Identification and Molecular Characterization of YsaL (Ye3555): A Novel Negative Regulator of YsaN ATPase in Type Three Secretion System of Enteropathogenic Bacteria Yersinia enterocolitica

Type Three Secretion (T3S) ATPases are involved in delivery of virulent factors from bacteria to their hosts (through injectisome) in an energy (ATP) dependent manner during pathogenesis. The activities of these ATPases are tightly controlled by their specific regulators. In Yersinia enterocolitica, YsaN was predicted as a putative ATPase of the Ysa-Ysp Type Three Secretion System (T3SS) based on sequence similarity with other T3S ATPases. However detailed study and characterization of YsaN and its regulation remains largely obscure. Here, in this study, we have successfully cloned, overexpressed,purified and characterized the molecular properties of YsaN from Yersinia enterocolitica. YsaN acts as a Mg2+ dependent ATPase and exists in solution as higher order oligomer (dodecamer). The ATPase activity of oligomeric YsaN is several fold higher than the monomeric form. Furthermore, by employing in silico studies we have identified the existence of a negative regulator of YsaN- a hypothetical protein YE3555 (termed ‘YsaL’). To verify the functionality of YsaL, we have evaluated the biochemical and biophysical properties of YsaL. Purified YsaL is dimeric in solution and strongly associates with YsaN to form a stable heterotrimeric YsaL-YsaN complex (stoichiometry- 2:1). The N terminal 6–20 residues of YsaN are invariably required for stable YsaL-YsaN complex formation. YsaL inhibited the ATPase activity of YsaN with a maximum inhibition at the molar ratio 2:1 (YsaL: YsaN). In short, our studies provide an insight into the presence of YsaN ATPase in Yersinia enterocolitica and its regulator YsaL. Our studies also correlate the functionality of one of the existing protein interaction networks that possibly is indispensable for the energy dependent process of Ysa-Ysp T3SS in pathogenic Yersinia enterocolitic

Schematic representation of functionality of YsaN and its regulation by YsaL.

<p>YsaN exists in solution as mixture of monomer and higher order oligomer (dodecamer) and act as Mg ²⁺ dependent ATPase. The oligomeric form of YsaN is highly active compared to monomeric form. Computational studies predicted YsaL as a putative ATPase regulator. YsaL exists as dimer in solution and form stable heterotrimeric complex with monomeric YsaN. This results in significantly loss of ATPase activity of YsaN, probably due to loss of oligomeric state. The complex is unstable at high salt concentration (>500 mM NaCl). Furthermore, N-terminal (1–20) residues of YsaN are involved in YsaL-YsaN interaction, as revealed from interaction studies of deletion mutants.</p

Binding propensity of YsaL with YsaN wild type and different deletion mutants using Surface Plasmon resonance.

<p>Interactions of untagged (UT) YsaL with (<b>A</b>) YsaN-His (<b>B</b>) YsaN_flr-His (<b>C</b>) YsaN Δ_(1–5) -His (<b>D</b>) YsaN Δ_(411–430) -His and (<b>E</b>) YsaN Δ _(426–430)-His. Untagged YsaL was used as an analyte in the concentration 5 nM, 10 nM, 20 nM and 50 nM.</p

Purification, stoichiometric characterization and secondary structure analysis of YsaL/Ye3555.

<p>(<b>A</b>) SDS PAGE analysis of refolded YsaL-His after Ni-NTA affinity chromatography: M-Marker, UI- Uninduced, I- induced, E1 and E2- Ni-NTA eluates. (<b>B</b>) Chemical crosslinking profile of refolded YsaL-His in SDS-PAGE using 1.5% Gluteraldehyde - M- Marker Ctrl- Control, C- crosslinking of YsaL-His using 1.5% Gluteraldehyde incubated for 5 min. (<b>C</b>) Size exclusion chromatography (SEC) profile of refolded YsaL-His in pre-calibrated Superdex 200 hi-load16/60 column (GE Healthcare). Molecular weight standards are indicated with triangles: Thyroglobulin (T) –669 kDa, Ferritin (F) –440 kDa, Aldolase (A) –158.4 kDa, Ovalbumin (O) –48 kDa, Carbonic anhydrase (C) –29 kDa and Ribonuclease A (R) –14 kDa. SDS PAGE analysis of Gel filtration profile: M – marker, P1 (peak1) - YsaL-His <b>(inset)</b>. (<b>D</b>) Molecular mass estimation from size exclusion profile of refolded YsaL-His using the above known standards. Refolded YsaL-His was dimeric (∼48 kDa). (<b>E</b>) Far UV-CD (Circular dichroism) spectra of refolded YsaL-His (HT) and refolded untagged (UT) YsaL.</p

Identification of critical residues of YsaN for stable YsaL-YsaN complex formation.

<p>(<b>A</b>) Schematic diagram of deletion mutant constructs of YsaN. (<b>B</b>) Far-UV CD spectra of his tagged deletion mutants of YsaN [YsaN Δ_(1–5), YsaN Δ_(1–20), YsaN Δ_(426–430), YsaN Δ_(411–430) and YsaN_(21–410)] compared with full length YsaN (YsaN-His) and refolded YsaN full length (YsaN_flr-His). (<b>C</b>) Relative ATPase activity (%) of deletion mutants of YsaN with YsaN_flr-His as a control. (<b>D</b>) Relative ATPase activity (%) of YsaN-His with YsaN_flr-His. (<b>E</b>) SDS-PAGE profile of Ni-NTA pull-down assay of deletion mutants of YsaN (His tagged) with untagged YsaL; YsaN _Δ(1–20) and YsaN_(21–410) does not bind to untagged (UT) YsaL. M-denotes molecular weight marker.</p

Pfam analysis of BLASTp hits with YscL as a query.

