Specific Substates of Ras To Interact with GAPs and Effectors: Revealed by Theoretical Simulations and FTIR Experiments.

The oncogenic Ras protein adopts various specific conformational states to execute its function in signal transduction. The large number of Ras structures obtained from X-ray and NMR experiments illustrates the diverse conformations that Ras adopts. It is difficult, however, to connect specific structural features with Ras functions. We report the free-energy landscape of Ras·GTP based on extensive explicit solvent simulations. The free-energy map clearly shows that the functional state 2 of Ras·GTP in fact has two distinct substates, denoted here as "Tyr32in" and "Tyr32out". Unbiased MD simulations show that the two substrates interconvert on the submicrosecond scale in solution, pointing to a novel mechanism for Ras·GTP to selectively interact with GAPs and effectors. This proposal is further supported by time-resolved FTIR experiments, which demonstrate that Tyr32 destabilizes the Ras·GAP complex and facilitates an efficient termination of Ras signaling.

Functional display of heterotetrameric human protein kinase CK2 on Escherichia coli: a novel tool for drug discovery.

BACKGROUND: Human protein kinase CK2 represents a novel therapeutic target for neoplastic diseases. Inhibitors are in need to explore the druggability and the therapeutic options of this enzyme. A bottleneck in the search for new inhibitors is the availability of the target for testing. Therefore an assay was developed to provide easy access to CK2 for discovery of novel inhibitors. RESULTS: Autodisplay was used to present human CK2 on the surface of Escherichia coli. Heterotetrameric CK2 consists of two subunits, α and ß, which were displayed individually on the surface. Co-display of CK2α and CK2ß on the cell surface led to the formation of functional holoenzyme, as demonstrated by NaCl dependency of enzymatic activity, which differs from that of the catalytic subunit CK2α without ß. In addition interaction of CK2α and CK2ß at the cell surface was confirmed by co-immunoprecipitation assays. Surface displayed CK2 holoenzyme enabled an easy IC50 value determination. The IC50 values for the known CK2 inhibitors TBB and Silmitasertib were determined to be 50 and 3.3 nM, respectively. CONCLUSION: Surface-displayed CK2α and CK2ß assembled on the cell surface of E. coli to an active tetrameric holoenzyme. The whole-cell CK2 autodisplay assay as developed is suitable for inhibition studies. Furthermore, it can be used to determine quantitative CK2 inhibition data such as IC50 values. In summary, this is the first report on the functional surface display of a heterotetrameric enzyme on E. coli.