Date of Award

5-2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Molecular Sciences

Research Advisor

Richard Kriwacki, Ph.D.

Committee

Seema Khurana, Ph.D. Susan Senogles, Ph.D. Stephen White, D.Phil Jie Zheng, Ph.D.

Keywords

p27, cyclin-dependent kinase inhibitors, isothermal titration calorimetry, intrinsically unstructured proteins, cell cycle, thermal denaturation, nascent helix, entropic cost, cell cycle arrest, circular dichroism

Abstract

p27kip1 binds to and regulates the activity of cyclin-dependent kinases (Cdks) which are the master timekeepers of the cell division cycle. Members of the p27 family of proteins, also including p21 and p57, are called cyclin-dependent kinase inhibitors (CKIs). The amino terminal domain of p27 inhibits Cdk activity and is referred to as the kinase inhibitory domain (KID). The KID is comprised of a cyclin-binding domain (D1) and a Cdk binding domain (D2) joined by a 22 residue linker domain (LH). Structural analysis of the KID in solution before binding its Cdk targets revealed that D1 and D2 are largely unstructured and that the linker domain exhibits nascent helical characteristics. The nascent helical structure of the linker domain is conserved amongst the CKI proteins and I hypothesize that it is an important determinant of their functional properties. To test this hypothesis, I probed the interactions of peptides corresponding to D1 and D2 (LH domain deletion mutant) with the Cdk2/cyclin A complex. Results from isothermal titration calorimetry (ITC) and kinase inhibition assays, show that the interaction and of either D1 or D2 with the Cdk2/cyclin A complex is less favorable relative to that of the p27-KID and that the D2 peptide cannot completely inhibit Cdk2/cyclin A kinase activity. These results indicate that the LH domain that couples D1 and D2 is necessary for the function of p27 as a Cdk inhibitor.

Despite having a conserved structure, the linker domains of the Cip/Kip proteins do not have a conserved sequence. However, the sequence of this domain in each Cip/Kip protein in different species is well conserved suggesting that sequence may play a role in the function of these proteins. I explored this sequence-divergence/structure-conservation relationship of the linker domains of the Cip/Kip proteins by constructing chimeric p27-KID molecules in which the p27 linker domain was replaced with the corresponding segments of either p21 or p57. ITC and kinase inhibition assay results show that the chimeric molecules bind and inhibit Cdk2/cyclin A in a manner similar to that of p27-KID. Moreover, thermal denaturation studies show that the complexes formed by these proteins bound to Cdk2/cyclin A have comparable melting temperatures. Taken together, these results indicate that the different linker domains, despite their structural differences, play similar structural roles in p27 binding to Cdk2/cyclin A.

In a third study, I investigated the consequences of perturbing the conserved structure of the linker domain. I successfully designed and prepared p27 variants in which domain LH was either more or less helical with respect to the wild-type protein. The secondary structural properties of the mutants were characterized by circular dichroism spectropolarimetry (CD). Thermal denaturation experiments showed that the ternary complexes of the p27 variants bound to Cdk2/cyclin A were less stable compared to the ternary complex formed by wild- type p27(p27-KIDwt). Thermodynamic analysis showed that there was a decrease in the enthalpy of association of the mutants with Cdk2/cyclin A relative to p27-KIDwt. The free energies of binding varied within a much narrower range. In addition, in vitro Cdk2 inhibition assays showed that the p27 variants exhibited disparate inhibitory potencies. Further, when we over-expressed the p27 variants in mouse fibroblasts, they were less effective in causing cell cycle arrest relative to wild-type p27. These results indicate that the conserved structure of the linker domain is important for p27 function. It seems that nature has selected the sequence of the p27 LH domain to be partially structured and that bolstering or eliminating this structure is deleterious to function; intrinsic structure is critical for function.

DOI

10.21007/etd.cghs.2007.0231

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