The vast majority of biological processes ranging from signal transduction to gene expression depend on macromolecular interactions (protein-protein or protein-nucleic acid). We are investigating the structural basis and functional relevance of macromolecular interactions involving bacterial translation elongation factors and toxin-antitoxin pairs in order to obtain a better understanding of their roles in normal as well as in stressed or diseased states. Examples of potential projects are provided below.
1. Single- molecule studies of the dynamics of EF-Tu
EF-Tu belongs to the superfamily of G-proteins, which act as molecular switches for a regulatory purpose. Many diseases such as cancer and memory disorders are associated with the malfunctioning of G-proteins. The normal role of EF-Tu is to carry aminoacyl-tRNA (aa-tRNA) to the A-site of the mRNA-programmed ribosome in a GTP-dependent manner. Upon correct interaction between the codon exposed in the ribosomal A-site and the anticodon of the incoming aa-tRNA, GTP-hydrolysis is triggered. The resulting EF-Tu·GDP needs to be recycled by its guanine-nucleotide exchange factor, EF-Ts, which promotes the exchange of GDP with GTP thus enabling a new round in the elongation cycle to take place.
During its functional cycle, EF-Tu undergoes dramatic structural changes as seen by comparing the active, GTP-bound form and the inactive, GDP-bound form of the factor (Figure 1). We are exploring the dynamical aspects of these changes, using advanced single-molecule fluorescence microscopy based on fluorescence resonance energy transfer (FRET). In contrast to the general view of EF-Tu, we have shown that the conformational change of EF-Tu initiates on the ribosome, but is not completed until after dissociation. We will produce more fluorescently labelled EF-Tu variants to explore these findings in detail and gain information about the functional consequences of our results. We are using site-directed mutagenesis, protein expression and purification followed by protein labelling to produce EF-Tu variants suitable for our studies. The mutated and labelled EF-Tu’s are tested in functional assays prior to single-molecule fluorescence microscopy, which takes place in collaboration with Professor Yale E. Goldman at Pennsylvania University.
Figure 1. Comparison of the active (EF-Tu:GDPNP) and inactive (EF-Tu:GDP) conformations of EF-Tu
In the inactive conformation, the three domains of EF-Tu form an open structure with a hole in the middle. Upon binding of GTP, domains 2 and 3 rotate by 90° relative to domain 1 causing closure of the hole. Also parts of domain I, switch region I (shown in magenta) and helix B, undergo structural rearrangements upon switching between the two conformations.
2. The role of host proteins during viral infection
Viruses are obligate parasites that depend on host proteins and pathways during most steps of an infection. In particular, the replication of the genomes of RNA viruses has been found to require the "kidnapping" of host proteins by the virally encoded RNA-dependent RNA polymerase (RdRP; also known as replicase). Qb is a bacteriophage that infects E. coli (Figure 2). Upon entry of its (+)-stranded RNA genome into the host, the genome gets translated and the resulting RdRP forms a complex with EF-Tu, EF-Ts and ribosomal protein S1 (and eventually host factor, Hfq). We have solved the three-dimensional structure of this complex using X-ray crystallography. The structure has revealed features of potential importance during genome replication i.e. in template binding, initiation, elongation and unwinding of the helix between template and product. We are subjecting the relevant amino acid residues or structural elements to structure-function analysis (see figure 3).
Our structure indicates that disruption of the interactions between the viral RdRP and host proteins may be a viable strategy in combating a viral infection. We will explore this possibility by developing a selection system that allows the identification of inhibitors of viral infection. Potential inhibitors will be selected from a library of peptide aptamers. Next, the mechanisms of the isolated inhibitors will be explored in binding- and activity assays.
The replication mechanism of Qb resembles the strategy applied by pathogenic viruses such as hepatitis C virus and polio virus. Thus, new information about the molecular details of replication of the Qb genome may be directly applicable to other (+)-stranded RNA viruses of medical relevance and lead to the identification of novel drug targets.
Figure 2. Structural illustration of the Qb infection cycle
The virus particle binds to the surface of a host cell and injects the RNA genome (green) into the cytoplasm of the bacterium (1). Host ribosomes translate the (+)-stranded Qb genome into protein (2) resulting in the production of the viral replicase subunit (the RdRP; lime). The replicase subunit forms a complex with the host proteins EF-Tu (blue) and EF-Ts (magenta) resulting in an active Qb replicase complex capable of copying the RNA genome (3). The cycle is completed when the genome copies are packaged into coat proteins to form virions.
Figure 3. Flow of structure-function studies
The three-dimensional structure of the studied protein forms the basis for designing mutations, which are predicted to affect the functional property of interest. The mutation is introduced into the protein-coding gene by site-directed mutagenesis. Subsequently, the mutant protein is expressed and purified. Finally, a functional characterization is carried out to test the working hypothesis.
3. Improvement of antibiotic treatment
The translation apparatus is the target of a number of bacterial toxins, which bring the cell into a dormant state, when environmental challenges are met. This allows the bacterium to survive during stress conditions such as antibiotic treatment. In the cell, the toxins are neutralized by antitoxins, which are targets of the Lon protease (Figure 4). We aim at reducing the action of bacterial toxins to increase the success rate of antibiotic treatments by inhibiting the Lon protease. Possible projects include (1) establishment of a selection system enabling the detection of Lon inhibitors, (2) establishment of a peptide library encoding potential inhibitor candidates, (3) screening of the library to identify Lon inhibitors and (4) characterization of the identified Lon inhibitors including their effect during antibiotic treatment.
Figure 4. Regulation of bacterial growth by toxin-antitoxin pairs
The action of the toxin (green) brings the bacterium into a dormant state. The antitoxin (blue) neutralizes the toxin to promote growth. Various stress conditions induce the hexameric Lon protease to degrade the antitoxin. Thus, the toxin is released and growth arrest results.