Cell division control protein 4/Suppressor of kinetochore protein 1 created by Jacob Davis
Cell division control protein 4/Suppressor of kinetochore protein 1(Cdc4/Skp1; pbd ID = 3MKS) of Saccharomyces cerevisiae is an essential E3 ubiquitin ligase that plays a key role in the final stage of the proteolysis of the cyclin dependent kinase (CDK) inhibitor Sic1. Cell cycle progression is highly dependent on the elimination of CDK inhibitors. For example, Sic1 degradation is necessary for entrance into the S phase. Ubiquitination and proteolysis of Sic1 must be properly timed in order for the appropriate cells to begin DNA replication. (1) This class of proteins can exert control over the cell cycle, signal transduction, transcription and development with its ability to monitor the intracellular concentrations of molecules. (2) E3s provide both stability and specificity to the ubiquitination reaction. Recent studies have shown the importance of investigating this protein in yeast, as there are many structural similarities between it and the human equivalent, Fbw7. Allosteric inhibition of the protein has potential for drug therapy. (3) The molecular weight is 144,491.65 Da and its isoelectric point (pI) is 5.77. (4)
Cdc4/Skp1 is divided into four subunits and exists as a heterotetramer. The secondary structure is comprised of 21% α-helices, 33% β-sheets and roughly 45% random coils. Units A and C are identical and represent Skp1, a polypeptide extending 169 residues that is primarily helical, consisting of 8 helices including 40% of the residues and 7% β-sheets. Units B and D represent Cdc4, which extends 464 residues and contains 15% helices and 43% β-sheets. (3) In complex, Skp1 is referred to as the adaptor protein, and Cdc4 as the scaffold protein. Units B and D are divided into three distinct domains: 1) a WD40 β-propeller, 2) a helical linker, and 3) the F Box. The WD40 domain covers residues 373-744 and serves as the primary active site. The helical linker consists of two helices separated by random coils that span residues 324-372 and serves as the bridge between WD40 and the F Box. The F box contains residues 270-323 and is responsible for binding Skp1 to Cdc4. (5) Subunits A and C represent invariable components essential for SCF complex formation. (6)
The helical linker, which is comprised of two helices (α5 and α6), plays a critical role in the functionality of Cdc4/Skp1 by bridging the WD40 domain to the F box. The domain expresses moderate flexibility, but is invariant at helix α6. The N-terminus of α6 has extensive hydrophobic interactions with the F Box between Phe-335 and Leu-356 of α6 and Ile-295, Ile-296, Leu-315, Trp-316 and Leu-319 of the F Box. The C-terminus is anchored between the seventh and eighth blade of the β-propeller. Hydrophobic interactions exist between Trp-365 and Ile-361 of α6 and Val-687, Ile-696, Leu-276 and Phe-743 of the WD40 domain. The C-terminus is further routed to the propeller through a single hydrogen bond between Asn-364 and Phe-743. (2)
TheF Box domain acts as the bridge between the Cdc4 and Skp1. It is composed of 5 α-helices. α0 to α3 of the F Box domain of Cdc4 associate with α5 to α8 of Skp1. F Box domains often consist of roughly 40-50 residues and tend to appear on proteins containing WD40 repeats. Leu-278, Pro-279, Ile-286, Leu-290 and Trp-316 are critical residues for direct Skp1 binding. (5)
The WD40 domain is the primary active site of Cdc4/Skp1. It consists of 8 copies of 40-residue segments ending in Trp-Asp (WD) arranged as a β-propeller. Each repeating segment extends from the outer band of one blade to the inner 3 blades of the adjacent blade. Each blade consists of 4 antiparallel β-strands. The top of the propeller serves as the Cdc4 phosphodegron (CPD) binding site, and the bottom is linked to the F Box domain via the helical linker. The propeller recognizes the core motif of Sic1, Ile/Leu-Ile/Leu-pThr-Pro. The P0 pocket hydrogen bonds to the pThr residue of Sic1 at Tyr-548 and form guanadinium bonds to Arg-534, Arg-462 and Arg-485. The three arginine residues are essential for Cdc4/Skp1 function. The P+1 pocket houses the Pro of Sic1 and is stabilized from behind by hydrogen bonding between Arg-485 and the C-terminal Proline of Sic1. The P-1 and P-2 pockets show strong preference for hydrophobic residues, and are characterized by the invariant residues Trp-426, Trp-717, Thr-386 and Tyr-574. (2) It should also be noted that 6 of the 9 CPD sites on Sic1 must be phosphorylated in order to achieve sufficient affinity for Cdc4. (1)
As the most essential pocket of the binding complex, P0 must be properly oriented to receive Sic1. It is possible to target this pocket as a means of drug therapy with 1-(2-carboxynaphth-1-yl)-2-napthoic acid, SCF-I2. SCF-I2 contains two naphthalene rings and two carboxyl groups. The top ring undergoes hydrophobic interactions with Leu-628, Ile-594, Leu-634, Trp-657 and Ala-649. The top carboxyl hydrogen bonds to the nitrogen of Trp-657. The bottom ring stacks with Arg-664 and has van der Waal’s interactions with Ser-667 and Arg-655. The bottom carboxyl hydrogen bonds with Arg-655 and His-631. Each of these interactions critically alter the orientation and functionality of both the P0 and P-2 pockets, compromising the ability of Cdc4/Skp1 to bind Sic1. (3)
SCFbeta-TrCP1 ubiquitin ligase (pdb ID =1P22) is an E3 that is present in the destruction process in Homo sapiens. Results of the DALI search (Z=16.2) indicate similar tertiary structure between SCFbeta-TrCP1 and Cdc4/Skp1. (7) The BLAST search (E=7e-87) indicated primary structure similarities between SCFbeta-TrCP1 and Cdc4/Skp1. (8) The WD40 β-propeller is conserved between the two proteins, but the SCFbeta-TrCP1 propeller has 7 blades, where as Cdc/Skp1 has 8 likely due to an unidentifiable change in the fifth blade. Additionally, the Skp1 component is similar between the two proteins: 40-44% of the chain is helical, and 8% is comprised of β-sheets. Both fall under the category of F Box proteins, which are classified by mediating protein-protein interactions, and are involved in the ubiquitination pathway. SCFbeta-TrCP1 selectively binds to β-catenin at pSer-33 in a similar manner to Cdc4/Skp1. The interaction occurs in the essential pockets A2 and B+1. (9)