The MECP2 TRD domain interacts with the DNMT3A ADD domain at the H3 tail binding site

The DNMT3A DNA methyltransferase and MECP2 methylation reader are highly expressed in neurons. Both proteins interact via their DNMT3A ADD and MECP2 TRD domains, and the MECP2 interaction regulates the activity and subnuclear localization of DNMT3A. Here, we mapped the interface of both domains using peptide SPOT array binding, protein pull down, equilibrium peptide binding assays, and structural analyses. The region D529 D531 on the surface of the ADD domain was identified as interaction point with the TRD domain. This includes important residues of the histone H3 N terminal tail binding site to the ADD domain, explaining why TRD and H3 binding to the ADD domain is competitive. On the TRD domain, residues 214 228 containing K219 and K223 were found to be essential for the ADD interaction. This part represents a folded patch within the otherwise largely disordered TRD domain. A crystal structure analysis of ADD revealed that the identified H3 TDR lysine binding pocket is occupied by an arginine residue from a crystallographic neighbor in the ADD apoprotein structure. Finally, we show that mutations in the interface of ADD and TRD domains disrupt the cellular interaction of both proteins in NIH3T3 cells. In summary, our data show that the H3 peptide binding cleft of the ADD domain also mediates the interaction with the MECP2 TRD domain suggesting that this binding site may have a broader role also in the interaction of DNMT3A with other proteins leading to complex regulation options by competitive and PTM specific bindin

Optimization of the Lead Compound NVP BHG712 as Colorectal Cancer Inhibitor

The ephrin type A receptor 2 EPHA2 kinase belongs to the largest family of receptor tyrosine kinases. There are several indications of an involvement of EPHA2 in the development of infectious diseases and cancer. Despite pharmacological potential, EPHA2 is an under examined target protein. In this study, we synthesized a series of derivatives of the inhibitor NVP BHG712 and triazine based compounds. These compounds were evaluated to determine their potential as kinase inhibitors of EPHA2, including elucidation of their binding mode X ray crystallography , affinity microscale thermophoresis , and selectivity Kinobeads assay . Eight inhibitors showed affinities in the low nanomolar regime K D lt;10 nM . Testing in up to seven colon cancer cell lines that express EPHA2 reveals that several derivatives feature promising effects for the control of human colon carcinoma. Thus, we have developed a set of powerful tool compounds for fundamental new research on the interplay of EPH receptors in a cellular contex

Photochemistry of Transition Metal Complexes Induced by Outer-Sphere Charge Transfer Excitation

The intermolecular (outer sphere, OS) interaction of a reducing and an oxidizing metal complex generates a new optical transition involving charge transfer (CT) from the electron donor to the acceptor. OS CT transitions are classified according to the redox site (metal or ligand). Generally, the interaction between donor and acceptor is facilitated by ion pairs consisting of an oxidizing complex cation and a reducing complex anion. There are also ion pairs which are composed of a metal complex and an organic counter ion as electron donor or acceptor. In addition, the review includes examples of OS CT interaction which do not involve ion pairs at all. — A short introduction into the theory is followed by the discussion of the spectroscopy of OS CT of transition metal complexes. Finally, photoreactions induced by OS CT transitions are reviewed. The optical transfer is frequently followed by a rapid back electron transfer which regenerates the starting complexes. In many cases the primary products are kinetically labile and substitution reactions compete successfully with back electron transfer. As a result stable redox products may be formed. As an alternative, the substitution can be followed by back electron transfer. Product formation appears then as a substitution of the starting complexes. The various possibilities are illustrated by appropriate examples

Vinorine synthase from Rauvolfia: the first example of crystallization and preliminary X-ray diffraction analysis of an enzyme of the BAHD superfamily

Crystals of vinorine synthase (VS) from medicinal plant Rauvolfia serpentina expressed in Escherichia coli have been obtained by the hanging-drop technique at 305 K with ammonium sulfate and PEG 400 as precipitants. The enzyme is involved in the biosynthesis of the antiarrhythmic drug ajmaline and is a member of the BAHD superfamily of acyltransferases. So far, no three-dimensional structure of a member of this enzyme family is known. The crystals belong to the space group P212121 with cell dimensions of a=82.3 A, b=89.6 A and c=136.2 A. Under cryoconditions (120 K), a complete data set up to 2.8 A was collected at a synchrotron source

High resolution crystal structure of cAMP dependent protein kinase from Cricetulus griseus

Protein kinases (PKs) are dynamic regulators of numerous cellular processes. Their phosphorylation activity is determined by the conserved kinase core structure, which is maintained by the interaction and dynamics with associated domains or interacting proteins. The prototype enzyme for investigations to understand the activity and regulation of PKs is the catalytic subunit of cAMP-dependent protein kinase (PKAc). Major effects of functional regulation and ligand binding are driven by only minor structural modulations in protein–protein interactions. In order to resolve such minor structural differences, very high resolution structures are required. Here, the high-resolution X-ray structure of PKAc from Cricetulus griseus is reported