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Structure of KI67 FHA domain and its binding to HNIFK

Li, Hongyuan
http://rave.ohiolink.edu/etdc/view?acc_num=osu1060882032

Abstract Details

2003, Doctor of Philosophy, Ohio State University, Biochemistry.
The Ki67 protein is a 359 kDa nuclear protein whose existence is strictly associated with the cell cycle and has been used extensively as a marker for proliferating cells. The exact role of Ki67 has been elusive despite its great biological significance. The FHA domain has been reported to be a phosphopeptide recognition domain. In many cases, it has been shown to be a signal transduction module. In this study, the gene encoding the N-terminus FHA domain of Ki67 was cloned into an expression vector to express in Escherichia coli BL21(DE3) CodonPlus strain and the recombinant protein was purified. The binding specificity of this FHA domain was studied extensively using library screening and NMR titration methods. The structure of this domain was solved in collaboration with Dr. In-Ja Byeon using NMR. The phosphorylation site and binding property of its biological target protein hNIFK were studied. Unlike the FHA1 domain in Rad53, which can bind small phosphopeptides whose consensus sequence can be easily determined by library screening methods, the Ki67 FHA domain only shows binding interaction with phosphopeptides whose length is much greater than the local region near the phosphorylation site. This suggests that higher order structure within the binding target is required for productive binding by Ki67 FHA domain. While non-phosphorylated hNIFK(226-269) fragment can bind to Ki67 FHA domain weakly with a Kd ~106 µM, site-specific phosphorylation at T234 increases the affinity by more than 60-fold. HSQC titration analyses showed that this phosphorylation-dependent binding involves an interacting surface composed of a phospho-dependent binding center and additional phospho-independent contacts. The coordination of these two regions may be key to understanding the specific, tight binding of Ki67 FHA domain to hNIFK. The results presented here provide the first example of an in vitro study of the binding of FHA domain to its biological target. It shows that while the binding pattern of FHA domain around phospho-interacting center can be similar to those demonstrated by complex structures using small synthetic phosphopeptides from library screening, additional selectivity and/or functionality can be provided through additional contacts.
Ming-Daw Tsai (Advisor)
FHA domain; NMR structure; protein-protein interaction; phospho-protein recognition

Recommended Citations

Citations

  • Li, H. (2003). Structure of KI67 FHA domain and its binding to HNIFK [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1060882032

    APA Style (7th edition)

  • Li, Hongyuan. Structure of KI67 FHA domain and its binding to HNIFK. 2003. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1060882032.

    MLA Style (8th edition)

  • Li, Hongyuan. "Structure of KI67 FHA domain and its binding to HNIFK." Doctoral dissertation, Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=osu1060882032

    Chicago Manual of Style (17th edition)

osu1060882032
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© 2003, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.