Team:NUDT CHINA/Basic Parts
BASIC PARTS
CDK4 αCHelix (BBa_K4016009)
Our project this year is centered on the degradation of the cyclins. The mammalian cell cycle is driven by a complex of cyclins and their associated cyclin-dependent kinases (CDKs). The activation of CDK4 is a key to influence the cell’s division in G1 of cell proliferation in many cell types1. CDK4 and its close homolog CDK6 are serine/threonine kinases that form heterodimers with D-type cyclins D1, D2, and D3 (cyclin D) and are central regulators of G1–S transition2.
This part is the αCHelix domain CDK4. CDK4 is the known endogenous binding parter of Cyclin D1. Reports have shown that CDK4 binds Cyclin D1 via its C terminus α Helix domain3. Therefore, we removed other protein domains of cyclin D1 to only obtain the binding afinity. In addition, to improve binding affinity, our part contains two copies of CDK4 αCHelix linked with 5xGS Linker.
Figure 1. Schematic diagram of peptide chain structure of CDK4 and the mechanism of how CDK4 αCHelix achieve its function on targeting cyclinD1
Our favourite part BBa_K4016009 is an important member of our functional parts. Based on its feature on combination with cyclin D specifically, we fused it with CIB1 (2×CDK4 αCHelix-CIB1, BBa_K4016027) and Coh2 (2×CDK4 αCHelix-Coh2,BBa_K4016028) to act as the target module of the cyclinD, achieving precisely inhibiting the G1–S cell-cycle transition by using blue-light to regulate the degradation process.
Figure 2. Schematic representation of the CDK4 αCHelix’s function on targeting cyclinD1 in the CycleBlue System
Research shows the cyclin D-Cdk4,6 phosphorylates and inhibits Rb via a C-terminal helix and that this interaction is a major driver of cell proliferation. Considering other protein domains in CDK4 may play unpredictable roles, the CDK4 was truncated to maintain only its αCHelix to exclude the effects of other factors as far as possible.
Our part contains two copies of CDK4 αCHelix (to ensure the expression of CDK4 and remove the influence of other parts of CDK4 on the experiment) and two copies of GS Linker (5xGS Linker & 2xGS Linker).
| 01 | BBa_K4016000 | PixE | 02 | BBa_K4016001 | PixD | 03 | BBa_K4016002 | Bcl-xl |
| 04 | BBa_K4016003 | LD3 | 05 | BBa_K4016004 | asLOV2 | 06 | BBa_K4016005 | Zdk |
| 07 | BBa_K4016006 | VH_cyclinE1 | 08 | BBa_K4016007 | VK_cyclinE1 | 09 | BBa_K4016008 | ScFv_cyclinE1 |
| 10 | BBa_K4016009 | 2×CDK4 αCHelix | 11 | BBa_K4016010 | 2×Rb αChelix | 12 | BBa_K4016011 | TCE |
| 13 | BBa_K4016038 | 3×GSlinker | 14 | BBa_K4016039 | 5×GSlinker |