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-element upstream from the c-myc promoters is the CT element. This is a CT rich sequence which is located 100 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 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 this has been determined by NMR spectroscopy using the liquid crystal technique in addition to the conventional protein NMR method. The liquid crystal environment produces a slight order in the system which reintroduces dipolar coupling providing 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, calculations carried out without the dipolar couplings has an RMSD of 0.32 Angstrom. These provide a quantitative evaluation of the precision of the structures with the smaller RMSD value implying higher precision. This comparison was made under ideal conditions: good signal to noise for all of the NMR experiments, some NMR experiments repeated twice for error estimates as well as consistency check, and all distance estimates 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 straight forward 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. One flexible loop (L52-R56) was identified in the KH3 domain which undergoes rapid (ps) fluctuation. In contrast to a previous study of a similar KH domain of FMR1 the conserved loop (G30-G33) does not show any flexibility. In order to get a better understanding of c-myc regulation through the CT element, a parallel project has been initiated to determine the structure of the cellular nucleic acid binding protein (CNBP). This protein binds the strand opposite to the hnRNPk binding site. CNBP upregulates the CT element activity. A construct of the full length (176 residues) 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 mutant KH3. We have also done titration studies on the KH3+DNA complex to map the DNA binding site. At this point, we have access to all dynamic parameters and maps of the DNA binding site. We are currently comparing all of these parameters to characterize the KH3 ss-DNA interaction based on structure as well as dynamic information. We also have initiated a study on side chain dynamics using KH3 as a model system. We have developed an experiment where we can probe the NH2 moiety on the side chain of Gln and Asn. Comparison of the dynamics 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, and CH3 moieties in the protein. This last year we have been trying to carry out experiments to identify inter-domain motions in KH3-containing protein as well as to finish our project on the side chain motion in KH3 of hnRNPk. In order to study the inter-domain motions we have used a model system that has been studied extensively to test our analysis model. The model system chosen was a calcium binding protein, calmodulin. We have been able to confirm that in order to quantify slow inter-domain motions a series of NMR backbone relaxation data obtained at different field strengths would be required. Furthermore, our simple model that represents a first order approach to the problem, seems to be adequate to provide the amplitude as well as the time scale of this motion. The estimated amplitude of motion has been confirmed by sterically modeling the domain motion in calmodulin. We are now applying this simple motional model to study KH3 motion. We have refined the motional model that can be used to describe slow internam domain motions in protein. Our latest model use a Pade approximation of wobbled in a cone which results in a description of the internal motion by three exponential terms. So far our tests have shown this model to be a more suitable model when the motion is large (angle of inflection of 50 degrees or larger). In the limit of small amplitude slow motion the earlier and mathematically simpler model is sufficient.
| 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 |
