The capabilities of ligand-based and protein-based NMR methods for the identification and characterization of ligand-protein interactions were presented using the results of our latest studies of ligand binding to the protein target muramyl ligaze D (MurD). Special attention was given to the unique ability of NMR spectroscopy to provide a combined structure-dynamic insight into ligand-protein binding. We applied advanced isotopic labelling of MurD for protein-based NMR studies. The selectively 13CH3 labelled MurD have provided a solid basis for the effective NMR characterization of a site specific MurD ligand binding, as was demonstrated for the various types of novel MurD inhibitors. We discovered fast domain motions of MurD, which affect the conformation and flexibility of bound ligands, stability of binding interactions and the adaptability of the MurD binding site. We further investigated these dynamic processes using the NMR spin relaxation experiments and the normal mode analysis based on MD trajectories. The addition of perdeuteration was crucial for the amide 1H and 15N backbone assignment of opened and closed MurD state, which was essential for 15N relaxation NMR studies of MurD domain motions in relation to ligand binding. By analysing the distribution of the low frequency modes during the binding process we elucidate the impact of the protein internal dynamics on the change of the free energy of ligand binding.
B.04 Guest lecture
COBISS.SI-ID: 6691610S. Golič Grdadolnik was invited as guest-editor. The aim of this Special Issue of the international journal Molecules was to attract contributions on all aspects of the application of NMR spectroscopy in the design and discovery of drug candidates, with special emphasis on the NMR studies addressing molecular flexibilities in relation to the biological profile of drug candidates or the druggability of targets.
C.03 Guest-associated editor
COBISS.SI-ID: 18462981Prof. Simona Golič Grdadolnik talks about her group's latest study of proteins' dynamic properties for the design of novel inhibitors towards new and better antibiotics. The research is based on the use of NMR techniques.
E.03 Other
COBISS.SI-ID: 6399002To describe the molecular determinants of the ATP binding to rhNGF and define its functional roles, the solution NMR structure of rhNGF in the unbound state was determined. The NOEs obtained from the 3D 15N- and 13C-NOESY-HSQC spectra were used for the 3D structural model construction and validation. The solution structure closely resembles those determined by X-ray crystallography for rhNGF-ligand complexes especially as to the topology of the Cys-knot, whereas it exhibits notable differences within the flexible loop regions. Furthermore, in the present NMR structure of unbound rhNGF, the N-terminus is flexible and lacks a specific secondary structure propensity, whereas in the X-ray crystal structures of the rhNGF-TrkA complex, it is involved in the interacting surface and adopts a helicoidal conformation. It is to be noted that this NMR structure represents not only the first solution structure of rhNGF, but the first structure ever for unbound rhNGF.
F.02 Acquisition of new scientific knowledge
COBISS.SI-ID: 14902275co-mentor for master thesis Lanosterol 14?-demethylase (CYP51A1), a member of the cytochromeP450 superfamily, is an essential enzyme in cholesterol biosynthesis. The catalytic activity and structure of CYP51 is evolutionarily conserved and the gene has a low number of polymorphisms. In a database of 1000 human genomes we identified natural single-nucleotide polymorphisms(SNP) that cause missense amino acid substitutions with damaging SIFT and PolyPhen-2 predictions. We chose the mutations R271L and R425H (Q16850, UniProt) for the experimental analysis and prepared them with a site-directed mutagenesis. We expressed the native and mutant proteins in Esherichia coli and isolated them from the spheroplasts fractions. We isolated a catalytically active native protein with an absorbance peak inCO-difference spectra at 447 nm, but failed to isolate the active mutantshCYP51-R271L and hCYP51-R425H, which had an absorbance peak at 417nm. To evaluate the substitution effect of the amino acid on enzyme substrate binding and interaction with POR (cytochrome P450 oxireductase), we also performed molecular dynamics simulations with polymorphism D146G. The results show that the mutations R425H andD146D have damaging effects on the interaction between hCYP51 and the substrate, whereas the mutations R271L and D146G prevent the interaction between hCYP51 and the redox partner POR. However, we were unable to produce active mutants and were therefore unable to test the catalytic efficiency of the mutants in vitro. We presume that the changes R271L and R425H have a damaging effect on conserved structure of hCYP51. The mutations effect the protein structure in such away that the heme-thiolate bond cannot form, thus making the mutants inactive.
D.10 Educational activities
COBISS.SI-ID: 1537489347