Protein Structure, Stability, and Amyloid Formation

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Below, we provide a few examples of our accomplishments in this project. In the first, cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) are key regulators of the G1-S phase transition in the cell cycle. In cancer cells, CDK6 overexpression often outcompetes CDK4 in driving cell cycle progression, contributing to resistance against CDK4/6 inhibitors (CDK4/6i). This suggests distinct functional and conformational differences between these two kinases, despite their striking structural and sequence similarities. Understanding the mechanisms that differentiate CDK4 and CDK6 is crucial, as resistance to CDK4/6i-frequently linked to CDK6 overexpression-remains a significant therapeutic challenge. Notably, CDK6 is often upregulated in CDK4/6i-resistant cancers and rapidly proliferating hematopoietic stem cells, underscoring its unique regulatory roles. We hypothesize that their distinct conformational dynamics explain their differences in phosphorylation of retinoblastoma protein, Rb, inhibitor efficacy, and cell cycle control. This leads us to question how their dissimilar conformational dynamics encode their distinct actions. To elucidate their differential activities, molecular mechanisms, and inhibitor binding, we combine biochemical assays and molecular dynamics (MD) simulations. We discover that CDK4 and CDK6 have distinct allosteric networks connecting the beta3-alphaC loop and the G-loop. CDK6 exhibits stronger coupling and shorter path lengths between these regions, resulting in higher kinase activity upon cyclin binding and impacting inhibitor specificity. We also discover an unrecognized role of the unstructured CDK6 C-terminus, which allosterically connects and stabilizes the R-spine, facilitating slightly higher activity. Our findings bridge the gap between the structural similarity and functional divergence of CDK4 and CDK6, advancing the understanding of kinase regulation in cancer biology. The second relates to ERK, a key kinase in the MAPK pathway. ERK, a coveted proliferation drug target, is a pivotal kinase in the Ras/ERK signaling cascade. Despite this, crucial questions about its activation have not been fully explored on the foundational, conformational level. Such questions include (i) Why ERK's activation demands dual phosphorylation; (ii) What is the role of each phosphorylation site in the activation loop; and (iii) Exactly how the (ordered) phosphorylation steps affect the conformational ensembles of the activation loop, their propensities and restriction to a narrower range favoring ERK's catalytic action. Here we used explicit molecular dynamics simulations to study ERK's stability and the conformational changes in different stages along the activation process. The initial monophosphorylation event elongates the activation loop to enable successive phosphorylations, which reintroduce stability/compactness through newly formed salt bridges. The interactions formed by monophosphorylation are site-dependent, with threonine's phosphorylation presenting stronger electrostatic interactions compared to tyrosine's. Dual phosphorylated ERKs revealed a compact kinase structure which allows the HRD catalytic motif to stabilize the ATP. We further observe that the hinge and the homodimerization binding site responded to a tri-state signaling code based solely on the phosphorylation degree (unphosphorylated, monophosphorylated, dual phosphorylated) of the activation loop, confirming that the activation loop can allosterically influence distant regions. Last, our findings indicate that threonine phosphorylation as the second step is necessary for ERK to become effectively activated and that activation depends on the phosphorylation order. Collectively, we offer ERK's dual allosteric phosphorylation code in activation and explain why the phosphorylation site order is crucial. The third example relates to mTOR and its pharmacology. mTOR plays a crucial role in PI3K/AKT/mTOR signaling. We hypothesized that mTOR activation mechanisms driving oncogenesis can advise effective therapeutic designs. To test this, we combined cancer genomic analysis with extensive molecular dynamics simulations of mTOR oncogenic variants. We observed that conformational changes within mTOR kinase domain are associated with multiple mutational activation events. The mutations disturb the alpha-packing formed by the kalphaAL, kalpha3, kalpha9, kalpha9b, and kalpha10 helices in the kinase domain, creating cryptic pocket. Its opening correlates with opening of the catalytic cleft, including active site residues realignment, favoring catalysis. The cryptic pocket created by disrupted alpha-packing coincides with the allosteric pocket in PI3Kalpha can be harmoniously fitted by the PI3Kalpha allosteric inhibitor RLY-2608, suggesting that analogous drugs designed based on RLY-2608 can restore the packed alpha-structure, resulting in mTOR inactive conformation. Our results exemplify that knowledge of detailed kinase activation mechanisms can inform innovative allosteric inhibitor development.