• EthR
• References
• Protein Monogram
• Slides

Transcriptional Regulatory Repressor Protein (EthR) in complex with fragment 7E8 (PDB ID: 5MWO) found in Mycobacterium Tuberculosis

Created by: Anna Carey

Transcriptional Regulatory Repressor Protein (EthR) in complex with fragment 7E8(PDB ID: 5MWO) is a biomolecule bound to the ligand (5-methyl-1-benzothiophen-2-yl) methanol with the intention of inhibiting the function of DNA binding. Tuberculosis (TB) is one of the deadliest virus’ in the world and is caused by the bacteria Mycobacterium Tuberculosis. EthR found in this virus and it is partially responsible for protecting the virus via DNA binding, making it a multi-drug resistant bacterium. The goal of adding ligands such as (5-methyl-1-benzothiophen-2-yl) methanol to this biomolecule is to determine the best way of disrupting the DNA-EthR complex, thus making the virus more susceptible to lower doses of drugs like ethionamide (ETH) (1, 2).

Protecting the DNA from degradation is EthR’s primary function. The EthA (another protein in Mycobacterium Tuberculosis, PDB ID: unknown) activates the drug administered, in this instance ETH, and NAD forming the complex ETH-NAD. The activated ETH-NAD then binds to 2-trans-enoyl reductase enzyme Inhibitor-A which deactivates the cell and its viral effects. EthR represses the expression of the EthA gene because of where the EthR binds on the DNA molecule, therefore the EthA protein is not expressed. The ETH, as a result, is not activated unless present in large quantities within the cell (1, 2).

The Expasy database was used to determine that EthR has a molecular weight of 25136.18 Da and an isoelectric point of 5.57 (3). Containing two homodimeric subunits formed from the interaction of residues 167-207, the protein is a symmetric biomolecule which has been crystallized at a resolution of 1.96 Å (4). Although the structure exists as a homodimer when free in the nucleus, when bound to its substrate, DNA, three of these homodimers bind together around the 62 bp operator sequence of DNA to perform its function, creating a complex composed of six identical subunits and DNA₆₂. For the purpose of identifying binding inhibitor ligands a complex of four subunits and DNA₃₁, instead of the more biologically accurate six EthR-DNA₆₂ was used in the fragment analysis (1).

EthR from Mycobacterium Tuberculosis was expressed in Escherichia coli (E. Coli) for purification and investigation. It was crystallized by the vapor diffusion hanging drop technique carried out under the conditions of 1.7 to 2.1 M ammonium sulfate 0.1 M MES (pH 6 to 7) 5 to 15%(v/v) glycerol 7 to 12%(v/v) 1,4-dioxane at 291.0 K. The ligand ethylene glycol was used for crystallization (4). Fragment screening by native mass spectroscopy experiments were used to identify ligands of EthR that disrupt DNA binding. This screening has led to the conclusion that (5-methyl-1-benzothiophen-2-yl) methanol and [N]-(5-oxidanylidene-7,8-dihydro-6-[H]-naphthalene- 2-yl) ethanamide are the effective inhibitors; the second compound is the fragment denoted 7E9 and can be found in complex with EthR (PDB ID: 5MXK) (1).

The primary structure of EthR contains 228 residues per subunit with an arrangement of both hydrophobic and hydrophilic amino acids. The secondary structure of this protein is composed of 9 α-helices, 10 segments of random coils, and one β-turn (5). The α-helix-dominated structure is important because of the mixed polarities of the sequence; the α-helix allows the hydrophobic residues to be pushed to the inside of the globular structure of the protein, while still allowing the polar residues to face outwards.

The tertiary structure of EthR, due to the hydrophobic residues from the helical structures facing inwards towards the core of the protein, contains a hydrophobic channel that acts as the active site in ligand binding (1). The domain segment of α-helix, β turn, α-helix (also known as helix-turn-helix or HTH domain) is present in the second two helical secondary structures (residues 45-54 and 56-64) which are stabilized by the first α-helical structure (residues 23-38). The HTH domain contains the DNA binding site from residues 45-64(2, 5). The length of a single subunit of EthR is 121.93 Å long and 33.73 Å in width at the largest diameters (3).

As stated previously, the quaternary structure of EthR is made of a homodimer formed from the interactions of the eighth and ninth α-helices and random coil from residues 168-207. The eighth α-helix (residues 168-188) is also involved in the intramolecular hydrophobic channel on the opposite side from these inter-subunit interactions. Both subunits within the biomolecule have the same DNA binding function. When bound to DNA, the 6 subunits that are found in complex are considered to be a quaternary structure, although this structure is not found outside of the DNA complex (1).

