An emerging theme in structural biology is that disordered proteins play important roles in biological systems. For example, some disordered proteins adopt structure during important signaling or regulatory events. The structural determinants of these events, however, are unknown, precluding the correlation of structure and function. The cyclin-dependent kinase (Cdk) inhibitor, p21, is a regulatory target of two important tumor suppressors, p53 and BRCA1. In humans, p53-dependent tumor suppression involves activation of p21 that causes cell cycle arrest. We have shown that p21, and a related protein named p27, are dynamically disordered in solution. Despite this, p21 and p27 bind to cyclin/Cdk complexes, the timekeepers of the cell cycle, with high affinity and specificity. In the past, p21 and p27 were considered to be universal inhibitors of Cdks; recent studies, however, have shown that p21 and p27 inhibit only a subset of Cdks (i.e. Cdk2/cyclin A) and that they stabilize and activate others (i.e. Cdk4/cyclin D). One important aim of our studies is to uncover the physical basis for the dual functions of p21 and p27 through studies of protein structure and dynamics using NMR spectroscopy and studies of binding thermodynamics using isothermal titration calorimetry (ITC). The PI's laboratory has shown using NMR that p21 and p27 possess a transiently populated -helix in solution - the """"""""linker helix"""""""" - and suggests that this structural feature is an important determinant of function. This hypothesis will be tested using protein engineering to both stabilize and destabilize the """"""""linker helix"""""""" followed by the determination of binding parameters and activity. Future studies with p21 homologs will determine whether this structural feature is evolutionarily conserved. Also, NMR studies will be extended to p21 and p27 within cyclin/Cdk complexes to uncover the structural determinants of Cdk inhibition versus activation. Finally, ITC and other techniques are being used to elucidate the structural and thermodynamic basis for the specificity of p21 and p27 for cell cycle Cdks. This work is important because p21 and p27 regulate the cell growth arrest mechanism that is most often disrupted in human cancer and because the relationship between the structure and function of these proteins is not well understood.
| Phillips, Aaron H; Ou, Li; Gay, Alexandre et al. (2018) Mapping Interactions between p27 and RhoA that Stimulate Cell Migration. J Mol Biol 430:751-758 |
| Follis, Ariele Viacava; Llambi, Fabien; Kalkavan, Halime et al. (2018) Regulation of apoptosis by an intrinsically disordered region of Bcl-xL. Nat Chem Biol 14:458-465 |
| Ragavan, Mukundan; Iconaru, Luigi I; Park, Cheon-Gil et al. (2017) Real-Time Analysis of Folding upon Binding of a Disordered Protein by Using Dissolution DNP?NMR Spectroscopy. Angew Chem Int Ed Engl 56:7070-7073 |
| Ban, David; Iconaru, Luigi I; Ramanathan, Arvind et al. (2017) A Small Molecule Causes a Population Shift in the Conformational Landscape of an Intrinsically Disordered Protein. J Am Chem Soc 139:13692-13700 |
| Banerjee, Priya R; Mitrea, Diana M; Kriwacki, Richard W et al. (2016) Asymmetric Modulation of Protein Order-Disorder Transitions by Phosphorylation and Partner Binding. Angew Chem Int Ed Engl 55:1675-9 |
| Das, Rahul K; Huang, Yongqi; Phillips, Aaron H et al. (2016) Cryptic sequence features within the disordered protein p27Kip1 regulate cell cycle signaling. Proc Natl Acad Sci U S A 113:5616-21 |
| Grace, Christy R; Ban, David; Min, Jaeki et al. (2016) Monitoring Ligand-Induced Protein Ordering in Drug Discovery. J Mol Biol 428:1290-1303 |
| Csizmok, Veronika; Follis, Ariele Viacava; Kriwacki, Richard W et al. (2016) Dynamic Protein Interaction Networks and New Structural Paradigms in Signaling. Chem Rev 116:6424-62 |
| Huang, Yongqi; Yoon, Mi-Kyung; Otieno, Steve et al. (2015) The activity and stability of the intrinsically disordered Cip/Kip protein family are regulated by non-receptor tyrosine kinases. J Mol Biol 427:371-386 |
| Follis, Ariele Viacava; Llambi, Fabien; Merritt, Parker et al. (2015) Pin1-Induced Proline Isomerization in Cytosolic p53 Mediates BAX Activation and Apoptosis. Mol Cell 59:677-84 |
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