Browsing by Subject "Kinase"
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Item On The Allosteric Mechanisms Of Paradoxical Activation By Raf Inhibitors(2024-04) Rasmussen, DamienThe type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.Item On the Role of Allosteric Cooperativity in the Regulation of Protein Kinase A and its Implications in Disease(2021-05) Walker, CaitlinFirst articulated half a century ago, allostery has remained a universal phenomenon and is essential in understanding processes beyond the molecular level, such as cellular signaling and disease. Allostery also referred to as allosteric regulation, is a process by which biological macromolecules transmit the effect of binding at one site to an often distal, functional site, allowing for regulation. To facilitate the modulation of function between sites, allosteric signal is propagated through conserved amino acid residues, often comprising various structural elements of a protein. In general, allosteric communication is of fundamental interest and potentially of high relevance for drug design and protein engineering. Furthermore, the dysfunction of allosteric networks has been implicated in the etiology of human diseases. However, defining these networks of residues that mediate crosstalk between distal sites remains experimentally challenging and thus, poorly characterized. Since allosteric signal propagation relies on subtle conformational rearrangements, nuclear magnetic resonance (NMR) has emerged as an instrumental tool in investigating allosteric communication. This thesis aims to map allosteric networks at atomic resolution to understand how mutations in protein kinase A (PKA) influence allosteric communication to elicit the progression of various disease states. In this work we demonstrate how disease mutations associated with Cushing’s Syndrome and Fibrolamellar Hepatocellular Carcinoma attenuate the allosteric network of PKA, thereby disrupting the finely tuned regulation, specificity, and activation of PKA to generate dysfunctional signaling. The findings of this thesis provide critical insights into the importance of intramolecular allostery in facilitating functional signaling, directly showing how changes in allosteric networks of proteins lead to dysfunction.