Researchers are currently testing fragment 7E8 also known as (5-methyl-1-benzothiophen-2-yl) methanol and other fragments for the disruption of the DNA/EthR complex. These ligands bind in distinct places within the hydrophobic channel. More specifically 7E8 binds to Phe-114, Phe-110, Trp-138, and Trp-145 or Trp-103, Trp-145, Trp-207, and Tyr-148 within the channel for position A or B respectively. The ligand is held in place through π interactions with the benzyl ring of the ligand side chain of the aromatic amino acids tryptophan and phenylalanine. The ligand binds to these sites in the channel to trigger a conformational change in the biomolecule leading to a structure incompatible for DNA binding (1). There has also been a trend of significant and successful inhibitor binding at residues Asn-176 and Asn-179 by other ligands (5).

Differences in ligand binding induce many different conformations of EthR. Two such ligands that interact differently than (5-methyl-1-benzothiophen-2-yl) methanol are found in the EthR complex with ~{N}-(5-oxidanylidene-7,8-dihydro-6~{H}-naphthalen- 2-yl)ethanamide (PDB ID: 5MXK) and 3-[1-[4-(methylaminomethyl)phenyl]piperidin- 4-yl]-1-pyrrolidin-1-yl-propan-1-one (PDB ID: 5EYR) (4). The first of these ligands has shown considerable EthA expression, changing the conformation in a way similar to (5-methyl-1-benzothiophen-2-yl) methanol. These two compounds have been determined very useful in promoting EthA expression and will continue to be used in the treatment and deactivation of the Mycobacterium Tuberculosis virus (1). Another important conformation is the unliganded form of the protein (PDB ID: 5N1I) (4). This is the structure of the protein before ligands were added to change the conformation and disrupt binding. Therefore this molecule will bind to DNA and inhibit EthA expression.

FadR in complex with E. coli-derived dclick hereodecyl-CoA(PDB ID: 3ANG) is derived from Thermus Thermophilus and has similar primary and tertiary structures as EthR (4, 6, 7). The Basic Local Alignment Search Tool (BLAST) database compares the primary structures of proteins, assigning an E value for every comparison it makes between the sequences. This E value is determined by the presence of gaps, or lack of homology, between to primary structures. A lower E value represents more closely related primary structures, i.e. fewer gaps. E values below 0.05 are considered significant. The E value of FadR and EthR in their respective complexes was 0.001, indicating very similar sequences (6). The Dali server compares tertiary structures of proteins and provides a quantitative Z score to signify the degree of structural similarity. A Z-score over 2 is considered significant; the comparison of FadR and EthR yielded a Z-score of 14.1, which indicates the tertiary structures are very similar (7). 

The biggest similarities between FadR and EthR in their respective ligand complexes are the dominant helical structure, DNA binding sites at the HTH domain found in α-helices 2-3, and the existence of a hydrophobic channel (2, 8). Although very similar, there are structural differences that exist between the two biomolecules.FadR's quaternary structure is a homotetramer, composed of 4 homologous subunits, whereas EthR is a homodimer with only 2. The HTH substrate binding site, while composed of α-helices 2-3 in each, differs in size and position within the two primary structures; in EthR it is composed of residues 45-64, whereas FadR’s binding site is at residues 32-49 (2, 8). The FadR exists as a homotetramer, differing from the homodimeric structure of EthR (8). FadR does not bind its DNA binding inhibitor, dodecyl-CoA, in the hydrophobic channel like EthR, instead it binds on the more accessible surface of the protein (8). This channel effects the molecules structurally as it prohibits EthR from binding large molecules due to the size restrictions and the longer hydrophobic residues that extend into the channel (1). FadR has a higher affinity for larger, more polar biomolecules due to hydrogen bonding and attractive forces of the more polar surface residues, whereas EthR binds smaller, hydrophobic molecules via π interactions and hydrophobic interactions (8).

A major functional similarity between the two is the effect of the ligand on the binding properties of the protein to DNA; as the ligand is introduced to the protein, it inhibits binding via non-competitive inhibition and distortion of the binding site in a trend shown by the Michaelis Menten plot (8). While the structures of these proteins do hold many similarities, they engage in different functions within the cell. FadR is a regulator of fatty acids in cells whereas EthR represses the expression of the protein EthA. FadR regulates fatty acids through inhibition of the fatty acid degenerating gene on DNA when there is a lack of acyl-CoA and long chain fatty acids. When there is an excess of acyl-CoA and long fatty acid chains, these groups bind to the surface of FadR, acting as ligands, and change its conformation. This renders the binding site incompatible for DNA which allows the DNA to express genes which degrade acyl-CoA and fatty acids. The ligand binding site on the surface of FadR allows the ligand to temporarily attach and detach in order fit the needs of the cell (8). This differs from the function of the ligand in EthR where the permanent conformation change is desired to allow EthA to fully activate the ETH drug complex.