RNA and DNA Binding Epitopes of the Cold Shock Protein TmCsp from the Hyperthermophile Thermotoga maritima.

Prokaryotic cold shock proteins (CSPs) are considered to play an important role in the transcriptional and translational regulation of gene expression, possibly by acting as transcription anti-terminators and "RNA chaperones". They bind with high affinity to single-stranded nucleic acids. Here we report the binding epitope of TmCsp from Thermotoga maritima for both single-stranded DNA and RNA, using heteronuclear 2D NMR spectroscopy. At "physiological" growth temperatures of TmCsp (≥ 343 K), all oligonucleotides studied have dissociation constants between 1.6 ((dT)7) and 25.2 ((dA)7) µM as determined by tryptophan fluorescence quenching. Reduction of the temperature to 303 K leads to a pronounced increase of affinity for thymidylate (dT)7 and uridylate (rU)7 heptamers with dissociation constants of 4.0 and 10.8 nM, respectively, whereas the weak binding of TmCsp to cytidylate, adenylate, and guanylate heptamers (dC)7, (dA)7, and (dT)7 is almost unaffected by temperature. The change of affinities of TmCsp for (dT)7 and (rU)7 by approximately 3 orders of magnitude shows that it represents a cold chock sensor that switches on the cold shock reaction of the cell. A temperature dependent conformational switch of the protein is required for this action. The binding epitope on TmCsp for the ssDNA and RNA heptamers is very similar and comprises ß-strands 1 and 2, the loop ß1-ß2 as well as the loops connecting ß3 with ß4 and ß4 with ß5. Besides the loop regions, surprisingly, mainly the RNA-binding motif RNP1 is involved in ssDNA and RNA binding, while only two amino acids, H28 and W29, of the postulated RNA-binding motif RNP2 interact with the uridylate and thymidylate homonucleotides, although a high affinity in the nanomolar range is achieved. This is in contrast to the binding properties of other CSPs or cold shock domains, where RNP1 as well as RNP2 are involved in binding. TmCsp takes up a unique position since it is the only one which possesses a tryptophan residue instead of a usually highly conserved phenylalanine or tyrosine residue at the end of RNP2. NMR titrations suggest that neither (dT)7 nor (rU)7 represent the full binding motif and that non-optimal intercalation of W29 into these oligonucleotides blocks the access of the RNP2 site to the DNA or RNA. NMR-experiments with (dA)7 suggest an interaction of W29 with the adenine ring. Full binding seems to require at least one single purine base well-positioned within a thymine- or uracil-rich stretch of nucleic acids.

Distinct binding mode of multikinase inhibitor lenvatinib revealed by biochemical characterization.

Lenvatinib is an oral multikinase inhibitor that selectively inhibits vascular endothelial growth factor (VEGF) receptors 1 to 3 and other proangiogenic and oncogenic pathway-related receptor tyrosine kinases. To elucidate the origin of the potency of lenvatinib in VEGF receptor 2 (VEGFR2) inhibition, we conducted a kinetic interaction analysis of lenvatinib with VEGFR2 and X-ray analysis of the crystal structure of VEGFR2-lenvatinib complexes. Kinetic analysis revealed that lenvatinib had a rapid association rate constant and a relatively slow dissociation rate constant in complex with VEGFR2. Co-crystal structure analysis demonstrated that lenvatinib binds at its ATP mimetic quinoline moiety to the ATP binding site and to the neighboring region via a cyclopropane ring, adopting an Asp-Phe-Gly (DFG)-"in" conformation. These results suggest that lenvatinib is very distinct in its binding mode of interaction compared to the several approved VEGFR2 kinase inhibitors.

HTS reporter displacement assay for fragment screening and fragment evolution toward leads with optimized binding kinetics, binding selectivity, and thermodynamic signature.

Parameters such as residence time, kinetic selectivity, and thermodynamic signature are more and more under debate as optimization objectives within fragment-based lead discovery. However, broad implementation of these parameters is hampered by the lack of technologies that give rapid access to binding kinetics and thermodynamic information for large amounts of compound-target interactions. Here, the authors describe a technology--the reporter displacement assay--that is capable of opening this bottleneck and of supporting data-driven design of lead compounds with tailor-made residence time, kinetic selectivity, and thermodynamic signature.