Regulation of solvent and antibiotic tolerance in Gram-negative bacteria
One of the major mechanisms underlying multidrug resistance in both prokaryotes and eukaryotes is the active extrusion of numerous structurally unrelated cytotoxic compounds by membrane proteins known as multidrug-efflux pumps. The phenomenon of multidrug recognition is not exclusively confined to multidrug transporters, several transcription regulators have been demonstrated to promote transporter expression in response to structurally dissimilar toxic compounds. The idiosyncrasy of these systems is that the regulators controlling the expression of the pumps respond to the same compounds that the pumps extrude. Despite extensive studies, the molecular mechanism that underlies multidrug resistance remains obscure, primarily because of the lack of structural information on these efflux transporter proteins and their regulators. We are carrying out research using a structural approach to elucidate the mechanism of solvent and antibiotic tolerance in Gram-negative bacteria.
Figure 1: Genomic organization of efflux pumps found in Gram-negative bacteria. Between the regulator and the pump operon is the regulatory site that the regulator gene product binds to.
We are dissecting the molecular basis of the unusual ability of Pseudomona putida DOT-T1E to tolerate high concentrations of toxic organic solvents and antibiotics, especially the mechanism of controlled regulation of the efflux transporter expression. To investigate this phenomenon, we have solved the atomic structure of the gene repressor TtgR by X-ray crystallography in its apoform and bound to several small-molecule ligands. We were able to explain the unique ligand binding properties of TtgR. The protein contains two distinct and overlapping ligand binding sites; the first one is broader and consists of mainly hydrophobic residues, whereas the second one is deeper and contains more polar residues. For more information, please refer to Alguel et al., 2007.
Figure 2: Crystal structure of the efflux pump regulator TtgR. Left: Cartoon representation of the functional dimer. Right: Ligand binding pocket with two different ligands superimposed.
We have recently determined the crystal structure of TtgV on its own and in complex with its intact 42 bp DNA operator (Lu et al., 2010). TtgV exists as tetramers in solution and we obtained the full-length tetrameric structures. The TtgV monomer consists of an N-terminal DNA binding domain (DBD) that belongs to the winged-HTH group while its C-terminal ligand binding domain (CTD) belongs to the GAF family. The two domains are connected by a linker helix. TtgV undergoes dramatic conformational changes at the monomeric, dimeric and tetrameric levels upon binding to DNA. It binds to its DNA as a pair of highly asymmetric dimers. The asymmetry within the dimer originates from the differences in the linker helix that connects the DNA binding domain with the ligand binding domain. Our structure also reveals a highly distorted DNA that resembles a W-shape with an overall bend of 60 degrees. Our structures show that binding to two DNA sites imposes significant constraints on the protein assembly and likewise, interacting with the tetramer induces significant distortions in the DNA. This raises interesting questions as to many models proposed for tetrameric gene regulator/DNA assemblies that are based on partial structural information, in particularly those that consist of a protein dimer in complex with a single DNA site.
Figure 3: Crystal structures of TtgV alone (left panels) and in complex with its 42-bp intact DNA operator (right panels). Ellipses indicate "units" within the tetramer that move together as a rigid body during conformational changes. The DNA binding domains in the TtgV alone structure (ellipses, upper left panel) have to rotate downwards in order to bind to the DNA. Binding induces large distortions in the DNA (upper right panel, central axis indicated by black line). Consequently, the C-terminal domains (ellipses, lower left panel) have to slide against each other to form the asymmetric configuration in the DNA-bound structure (lower right panel).
Our structures of TtgV on its own and in complex with its DNA reveal a cooperative DNA binding model that resembles the concerted model for cooperative binding to ligands. The apo structure is more stable but unable to bind to the DNA, we therefore termed it the Tense (or T) state but the DNA structure is less stable but can bind to the DNA, hence we term it the R (Relaxed)-state. We propose that in the absence of DNA, TtgV can exist in an equilibrium of the T and R states. In order to bind DNA, the symmetric tetramer (T-state) has to release the two pairs of DNA-binding domains from the C-terminal domains. This has two conflicting effects: it costs favorable interaction energy between the CTDs and the DBDs while it gains energy through the favorable asymmetric CTD arrangement. The energy values are poised so that binding to a single DNA site does not provide sufficient energy to compensate for the loss of two pairs of CTD/DBD interactions but binding to two sites does. This explains the extremely weak binding affinity of TtgV to a single site as mutating one of the two operator sites reduces the ability of TtgV binding to its operator significantly while TtgV dimer is unable to bind to DNA. However, binding to two sites provides sufficient favorable interactions and allows the tight binding of the TtgV tetramer to its operator. (For a clearer picture of the conformational changes between unbound and bounds forms of TtgV and DNA, please see the explanatory movie.)
This area of research is funded by the Medical Research Council and is in collaboration with Juan Ramos and Mari-Trini Gallegos (Granada, Spain). Early work on this project was funded by an HFSP Young Investigators's Grant.