Distinct allosteric networks in CDK4 and CDK6 in the cell cycle and in drug resistance

Abstract

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 β3-αC 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.

Keywords: CDK inhibitor; G1 cell cycle phase; G1/S transition; allosteric drug discovery; cancer; cyclin-dependent kinases (CDKs); ligand binding.

Figure 1.. CDK4 and CDK6 domain structures and sequence alignments.

(A) Cell cycle and G₁-S phase signaling pathway. CDK4 and CDK6 are essential in regulating the G₁ phase of the cell cycle. When complexed with cyclin-D (Cyc D), they phosphorylate Rb, partially releasing E2F transcription factors to induce cyclin-E expression. Cyclin-E/CDK2 then completes Rb phosphorylation, fully releasing E2F, and driving cells into the S phase. The pathway shows the cooperative roles of CDK4/6 and CDK2 in promoting cell cycle progression through a positive feedback loop. (B) Domain structure of CDK4 and CDK6; both CDK4 and CDK6 share a kinase domain but differ in their N-terminal αB-helix and C-terminal αJ-helix regions. (C) Structural alignment of CDK4 and CDK6. The 3D structures of CDK4 (cyan, PDB ID: 7SJ3) and CDK6 (pink, PDB ID: 1XO2) are superimposed, revealing overall similarity with some differences in specific regions. The red circle highlights the β3-αC loop, where structural divergence is observed. (D) Sequence alignment of CDK4 and CDK6 highlights conserved residues (purple background) and divergent residues (white background). Key secondary structures, including α-helices (αB, αC, αD, etc.) and β-strands (β1, β2, etc.), are annotated. Differences in the N-terminal αB helix and C-terminal αJ helix are marked with orange boxes, while variations in the β3-αC loop are emphasized with dashed boxes, corresponding to structural discrepancies shown in panel C. CDK6 contains seven additional residues in the N-terminus and 23 extra residues in the C-terminus, including the αJ helix, which is absent in CDK4.

Figure 2.. CDK6^WT is more active than CDK4^WT, in part due to its lower local dynamics in β3-αC and β4-β5, and G-loops.

(A) Normalized kinase activity of cyclin D1-CDK6^WT and cyclin D1-CDK4^WT fusions toward RbC. (N = 5–6; ****P<0.0001) (B) Tube representations of the first principal component (PC1) from PCA of CDK4^WT and CDK6^WT. Color gradients represent mode mobility, from immobile (blue) to highly mobile (red). Notable dynamic regions include the β3-αC, β4-β5, and β2-β3, and G-loops. ATP is shown bound in the active site. The binding cyclins are not displayed for visual clarity. (C) PCA plot for the first and second principal component (PC1 and PC2) of CDK4^WT and CDK6^WT, show distinct clustering patterns. For CDK4^WT, PC1 and PC2 account for 27.78% and 11.23% of the total variance, respectively. For CDK6^WT, PC1 and PC2 explain 23.88% and 10.45% of the total variance, respectively. Each dot represents one snapshot from the trajectories in the plot. The color ranging from blue to red represents the trajectory frame from beginning to end. (D) PC1 loading plots for CDK4^WT and CDK6^WT. The fluctuation profiles identify regions of high conformational variability, particularly in the β3-αC and β4-β5 loops. β3-αC loop has more significant contribution to the PC1 than that of CDK6. (E) Dynamic cross-correlation maps for CDK4^WT and CDK6^WT. Positive correlations (magenta) and anti-correlations (cyan) highlight coupled motions between specific regions.

Figure 3.. Longer β3-αC loop of CDK4 is the key in the binding interface between CDK4 and cyclin-D and decouples N-lobe β-strands and αC-helix, negatively regulating their activities.

(A) Sequence alignment of the β3-αC loop region in CDK4 and CDK6. This panel shows the β3-αC loop of CDK4 has a significantly longer sequence of glycine residues than CDK6. (B) Structural overlay of flexible β3-αC loop region from the trajectories of CDK4^WT. The overlay demonstrates the highly mobile nature of the β3-αC loop in CDK4^WT. (C) Binding free energy (ΔG^CycD) of cyclin-D with CDK4^WT and CDK6^WT. The bars represent the calculated ΔG for the binding of cyclin-D to wild-type CDK4 (left, blue) and wild-type CDK6 (right, purple). More negative values indicate stronger binding, with error bars representing the standard deviations. Community network of (D) cyclin-D/CDK4^WT and (E) cyclin-D/CDK6^WT complexes. In the CDK4 complex (D), the G-loop and αC-helix are in different communities, whereas in the CDK6 complex (E), they are in the same community. Different colors of the cartoon representation of CDKs represent distinct communities. (F) Normalized kinase activity of cyclin D1-CDK6^WT and cyclin D1-CDK6^{β3-αC(CDK4)} fusions toward RbC. (N = 6; ****P<0.0001) (G) Probability of β3-Lys and αC-Glu salt bridge formation. This bar graph shows the higher probability of the formation of the β3-Lys and αC-Glu salt bridge in CDK6^WT compared to CDK4^WT. The higher probability in CDK6^WT suggests more stable active conformation. (H) Snapshot highlighting the Lys-Glu salt bridge in CDK6^WT. This conformation is characteristic of the active conformation of kinases.

