Structural and functional studies on the hypothetical protein TTHA1873 from Thermus thermophilus

Abstract

This thesis reports a detailed study on the structural and functional characterization of the hypothetical protein (TTHA1873) from Thermus thermophilus. In this study, both the structure and function of TTHA1873 were characterized. To elucidate the novel structure of this uncharacterized protein, the study employed a heavy atom derivative compound K2[HgI4] to obtain an anomalous signal using home source X-ray. The crystal structure has been determined using X-ray crystallography to a resolution of 1.78 Å using the single-wavelength anomalous diffraction method. The protein crystallized as a dimer in two space groups: P43212 and P6122. Structural analysis of the hypothetical protein revealed that the overall fold of TTHA1873 has a β-sandwich jelly-roll topology with nine β-strands. TTHA1873 is a dimeric metal-binding protein that binds to two Ca2+ ions per chain, with one on the surface and the other stabilizing the dimeric interface of the two chains. The structural information obtained was then used to infer the function of this protein. Biochemical experiments demonstrated that TTHA1873 acts as a nuclease, indiscriminately cutting methylated and non-methylated DNA in divalent metal ions and relaxing plasmid DNA in the presence of ATP. This activity is inhibited by EDTA. Structural analysis identified functionally important residues, and molecular dynamics simulations were conducted to investigate the effects of mutating two critical residues (R55A and R138A) on DNA binding. The MD analysis revealed that the WT-TTHA1873 demonstrated stable interactions with DNA. The calculated binding free energies imply more stability of the WT-TTHA1873-DNA complex, while the mutants showed lesser binding affinity toward its interacting partner, double-stranded DNA. Substituting single mutation R55A and R138A on TTHA1873 weakens their DNA-binding ability. It portrays the critical role of R55 and R138 from TTHA1873 as likely involved in DNA binding. The molecular dynamics study also explored the effects of high temperatures on this protein, identifying temperature-sensitive regions and providing insights into thermal denaturation. The results revealed that Loop 2, Loop 4, Loop 8, and the N-terminal and C-terminal regions are the most temperature-sensitive regions of the protein. The unfolding process of TTHA1873 was consistent with that of other thermophilic proteins, where loops surrounding the hydrophobic core open and expose it to the solvent, leading to denaturation. The use of heavy atom compound K2[HgI4] to obtain phases has been previously reported in the literature. Still, the protocols used to solve the three-dimensional structure of this protein may be particularly useful in challenging cases where molecular replacement is ineffective or when no similar structures are available in the Protein Data Bank (PDB) without relying on a synchrotron source. Overall, the structural and functional studies on TTHA1873 from Thermus thermophilus provided critical insights into the structure and function of this protein.