Thermodynamic parameters of Tipα with different lengths of ssDNA.

<p>Thermodynamic parameters of Tipα with different lengths of ssDNA.</p

Thermodynamic parameters for Tipα and its mutants binding to dsDNA.

<p>Thermodynamic parameters for Tipα and its mutants binding to dsDNA.</p

Crystal Structure of TNF-α-Inducing Protein from <em>Helicobacter Pylori</em> in Active Form Reveals the Intrinsic Molecular Flexibility for Unique DNA-Binding

<div><p>Tipα (TNF-α-inducing protein) from <em>Helicobacter pylori</em> is a carcinogenic effector. Studies on this protein revealed that a homodimer linked by a pair of intermolecular disulfide bridges (Cys25-Cys25 and Cys27-Cys27) was absolutely necessary for its biological functions. The activities of Tipα would be abolished when both disulfide bridges were disrupted. The crystal structures of Tipα reported to date, however, were based on inactive, monomeric mutants with their N-terminal, including residues Cys25 and Cys27, truncated. Here we report the crystal structure of <em>H. pylori</em> Tipα protein, TipαN²⁵, at 2.2<b> </b>Å resolution, in which Cys25 and Cys27 form a pair of inter-chain disulfide bridges linking an active dimer. The disulfide bridges exhibit structural flexibility in the present structure. A series of structure-based mutagenesis, biochemical assays and molecular dynamic simulations on DNA-Tipα interactions reveal that Tipα utilizes the dimeric interface as the DNA-binding site and that residues His60, Arg77 and Arg81 located at the interface are crucial for DNA binding. Tipα could bind to one ssDNA, two ssDNA or one dsDNA in experiments, respectively, in the native or mutant states. The unique DNA-binding activities of Tipα indicate that the intrinsic flexible nature of disulfide bridges could endow certain elasticity to the Tipα dimer for its unique bioactivities. The results shed light on the possible structural mechanism for the functional performances of Tipα.</p> </div

Structural comparison of TipαN²⁵, TipαN²⁸ and TipαN³⁴.

<p>(A), (B) Dimeric organizations. TipαN²⁵ takes a shoulder-to-shoulder assembly mode (A), while two TipαN²⁸ and TipαN³⁴-II from alkaline conditions adopt a different head-to-head dimeric mode (B). (C)- (E) Structural comparison of TipαN²⁵ and TipαN³⁴-I from acidic conditions. TipαN³⁴-I adopts a similar dimeric organization mode with that of TipαN²⁵ (C), however the dimeric β-sheets are oriented away from the dimeric interface by 10° (D) and some residue contacts involved in dimer interactions are instabilized (E) in TipαN³⁴-I. TipαN²⁵ (3VNC), TipαN²⁸ (3GIO), TipαN²⁸ (3GUQ), TipαN³⁴-I (2WCQ), and TipαN³⁴-II (2WCR) are coloured in red, blue, yellow, cyan and deepsalmon, respectively.</p

Dimeric interactions of TipαN²⁵.

<p>Two subunits of Tipα (Chain A and Chain B) are shown in purple and yellow, respectively. (A) Stereo view of hydrophobic interactions of Tipα. (B) Hydrogen bond network of β-sheet-mediated interface. (C) Top view of the potential DNA-binding site at the dimer interface.</p

SsDNA binding activities of Tipα and H60A in SPR assay.

<p>SsDNA binding activities of Tipα and H60A in SPR assay.</p

Chromatographic behaviors of TipαN²⁵ at different conditions.

<p>(A) Gel filtration analysis of TipαN²⁵ and TipαN²⁵ monomer (TipαN²⁵ treated with 50<b> </b>mM DTT) at pH 4 and pH 7. (B) SDS-PAGE gel showing TipαN²⁵ under different concentrations of DTT at pH 4.0. Lane 1, native TipαN²⁵; Lane 2, TipαN²⁵ with 10<b> </b>mM DTT; Lane 3, TipαN²⁵ with 50<b> </b>mM DTT; Lane 4, molecular-mass-marker proteins. The sample in Lane 2 was boiled before electrophoresis, whereas those in Lane 1 and 3 were incubated at room temperature instead.</p

Interactions of TipαN²⁵ and dsDNA.

<p>ITC experiments of 20 bp dsDNA with Tipα (A) and its mutants K65A/K66A (B), R77A (C), R77A/R81A (D) and H60A (E), respectively.</p

Thermodynamic parameters for Tipα and its mutants binding to ssDNA.

<p>Thermodynamic parameters for Tipα and its mutants binding to ssDNA.</p

Interactions of TipαN²⁵ and ssDNA.

<p>ITC experiments of Tipα with 20 nt (A), 40 nt (B), and 60 nt (C) oligomeric ssDNA. Autodock simulated model of Tipα binding an ideal ssDNA-d (GCGCG) through the dimer interface (D, E) with hydrogen bonds and salt bridges between Tipα and ssDNA (F).</p