2 kDa to 47.3 kDa, with mean value of 44.4 kDa (data not shown).</p

GST-EvpC pull-down assay for truncation mutants of EvpP.

<p>SDS-PAGE showing pull-down results using GST-EvpC and truncation mutants of EvpP. Lane 1: M.W. marker; lane2: GST-EvpC+EvpP; lane 3: GST+EvpP; lane 4: EvpP; lane 5: GST-EvpC+EvpP_1–168; lane 6: GST+EvpP_1–168; lane 7: EvpP_1–168; lane 8: GST-EvpC+EvpP_1–142; lane 9: GST+EvpP_1–142; and lane 10: EvpP_1–142. The region from residues Pro143 to Ile168 is essential for the interaction between EvpC and EvpP.</p

Heteronuclear NOE experiment on P143T EvpP.

<p>(A) Overlay spectra from heteronuclear NOE experiments showing cross-peaks from an experiment without proton saturation (red) and an experiment with proton saturation (black). The label next to each peak corresponds to sequential assignment of the residue. (B) A plot showing the relative ratio of peak intensities with proton saturation against those without proton saturation for all residues that can be assigned. Residue with a low value for the ratio is located at a disordered region. Boundaries of the three identified ordered regions are boxed by dashed lines and labeled Box 1, 2 and 3.</p

Limited trypsin digestion of EvpP and EvpP P143T.

<p>(A) Limited trypsin digestion of both wild type and P143T EvpP resulted in two protease-resistant fragments of 10 kDa and 4 kDa consistently within a digestion period of 20–60 min. (B) Gel filtration elution profile showed that the 10-kDa and 4-kDa fragments remain associated with each other after protease digestion.</p

¹H-¹⁵N HSQC spectra of wild type, P143T and trypsin digested EvpP.

<p>(A) Overlay of the ¹H-¹⁵N HSQC spectra of wild type EvpP (black) and P143T EvpP (red). (B) ¹H-¹⁵N HSQC spectrum of trypsin digested EvpP. (C) Sequence of P143T EvpP from <i>E. tarda</i> PPD130/91, with the boundaries of the 10-kDa (residues Met1 to Arg85) and 4-kDa (residues Ile106 to Gly141) protease-resistant fragments boxed. Residues that can be assigned by NMR experiments are shaded in grey. Residues that are predicted to be disordered by the software RONN <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110810#pone.0110810-Yang1" target="_blank">[33]</a> and PrDOS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110810#pone.0110810-Ishida1" target="_blank">[34]</a> are marked by black and red asterisks, respectively, above the sequence.</p

Residues at the hydrophobic interface between AscE and AscG_1–61.

<p>Ribbon representation of the AscE-AscG_1–61 crystal structure showing side chains of residues involved in the hydrophobic interface between AscE (blue) and AscG_1–61 (red), at two different views.</p

Thermal denaturation of AscE-AscG, AscE-AscG_1–61, AscE-AscG-AscF and AscE-AscG-AscF_53–87 complexes monitored by Far-UV CD.

<p>Thermal denaturation of the AscE-AscG_1–61 (closed circle), AscE-AscG full length (open circle), AscE-AscG-AscF_53–87 (closed square) and AscE-AscG-AscF full length (open square) complexes monitored by Far-UV CD at 220 nm from 10°C to 85°C.</p

Superposition of the structure AscE-AscG_1–61 with PscE-PscF-PscG and YscE-YscF-YscG.

<p>The crystal structure of AscE-AscG_1–61 was superpositioned with (A) PscE-PscF-PscG or (B) YscE-YscF-YscG at two different views. The AscE and AscG_1–61 proteins are colored blue and red, respectively. The E, F and G proteins in the PscE-PscF-PscG or YscE-YscF-YscG complexes are colored magenta, green and cyan, respectively.</p

Surface diagram of the AscE-AscG_1–61 complex and predicted AscF interacting residues.

<p>Surface diagram of the AscE-AscG_1–61 complex showing the tight interaction between AscE (blue) and AscG_1–61 (white), at two different views. The residues on AscG_1–61 predicted to interact with AscF based on the crystal structures of PscE-PscF-PscG and YscE-YscF-YscG are colored red. The surface diagrams were generated using the software Chimera <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019208#pone.0019208-Pettersen1" target="_blank">[44]</a>.</p