SmeT is a transcriptional repressor protein of 24.6 kDa, found in the pathogenic bacteria Stenotrophomonas maltophilia. SmeT is responsible for the regulation of the Multidrug Resistance (MDR) efflux pump, SmeDEF, that gives the bacteria resistance to several antibiotics including macrolides, TMP/SMX, tetracycline, chloramphenicol, quinolones, and erythromycin. SmeT is encoded 223 bp upstream of SmeDEF, with just 56 base pairs between their transcription start sites and an overlapping region between the promoters. The production of the SmeT protein downregulates its own transcription, along with that of the efflux pump by sterically hindering the binding of RNA Polymerase to the DNA. SmeDEF was the first MDR pump discovered in the S. maltophilia species. The pump is named by its different parts: SmeE, the transporter itself that spans the plasma membrane, SmeF, the protein on the outer portion of the membrane, and SmeD, a membrane fusion protein. On general purpose media and no selectors, the genes for MDR pumps are typically not expressed, and the repressor is found bound to the DNA. In fact, mutations in SmeT that lead to overexpression of SmeDEF can pose fitness challenges to the bacteria. However, this overexpression has been identified in the bacterium and may pose a threat to our health.
Origins Though a lot of recent antibiotic resistance in bacteria is due to mutations in genes for repressors such as SmeT, SmeDEF and SmeT are highly conserved within S. maltophilia. S. maltophilia are naturally found colonizing plant roots in water and soil, and SmeT has played an evolutionary role in the survival of the bacteria against plant-produced flavonoids that act as effectors to the repressor. Since many man-made antibiotics are plant products or related derivatives, this evolutionary role of SmeT also contributes to the characteristic resistance to antibiotics by the species. In other words, SmeT and SmeDEF are not recent traits acquired by horizontal gene transfer due to antibiotics but have arisen over time in S. maltophilia due to selective pressures in their natural habitats against plant agents.
Structure
SmeT is a homodimer, like many other proteins in the TetR family that the repressor is categorized in. The rmsd for the subunits is 0.811Å, a sign of their structural similarities. SmeT is made of 9 helices: α1, α2 and α3 and the beginning of α4 are responsible for DNA binding in the N-terminus domain, while the rest of α4, α5, α6, and α7 form the effector binding pocket in the C-terminus. α8 and α9 allow the dimerization of the protein. Hydrophobic interactions, Van der Waals forces, salt bridges, and hydrogen bonding between amino acid residues contribute to the stability of the N-terminus and its connection to the C-terminus. These interactions also contribute to the recognition of an effector by the protein since the subunits must interact in the ways mentioned above to induce conformational changes. On the surface of the protein, in the effector binding site, and in the DNA binding site, there are hydrophobic residues of amino acids. The surface of the protein is negatively charged, while the N-terminus has an overall positive charge. SmeT shows extended N and C termini and a much smaller binding site, about 630Å, compared to that of other proteins in the same family. Six amino acid residues that line the ligand pocket have multiple conformations, which allow different effectors in different orientations to bind to the protein, contributing to the range of effectors that the repressor is induced by.
Mechanism
SmeT binds to an operator region, a 28 bp pseudopalindromic site found in many Gram-negative bacteria, that overlaps both the promoter regions of SmeT and SmeDEF. This site is just upstream to the SmeD transcription start site. The sequence consists of 2 inverted and overlapping repeats, named IR1 in the coding strand and IR2 in its complement, that 4 SmeT proteins can bind to (2 on each repeat). The sequence TGTATGT in IR1 is necessary for the first homodimer to bind. This is the strongest bond between the dimers and the DNA, and the following homodimer binds to the DNA on the coding strand cooperatively. The Km of the protein for this region is about 1uM. A third homodimer then recognizes a similar sequence on the complementary strand and is stabilized by the interactions already made by the first homodimers to bind to the DNA. The fourth homodimer binds cooperatively. This results in the repression of both genes by the blocking of RNA Polymerase to the DNA.
The extension of amino acids on the N-terminus are negatively charged and maintain close interactions with the rest of the positively charged terminus. For DNA, which is negatively charged, to bind to the protein, this extension must not hinder the binding site. This is why, when the effector binds to the protein, one of the characteristic conformational changes is the movement of this extension. The expression of SmeDEF and SmeT initiates when an effector, such as tetracyclin, bile salts, or triclosan, binds to a pocket in the C-terminus domain, inducing conformational changes in the N-terminus DNA binding motif, stabilizing it. In the case of triclosan, 2 molecules are required to bind to the C-terminus domain in order for this change to occur. This conformation change leads to the release of the repressor from the operator so that expression of SmeDEF and SmeT initiates. Stabilizing of the N terminus is ultimately what leads to the dissociation of SmeT from the DNA. This region is highly disordered, but upon binding of the effector, new interacting structures are formed between α6 and α7, as well as α1 and α2. This changes the distance between the α3 helices that bind to the DNA, making them about 10Å longer than the grooves in the DNA that the helices bind to.
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