Perturbance of enzyme function by blocking dimer interface formation: Novel route to specific antibiotics
Zusammenfassung der Projektergebnisse
Drug research has started to focus on the interference with protein-protein interface formation as an attractive opportunity for therapeutic intervention. The tRNA-modifying enzyme TGT, a putative drug target to fight Shigellosis, is only functionally active if the protein assembles as a homodimer. To better understand the driving forces responsible for the assembly and stability of the formed homodimer interface we embarked onto a computational analysis and subsequent mutational study of the residues forming the interface and we launched spiking ligands into the interface region to perturb contact formation of the homodimer. These studies were done under the control of non-degrading nanoESI mass spectrometry indicating the actual ratio of the monomer-dimer equilibrium in solution and crystal structure analyses elucidating the geometrical changes resulting from the induced perturbance. The stability of the C2 symmetrical interface which spans over about 1600 Å2 is established by a patch of four aromatic amino acids (Trp, Tyr, His, Phe) contributed by both parts of the dimer interface. The residues arrange in a mutual stacking and achieve further stabilization by a network of hydrogen bonds using the donor functionalities of the heteroaromatic side chains to interact with backbone carbonyl groups on the adjacent dimer mate. This patch is embedded into a ring of hydrophobic amino acids which supposedly shields the aromatic interaction hot spot from solvent access. Mutation of any of the four aromatic residues by a non-aromatic amino acid reveals pronounced loss of the homodimer stability. Whereas the wild type is nearly exclusively in dimeric state, for some of the mutated variants only minor to hardly any dimer formation could be observed in solution. By labeling and recording the monomer exchange kinetics we found that the wild type exchanges monomer units very slowly over hours whereas a destabilized variant exchanges very fast. Similarly, the binding of the spiking ligands shift the monomer-dimer ratio towards increasing amount of monomer. Apart from the aromatic hot spot, the interface shows an extended loop-helix motif which exhibits remarkable flexibility. Even though this stretch comprises a fair number of polar residues which are involved in the wild type in several salt bridges and hydrogen bonds across the interface, this motif occurs in multiple conformations including a folding to helical geometry which could be characterized in the various crystal structures. In the destabilized mutant variants and in the complexes with the spiking ligands, the loophelix motif adopts deviating conformations in the interface region. This motivated us to follow a strategy to raise small molecule binders against this motif to mould the loop geometry in a conformation incompatible with the interface formation. Via the introduction of cysteine residues we were able to reinforce under oxidizing conditions a new covalently linked “dimer”. The crystal structure of this species presents the interface and the loop-helix motif in a geometry only in contact with disordered solvent molecules. Here the loop-helix motif adopts a conformation not compatible with the dimer interface formation. Furthermore, a DMSO molecule, picked up from the cryo-buffer, was entrapped in a small cavity beneath the loop suggesting a putative binding niche for interface perturbing binders. They will be followed-up in a subsequent fragment-based design strategy.
Projektbezogene Publikationen (Auswahl)
- Launching Spiking Ligands into a Protein-Protein Interface: A Promising Strategy to Destabilize and Break Interface Formation in a tRNA Modifying Enzyme. ACS Chem Biol, 8 (6) (2013) 1163–1178
F. Immekus, L.J. Barandun, M. Betz, F. Debaene, S. Petiot, S. Sanglier-Cianferani, K. Reuter, F. Diederich, G. Klebe
(Siehe online unter https://dx.doi.org/10.1021/cb400020b)