*<p>Belonged to same clan HrpE/YscL/FliH and V-type ATPase subunit E (CL 0255).</p

Strains, plasmid and constructs used in the work.

*<p>ATCC-American Type Culture Collection.</p

Oligomerization analysis and enzymatic characterization of YsaN ATPase.

<p>(<b>A</b>) SDS PAGE analysis of YsaN-His after purification by Ni-NTA affinity chromatography: M- Marker, UI- Uninduced, I- induced, E1 and E2- Ni-NTA eluates. (<b>B</b>) Chemical crosslinking profile of YsaN-His in SDS-PAGE using Sulfo - EGS (EGSS) - Ctrl-Control, C1 and C2- crosslinking of YsaN-His using 0.5 mM EGSS incubated for 2 min and 5 min respectively, M-Marker. (<b>C</b>) Size exclusion chromatography (SEC) profile of YsaN-His in pre-calibrated Superdex 200 hi-load16/60 column (GE Healthcare). Molecular weight standards are indicated with triangles: Thyroglobulin (T) –669 kDa, Ferritin (F) –440 kDa, Aldolase (A) –158.4 kDa, Ovalbumin (O) –48 kDa, Carbonic anhydrase (C) –29 kDa and Ribonuclease A (R) –14 kDa. SDS PAGE analysis of Gel filtration profile: P1 (Peak1) - YsaN-His Dodecamer and P2 (Peak2) - YsaN-His monomer, M - marker <b>(inset)</b>. (<b>D</b>) Molecular mass estimation from size exclusion profile of YsaN-His using the above known standards. Peak1 from SEC profile corresponds to ∼603 kDa (Dodecamer) and peak 2 corresponds to ∼49.5 kDa (Monomer). (<b>E</b>) Measurement of hydrodynamic diameter of YsaN-His using Dynamic Light Scattering. Hydrodynamic radii (R_H) of YsaN corresponded to monomer and dodecamer. (<b>F</b>) ATPase activity of Ni-NTA eluates of YsaN-His measured in terms of Phosphate (Pi) released per minute per mg of protein. Hill fit equation provided the kinetic parameters - V_max, K_0.5 and hill coefficient (n). (<b>G</b>) Relative ATPase activity of YsaN Dodecamer and YsaN Monomer separated after SEC. YsaN mixture (monomer and oligomer) was used as a control.</p

Primers used in the work.

<p>Letters in bold indicate restriction sites for NdeI (sense primer) and XhoI (Antisense primer).</p

Co-Purification, stoichiometry and functional analysis of YsaL-YsaN complex.

<p>(<b>A</b>) SDS-PAGE analysis of YsaL-YsaN complex after co-purification in Ni-NTA affinity chromatography: M-Marker, UI- Uninduced, I- induced, E1 and E2- Ni-NTA eluates of untagged YsaL-YsaN-His complex. (<b>B</b>) Size exclusion chromatography (SEC) profile of untagged YsaL-YsaN-His complex in pre-calibrated Superdex 200 hi-load16/60 column (GE Healthcare). Molecular weight standards are indicated with triangles: Thyroglobulin (T) –669 kDa, Ferritin (F) –440 kDa, Aldolase (A) –158.4 kDa, Ovalbumin (O) –48 kDa, Carbonic anhydrase (C) –29 kDa and Ribonuclease A (R) –14 kDa. SDS PAGE analysis of Gel filtration profile: M – marker, P1 (Peak1) - YsaN-His dodecamer P2 (Peak2) - untagged (UT) YsaL-YsaN-His heterotrimer and P3 (Peak3) - YsaN-His monomer <b>(inset)</b>. (<b>C</b>) Molecular mass estimation from size exclusion profile of untagged YsaL-YsaN-His complex using the known molecular weight standards. Peak1 corresponds to YsaN-His (Dodecamer) - ∼603 kDa, Peak2 corresponds to heterotrimeric assembly of untagged YsaL-YsaN-His complex (2∶1) - ∼102 kDa and Peak3 corresponds to YsaN-His (monomer) - ∼49.5 kDa. (<b>D</b>) Chemical crosslinking profile of untagged (UT) YsaL-YsaN-His complex in SDS-PAGE using Sulfo - EGS (EGSS) - Ctrl-Control, C1 and C2-crosslinking of untagged YsaL-YsaN-His using 1.0 mM EGSS incubated for 5 min and 10 min respectively, M-Marker. (<b>E</b>) Relative ATPase activity (%) with increasing molar ratio of refolded YsaL-His/Ye3555 and YsaN-His. Maximum inhibition of ATPase activity occurs in the ratio 2∶1 shown by arrow. Relative ATPase activity of untagged YsaL-YsaN-His complex (NL_C) and YsaN-His incubated with YsaL-His refolded (N +L_HR) in the molar ratio 2∶1. YsaN-His (N) was used as a control <b>(inset)</b>.</p