Thermus thermophilus HB8 TTHA1718 gene product
Thermus thermophilus HB8 (thermophilic bacteria)
The Thermus thermophilus HB8 TTHA1718 gene product is a putative heavy metal binding protein. The most significant point of this PDB entry (PDB ID: 2ROE) is that this is the world's first protein structure that was determined exclusively from the data obtained the samples inside living cells. Nowadays X-ray crystallography, Nuclear Magnetic Resonance (NMR) and electron microscopy are routinely utilized for determining three dimensional (3D) structures of proteins, which are usually over-expressed in the cells and purified. However, these methodologies cannot be used for the investigation of protein behaviours, structures, dynamics and various binding events, at work inside living cells, which can be largely affected by so called “macromolecular crowding”. Recent advances in measurement sensitivity have permitted heteronuclear multi-dimensional NMR spectroscopy of proteins inside living cells (in-cell NMR). Various intracellular events such as conformational changes, dynamics and interactions with other macromolecules have been investigated by this method. However, the low sensitivity and short life time of the samples have so far prevented the acquisition of sufficient structural information to determine protein structures by in-cell NMR. This is the first example of the protein structure determined in the living cells. The technologies we have applied for determining the structure opens new avenues for the investigation of protein conformations at atomic resolution and how they change in response to biological events in living environments.
The structure of TTHA1718 has been solved both inside living cell (PDB ID:2ROE, shown in three figures at the upper line) and by conventional in vitro approach (PDB ID:2ROG, shown in three figures at the bottom line). They are very similar (the backbone r.m.s.d. between the in-cell and in vitro structures is 1.16 Å), and are composed of a beta-sheet with 4 strands and two alpha-helices. Slight structural differences were found in the more dynamic loop regions, particularly in the putative heavy-metal binding site. The differences may be due to the effects of the molecular crowding as well as the interactions with metal ions in the E. coli cytosol. Indeed, C11S/C14S and C11A/C14A mutants, which lack the metal-binding activity, showed fairly small chemical shift changes in E. coli cells, while the significant changes were found in the region of wild type protein.
Protein Data Bank (PDB)
author: Teppei Ikeya and Yutaka Ito