Specificity and structural modeling of FHA domain of Chk2 and a general characterization of FHA domain of Caenorhabditis elegans Chk2.

摘要

This work combines the use of biochemical and structural modeling approaches to characterize the FHA domains from human Chk2 and Caenorhabditis elegans Chk2 proteins.; The DNA damage checkpoint pathway is vital to the maintenance of genomic integrity. Chk2 plays key roles on this pathway by phosphorylating several important cell cycle control modulators, such as tumor suppressor p53. The exact functions of the FHA domain of Chk2 are still not clearly known, while it has been generally considered as the regulator to the Chk2 kinase activities. In this dissertation, efforts have been put to express and purify the unstable Chk2 FHA domain. After fine tuning the conditions, great improvements have been observed on the NMR spectrums, which are the essentials for the future structural work. Well-resolved 2D and 3D spectra have been obtained. Seeking to identify binding substrates for Chk2 FHA domain, combinatorial peptide library screenings have been utilized to determine the consensus sequence of the target phosphopeptides. Chk2FHA was found to bind to phosphotyrosyl peptides containing pYXXXL motif. A phosphopeptide from p53 centered by (pY 107)GFRL was identified with mild affinity (K_D = 6.1μM) to Chk2 FHA domain. Chk2 complex structure with this pY-peptide was proposed by structural modeling. It features similar global structure as the recently solved Chk2FHA-pT-peptide complex, while the interaction details between Chk2FHA and phosphopeptides might be different. The consensus sequence identified by pT-peptide library screening displays strong selection of Ile at the +3 position. A phosphopeptide with sequence containing pT329LQI from p53 was shown to bind to Chk2FHA (K_D = 11.7 μM) and perturb its global structure, though the physiological impact of this binding event still needs to be elucidated. Another pT-peptide containing pT1851 YLI from tumor suppressor BRCA1 has also been investigated and it exhibits decent affinity (K_D = 1.2 μM) to Chk2FHA.; C. elegans Chk2 was recently discovered and very little is known about its functions. Ce-Chk2 has been demonstrated to play key roles in meiotic recombination, while it conserves the function of a DNA damage checkpoint at the pachytene stage. Investigation on Ce-Chk2 will shed more light on the properties of the Chk2 family. While no report on Ce-Chk2 at the protein level has been published to date, we have successfully expressed the full-length, FHA domain and kinase domain of Ce-Chk2 in E. coli . Purification of Ce-Chk2 FHA domain was achieved and combinatorial peptide library screenings were carried out to search for the binding targets of Ce-Chk2FHA. It was found that Ce-Chk2FHA, similar to Chk2FHA, strongly selects Leu at the +4 position in pY-peptides. The pT- and pS-peptide libraries have also been screened.