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

Tip alpha (TNF-alpha-inducing protein) from Helicobacter pylori 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 Tip alpha would be abolished when both disulfide bridges were disrupted. The crystal structures of Tip alpha 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 H. pylori Tip alpha protein, Tip alpha N-25, at 2.2 angstrom 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-Tip alpha interactions reveal that Tip alpha 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. Tip alpha 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 Tip alpha indicate that the intrinsic flexible nature of disulfide bridges could endow certain elasticity to the Tip alpha dimer for its unique bioactivities. The results shed light on the possible structural mechanism for the functional performances of Tip alpha.