Cul1-Rbx1-Skp1-F boxSkp2 SCF Ubiquitin Ligase Complex (1LDK) from Homo sapiens
Created by: Shahryar Farooq
Cul1-Rbx1-Skp1-F boxSkp2 SCF Ubiquitin Ligase Complex(PBD ID :1LDK) belongs to the largest family of E3 ubiquitin protein ligases. The SCF complexes govern ubiquitination of other regulatory and signaling proteins. The SCF complexes ubiquitinate a wide range of proteins involved in signal transduction, cell cycle advancement, and transcription. Protein accumulation is regulated through ubiquitin-dependent proteolysis, serving a critical role in physiological processes (1). Deregulation of SCF protein can result proteolysis of regulatory enzymes such as p27Kip1, a recurrent event with cancer. Subunit mutation of SCF complexes has been linked with Ovarian and Breast cancers (2).
Ubiquitination is the addition of ubiquitin after the protein has been translated. Ubiquitin operates through covalent interaction changing stability or activity levels of the target protein (3). Ubiquitin-protein ligases are known as E3s; the last step of a three enzyme cascade. The process begins with ubiquitin activating enzymes (E1) and proceeds to ubiquitin conjugating enzymes (E2) before being mediated by E3 enzymes. E3 enzymes operate by transferring ubiquitin to the substrate protein from E2 sites through formation of an isopeptide bond between the carboxyl terminus and the lysine side chains (1).
The molecular weight of the Cul1-Rbx1-Skp1-F boxSkp2 SCF complex is 118,481 Daltons (1). The isoelectric point of the SCF complex is 6.31 based on the Expasy Bioinformatics Resource Portal (4). PSI-Blast is a protein database search engine that determines protein similarity based off primary structure. The query protein is assigned an E value based of significant similarity between primary structures. Sequence homology decreases the E value, while gaps in sequencing will increase the E value. An E value below 0.5 will correspond to a close match in homology between proteins. Cul5 N-terminal domain (PDB ID :2WZK) has an E value of 9 x 10-143 with Cul1-Rbx1-Skp1-F boxSkp2 SCF Ubiquitin Ligase Complex, indicating significant homology (5).
The Dali server’s purpose is to find proteins with corresponding tertiary structure. The Dali server operates using a sum of pair method to measure similarity between proteins by comparing intramolecular distances. This measure is a Dali Z score. A Z score of greater than 2 indicates significant similarity. Cul5 N-terminal domain’s Z score is 30.1 indicating significant similarity with the query Cul1-Rbx1-Skp1-F boxSkp2 SCF Ubiquitin Ligase Complex (6).
Cul5 N-terminal domain is an E3 ligase found in Mus musculus The alternative design found in this structure enables for binding to the ubiquitin of E2 binding sites and bulky substrates due to suitable spacing (7). As an E3 ligase, Cul 5 N-terminal domain has an analogous function to the SCF complex. Both Cul5 N-Terminal domain and SCF complexes have leucine rich repeating units and WD40 repeats. These structures are an integral component in the binding to E2 ubiquitin (1, 7).
The structure of Cul1-Rbx1-Skp1-F boxSkp2 SCF Ubiquitin Ligase Complex is RING-type E3. Rbx1 composes the RING domain and the catalytic core complex is Cul1. This core recruits a cognate E2 while the F-box subunit complexes with the substrate. The Skp2 serves as a link between the F-box and the Cul1 subunits. There are several F-Box proteins in Eukaryotic genomes. These allow for the specific ubiquitination on a vast number of structurally and functionally varied substrates. Complex eukaryotes have a higher amount of homologous to SCF subunits. Homology to the Cul1 and Rbx1 SCF subunits has been found in the eleven-subunit Anaphase-Promoting Complex E3. This promotes the ubiquitination of several mitotic regulatory proteins. The Cul1–Rbx1–Skp1–F boxSkp2 complex has a highly elongated structure with Rbx1 and the Skp1–F boxSkp2 complex segregated to opposite ends (1).
Cul1 serves as scaffold and interacts with all three other subunits. Cul1 consists of a 415-amino-acid N-terminal helical region that adopts a long stalk-like structure and binds the Skp1–F boxSkp2 complex, and a 360-amino-acid C-terminal globular alpha/beta domain that binds Rbx1. Cullin repeat consists of two short helices and three long helices. The second and third helical repeats are similar, yet repeat 1 is more divergent due to shifts and rotations in the short helices, which function as an N-terminal cap and because of a two-helix insertion. These unique features of the structure account for Skp1 binding. The carboxyl terminus forms a globular protein with the use of several structural units. Cul1 carboxyl terminal domain forms a five-stranded intermolecular beta-sheet that yields a second beta-strand on the N-terminal sequence of Rbx1. The rest of Cul1 carboxyl terminus forms a wide groove. This is where the Rbx1 RING domain is rooted (1).
The sixteen residue Rbx1 beta strand with a kink in the middle forms beta-sheet backbone hydrogen bonds with the S1, S2 and S3 strands of Cul1. Rbx1 is involved in the creation of the alpha/beta hydrophobic core. The conserved Phe-22 and Trp-27 of Rbx1 have a central role, making multiple van der Waals contacts to side chains. The 70-residue Rbx1 RING domain implements the structure of the canonical RING (1).
This structure is stabilized by two zinc ions and contains a twenty-residue insertion. The insertion contains three zinc ligands (Cys-53, Cys-56, and Cys-68), which, together with a fourth zinc ligand from the RING (Cys-82), create a new zinc-binding site (1). The intermolecular beta-sheet is the primary apparatus of Rbx1 attachment. This is shown by the mutations in the zinc-chelating residues which did not interrupt the complex of Rbx1 and Cul1, even though SCF function was eliminated (1, 8).
The Skp2 leucine-rich repeat domain and E2 are on the same side of the SCF complex. The tip of the leucine-rich repeat domain points toward the active site cysteine of the E2, with a distance of 50 Å in between them. This distance could readily be bridged by the portion of p27 between phosphothreonine Thr-187, required for Skp1–Skp2–Cks1 binding, and the candidate ubiquitination sites at lysines 134, 153, and 165 (1).
The rigidity observed in the Cul1–Rbx1–Skp1–F boxSkp2 structure along with mutational data suggests that RING E3s may facilitate ubiquitin transfer by positioning the substrate protein in an optimal manner, such that the lysine side chains to be ubiquitinated are presented in the immediate vicinity of the E2 active site. Rigidity is critical for the preservation of function for the entire molecule in order to retain E3 capabilities (1).
The Cul5 exhibits an extensive stalk-like structure, analogous to other proteins within the Cullin family. Three consecutive repeat domains which have similar structures. The five helix fold is diverged in the first repeat domain. There is significant deviation in the Cul5 and other Cullin structures which indicate different relative orientation of the three repeat Cullin domains (7). The middle of the 4HB region is where Cullin homology region starts in the SCF complex.
The H24 helix, components of the beta-sheet, and WH-A domains are present. The structure suggests that the proteins that have the Cullin-homology sequence will also have the rest of the 360-amino-acid Carboxyl terminal domain structure; nonetheless no significant homology exists beyond the Cullin region (1). The structures of these two points of interest are similar and both have analogous functions in their use of the Cullin groups to form binding sites within the molecule. The prosthetic groups on the Cul5 N-terminal domain are 1,2- Ethanediol opposed to the zinc ions on SCF complex (1, 7)