The ability of a single cell to grow, replicate its genetic material, and divide into two identical daughter cells is a vital process to ensure the propagation of all life. This process is known as the cell division cycle (cell cycle) and is one of the most highly spatially and temporally regulated cellular processes. Misregulation of the cell cycle, particularly in ways that confer both a proliferative advantage and escape from ultimate growth control mechanisms like cellular senescence or apoptosis, can result in unrestrained cell division and tumor formation. In this study, I utilize structural biology, biochemistry, and biophysical data to demonstrate a novel mechanism regulating Rb phosphorylation state and to map pairwise interactions in a conserved cell-cycle regulatory complex called the MuvB Core. First, competition between CDK and PP1 for Rb access provides an efficient regulatory mechanism to dephosphorylate Rb during mitotic exit and times of genotoxic stress. Second, data is presented mapping domain contacts in the MuvB Core, and structure/function studies with MuvB Core suggest an atypical function in regulation of chromatin structure.

The study of the cell cycle, specifically, understanding mechanistic regulation is fundamental for the development of therapeutic treatment in the event of disease. Limitless cell proliferation is a hallmark of cancer and often tied to cell cycle control. My research studied Cyclin- dependent kinases (Cdks) and their role in cell proliferation at the G1-S phase restriction point. I was able to shed insight on cyclin binding kinetics using Biolayer Interferometry (BLI) and the influence different types of inhibitors have on association of Cdks and cyclins. I reveal that Type I inhibitors increase binding affinity of CycA for Cdk2, whereas Type II inhibitors decrease affinity but differ in how they alter the CycA kinetic profile. Moreover, I explore an allosteric binding site of Cdk4-CycD that may be responsible for structural rearrangement to an active kinase configuration. My research as a whole gives insight into the development of a Cdk2 specific inhibitor and highlight the use of allosteric sites to understand mechanistic control of the cell cycle for Cdk inhibitor treatment therapy.

The cell cycle is essential for the growth and development of all multi-cellular organisms and is temporally regulated to coordinate processes such as cell size growth, organelle expansion, DNA replication, expression of mitotic proteins, chromatin condensation, and mitosis. Cell cycle proliferation is tightly regulated by a number of mechanisms including the transcription of G1/S and G2/M genes. In mammals, DREAM and Rb-E2F complexes are considered master regulators of the cell cycle and differentiation yet the molecular mechanisms underlying how these complexes activate and repress genes are still emerging. DREAM and Rb-E2F complexes are promoter dominant transcription factors that bind to target genes near their transcription start sites and modulate their expression. Evidence suggests that these transcription factors interact with chromatin and chromatin-modifying enzymes to alter the chromatin state in promoters. This work explores how these transcription factors engage with chromatin to modulate gene expression.

Intrinsically disordered transcription factor transactivation domains (TADs) function through structural plasticity, adopting ordered conformations when bound to transcriptional co-regulators. Many transcription factors contain a negative regulatory domain (NRD) that suppresses recruitment of transcriptional machinery through autoregulation of the TAD. We report the solution structure of an autoinhibited NRD-TAD complex within FoxM1, a critical activator of mitotic gene expression. We observe that while both the FoxM1 NRD and TAD are primarily intrinsically disordered domains, they associate and adopt a structured conformation. We identify how Plk1 and Cdk kinases cooperate to phosphorylate FoxM1, which releases the TAD into a disordered conformation that then associates with the TAZ2 or KIX domains of the transcriptional co-activator CBP. Our results support a mechanism of FoxM1 regulation in which the TAD undergoes switching between disordered and different ordered structures.

Eukaryotic cells coordinate the processes of cell growth and division through the activity of cyclin-dependent kinases (Cdks). Cdks generate immense signaling specificity and complexity by modifying hundreds of substrates via reversible phosphorylation. Many Cdk substrates are multiphosphorylated, but the mechanisms underlying the enzymatic activity of Cdks toward such substrates are not fully understood. Cks1 is a small essential protein that physically associates with Cdks, possesses a phosphate-binding pocket, and acts as both a positive and negative regulator of Cdk activity. Cks1 has also been extensively studied for its ability to form domain-swapped homodimers, the formation of which is controlled by two conserved proline residues. This dissertation presents research that combined x-ray crystallography, enzyme kinetics, and biophysical methods to demonstrate a direct role for Cks1 in targeting Cdks to multiphosphorylated substrates and develop a new hypothesis for the conservation of a proline residue that mediates domain-swapping in Cks1.

Inappropriate cell division and aberrant cell cycles are the hallmarks of cancer. Cyclin dependent kinases (CDKs) are the master regulators of the cell cycle. CDK activity drives cells to divide and suppression of CDK activity inhibits cell division. It is crucial to understand mechanisms of CDK regulation, as many cancers are correlated with inactivating mutations to CDK inhibitors and upregulation of CDK activators. This dissertation presents research that utilizes X-ray crystallography, kinetic assays, and various biochemical and biophysical methods to examine two different aspects of CDK regulation. The first aspect focuses on the small protein called Cyclin-dependent kinases regulatory subunit (Cks). We’ve shown that Cks confers specificity to CDK toward substrates via a phosphate-binding pocket. This specificity is required for multi-site phosphorylation of these substrates as well as in vivo viability. The second aspect investigates the mechanism of CDK activation by the atypical cyclin-like protein Speedy A. Using kinetic assays and X-ray crystallography we have elucidated the molecular source of CDK activation by Speedy. The structure shown shows how Speedy is a potent CDK activator whose expression overrides normal inhibitory cell cycle signaling. These findings reveal the molecular source of CDK regulation by Cks and Spy1 proteins and can inform the development of these proteins as prognostic markers for cancers and design of design of cancer therapeutics.