Figure 4.. Allosteric signaling pathways between the β3-αC loop and the G-loop.

Optimal (red lines) and sub-optimal (blue lines) allosteric paths for (A) CDK4^WT, and (B) CDK6^WT. The allosteric signaling pathway starting from the β3-αC loop to G-loop of CDK4 follows a longer path than that of CDK6. The green spheres are α-carbon of residues and represent nodes in the allosteric pathways. (C) Schematic illustration of the optimal allosteric paths and (D) path length distribution between the source and sink residues for CDK4^WT. (E) Schematic illustration of the optimal allosteric paths and (F) path length distribution between the source and sink residues for CDK6^WT. Shorter path length suggests a stronger allosteric pathway between the source and sink. Overlay of simulation trajectories for (G) CDK4^WT and (H) CDK6^WT. G-loop (green) of CDK4 is longer than that of CDK6 and is more dynamic. (I) IC₅₀ values for palbociclib and atirmociclib mediated inhibition of cyclin D1-CDK6^WT (11.17 ± 1.51 nM and 79.69 ± 13.23 nM) and cyclin D1-CDK6^{β3-αC(CDK4)} (15.22 ± 4.66 nM and 12.18 ± 5.84 nM). (N = 3; **P<0.005)

Figure 5.. C-terminus modulates the C- and N-lobe opening and closing of CDK6 facilitating more efficient catalysis.

Dynamic cross-correlation maps of residue for (A) CDK6^WT and (B) CDK6^ΔCterm. The maps use a color scale from cyan (−1.0, anti-correlated) to magenta (+1.0, correlated) to show how different protein residues move in relation to each other. A key difference is highlighted in the circled region, showing distinct correlation patterns between N-lobe (residues 1–100) and C-lobe (residues >100) movements in the two kinases. Tube representations of the most correlated and anti-correlated motion in (C) CDK6^WT and (D) CDK6^ΔCterm. These panels show the regions of CDK6^WT and CDK6^ΔCterm with the most correlated (positive correlations, red) and anti-correlated (negative correlations, blue) motions, showing how the truncation of the C-terminus alters these dynamics. (E) Normalized kinase activity of cyclin D1-CDK6^WT and cyclin D1-CDK6^ΔCterm fusions toward RbC. (N = 6; ****P<0.0001) (F) Structural community analysis of CDK6^WT and CDK6^ΔCterm focusing on the regulatory spine (R-spine). In CDK6^WT, the R-spine residues (L79, L65, F164, H143) form a single cohesive community (orange), while in CDK6^ΔCterm, these residues are dispersed across three different communities (colored differently), suggesting compromised structural integrity. Allosteric pathway analysis in (G) CDK6^WT and (H) CDK6^ΔCterm. The graphs trace the communication pathway (highlighted in various colors) from the source residue E308 in the C-terminal region to the sink residue H143 in the R-spine. CDK6^WT shows a direct pathway through the αE-helix, while CDK6^ΔCterm exhibits a longer route through multiple helices (αH and αF), indicating altered internal dynamics due to C-terminal truncation.

Figure 6.. CDK4 and CDK6 dynamic divergence gives rise to their distinct activity.

(A) Schematic of cell cycle showing CDK-cyclin complex activity at different phases (G₁, S, G2, M). Gradient scales show that CDK4^WT exhibits higher flexibility but lower phosphorylation rate, while CDK6^WT shows lower flexibility but higher phosphorylation rate. This inverse relationship between flexibility and catalytic efficiency reflects the evolutionary adaptation of CDK6 for rapid cell division in stem-like cells like ST-HSC cells. (B) Structural comparison of CDK4^WT and CDK6^WT highlighting key differences in their regulatory mechanisms. CDK4^WT has a longer β3-αC loop that results in weak allosteric coupling between αC-helix and the G-loop, while CDK6 has a shorter β3-αC loop with a stronger allosteric coupling, and its C-terminus stabilizes the R-spine for more efficient phosphorylation.