The transcription of the human c-myc proto-oncogene is regulated by multiple cis-elements upstream as well as downstream of the promoter sites. One cis-elements upstream from the c-myc promoters is the CT element. This is a CT rich sequence which is located100 bases upstream from the P1 promoter. One protein that binds the coding strand of the CT element is hnRNPk. Binding of hnRNPk to the CT element upregulates c-myc transcription. There are three homology repeats in hnRNPk. These are called the KH domains. The 86 residue C-terminal segment of the hnRNPk protein comprises the third KH motif (KH3) in hnRNPk. The three-dimensional structure of the C-terminal domain of hnRNPk protein in solution has been determined by NMR spectroscopy. The structure of KH3 of hnRNPk was determined using the liquid crystal technique in addition to conventional protein NMR method. The liquid crystal environment produces a slight order in the system. The ordering of the molecule reintroduces dipolar coupling which contain useful structural information. The final family of structures calculated using the addition of dipolar coupling information results in root mean square deviation (RMSD)for the family of 0.17 Angstrom. In contrast, calculation carried out without the dipolar couplings has an RMSD of 0.32 Angstrom. The RMSD value provides a quantitative evaluation of the precision of the structures with smaller RMSD value being higher precision. This comparison was made under ideal conditions, i.e. : good signal to noise for all of the NMR experiments, some NMR experiments were repeated twice for error estimates as well as consistency check, all distance estimates were done conservatively to take into account any possible systematic error. Thus for a typical NMR structure under non-ideal conditions the increase of the RMSD value with the addition of dipolar coupling information should be much greater. In parallel to the KH3 structure determination we also developed a straightforward protocol of structure refinement using the dipolar coupling information. This protocol is being provided to any laboratory which wishes to take advantage of dipolar coupling information. A dynamic study of KH3 domain has also been carried out. There are two different purposes for carrying out the dynamic study. One is to get the detail information regarding the overall tumbling of the molecule as well as the local microscopic motion. Two is to use the hydrodynamic parameters derived from the relaxation data to cross check the quality of protein structure. The overall tumbling rate of the KH3 has been determined to be 6.87 ns. There is one flexible loop (L52-R56) in the KH3 domain which undergoes rapid (ps) fluctuation. In contrast to previous study of a similar KH domain of FMR1 the conserved loop (G30-G33) do not show any flexibility. This is suspected due to the different in pH under which the two studies were carried out. Thus points to the possibility of this particular loop undergoing chemical exchange which diminishes at low pH where the KH3 study was done. In addition to this local information the overall fit of the relaxation rates to the KH3structure has revealed that the molecule is non spherical with the ratio of the long diffusion axis to the short diffusion axis of 1.37 and the asymmetry (ratio of diffusion along the x and they axis) of 1.12. We pointed out that the quality of the fit of the relaxation data to the structure provides an independent quality assessment of the structure it self. When the dynamics data were fitted to the structure without the dipolar coupling one observed a residual error (chi squared) of 3.0 while using the dipolar refined structure the chi-squared is 1.5. This is suggesting that the structure refined using dipolar coupling is twice as accurate as the one without dipolar coupling. The next steps in the structural study of hnRNPk has been initiated. The construct of KH1+KH2 of hnRNPk has been made. The protein expression is currently being characterized. Single stranded DNA binding constant for the KH1+KH2 domain is also under investigation. In addition the C-terminal domain of hnRNPk contains 3 SH3 binding sites. A plasmid containing the Vav-SH3domain is being constructed to produce the target peptide for KH3domain.In order to get a better understanding of c-myc regulation through the CT element a parallel project has been initiated. In this particular project we would like to determine the structure of cellular nucleic acid binding protein (CNBP). This protein binds the non-coding strand of the CT element, thus the complimentary site to the hnRNPk binding site. CNBP upregulates the CT element activity. A construct of the full length (176residues) CNBP has been made. It consists of seven zinc fingers. A minimum construct of CNBP which still retains the nucleotide binding affinity is being probed. We have completed the NMR backbone dynamic studies of mutant(Gly26-Arg) KH3, wild type KH3, and KH3+ss-DNA complex. We have shown that there is no ss-DNA binding activity for the mutantKH3. We have also done titration study on the KH3+DNA complex to map the DNA binding site. At this point we have access to all dynamic parameters and map of the DNA binding site. We are currently comparing all of these parameters to characterize theKH3 ss-DNA interaction based on structure as well as dynamic information. We also have initiated a study on side chain dynamics using KH3as a model system. We have developed an experiment where we can probe NH2 moiety on the side chain of Gln and Asn. Comparison of the dynamic of this NH2 group in the free and bound form would provide information on side chain interaction with the ss-DNA target. This methodology will be extended to look at CH, CH2, andCH3 moieties in the protein.
| Xue, Qi; Nagy, Janice A; Manseau, Eleanor J et al. (2009) Rapamycin inhibition of the Akt/mTOR pathway blocks select stages of VEGF-A164-driven angiogenesis, in part by blocking S6Kinase. Arterioscler Thromb Vasc Biol 29:1172-8 |
