Cyclin K (PBD ID: 2I53) from Homo sapiens
Created by: Madison Meredith
The
protein cyclin K (PBD ID: 2I53) is a member of the cyclin family of proteins
which progress the cell through the cell cycle by binding to and activating cyclin-dependent
kinases (CDKs, PDB ID: 1UNL). The cell cycle has 4 distinct stages: G1, S, G2,
and M. Cyclin K was first discovered in Saccharomyces cerevisiae as a
substitute for a G1 cyclin (1,2). During G1, the cell grows in preparation for
mitotic split. CDKs bound to cyclins regulate this process to ensure protein
synthesis has occurred and DNA is intact for separation during the subsequent
stages of the cycle (2,3). In Saccharomyces cerevisiae, cyclin K
restores cell cycle progression by preventing cell death due to the deletion of
the G1 cyclin (acetate ions (1,8). Two thirds of the
alpha-helices of cyclin K are divided into two terminal domains, the C-terminal
and N-terminal domains. The alpha-helices of cyclin K are
conserved with critical residues for binding interactions between cyclin K and
CDKs (1).
The
primary structure of cyclin K is a sequence of 258 amino acids. Cyclin K's secondary structure consists of 15 alpha-helices, 10 of which make up the C-terminal and N-terminal
cyclin domains. The alpha-helices that make up the two cyclin domains are
H1’-H5’ (C-terminal domain) and H1-H5 (N-terminal domain). At the N terminus,
the 3/10 helix HNa (residues 28-32) and the α-helix HNb (residues 35-39)
precede the first cyclin domain. While both of the cyclin domains are similar
in structure, there are several notable differences. Helix H1 in the N-terminal
domain is eleven residues longer than H1′ in the C-terminal domain, whereas H2
is four residues shorter than H2′ and H5 is two residues longer than H5′.
Additionally, the N-terminal cyclin domain has two
helices, H4 (residues 113-123) and H4a (residues 126-132), whereas the
C-terminal box contains two short remnants of an H4′ helix with 3/10 geometry
(residues 222-224 and 234-237) (1).
The
cyclin domains are stabilized by hydrogen bonding between residues of different
helices. The N-terminal domain is stabilized by H-bonds between specific residues
across different alpha-helices such as Glu-52 and Arg-92 in the first and third
alpha helices, respectively. The C-terminal domain is stabilized by hydrogen
bonds formed between Tyr-161 (H1’) and Asn-190 (H2’), and between Tyr-212 (H3’)
and Glu-248 (H5’). Additionally, the overall folding of
cyclin K is stabilized by interdomain contact between three pairs of residues (1).
In
Homo sapiens, cyclin K is structurally similar to cyclin C. Both of the
cyclins have two terminal domains comprised of 5 alpha-helices each. Notably,
the residues involved in the stabilization of the cyclin fold, Thr-73, Lys-105,
Glu-144, Asn-190, and Leu-199, are invariant in cyclins K and C. Additionally, at
the beginning of H1 in both cyclin K and cyclin C there are additional
N-terminal helices with polypeptide chains. However, in the H4-H5 loop, cyclin
K has a short helix H4a and helix H4' disturbance via the insertion of residues
225-233, resulting in greater surface exposure of these residues. Cyclin C does
not have these (1).
Cyclin K and cyclin C can also be compared using the Dali Server in the Institute of
Biotechnology and the Position-Specific-Iterated Basic Local Alignment Search Tool
(PSI-BLAST). The Dali server uses a sum-of-pairs method to measure the similarity
of protein tertiary structure by comparing intramolecular distances. The similarity
in tertiary structure is measured by Dali Z-scores. When compared with cyclin
K, the Dali Z-score of cyclin C is 24.5, suggesting the two proteins have
similar folds (9). Furthermore, the PSI- BLAST looks at the total
sequence homology and assigns amino acids that exist in the comparison
protein’s sequence but not in the original protein’s sequence an E value.
Proteins with lower E values are more structurally similar. The E value of
cyclin C in the PSI-BLAST is 4?10-77, suggesting that the sequences
of cyclin C and K are similar (10). Functionally, both cyclin C and cyclin K
act as positive regulatory subunits of the cyclin-dependent kinases they bind
to. The CDK/cyclin complexes play a role in the regulation mRNA transcription
and the cell cycle (1, 5). However, the cyclin K gene also has a p53 binding
site in its first intron, suggesting cyclin K is also reactive to p53-dependent
stresses (1,3).
In
human cells, cyclin K is a CDK-9 regulatory subunit and participates in RNA Polymerase
II transcription. There are several residues of cyclin K that constitute the binding domain for CDK-9. Specifically, 15 cyclin K residues interact
with CDK-9 residues Glu-55, Glu-57, Arg-65, and Arg-172 through
hydrogen bonding and hydrophobic interactions. Thus, creating the CDK9/cyclin-
K complex (1). The CDK9–cyclin K complex phosphorylates the carboxyl-terminal
domain of RNA polymerase II (RNAPII), a reaction that is considered to be one
of the most important steps in transcription of many genes (3).
The
CDK9/cyclin-K complex is particularly important because it is required for
genome integrity maintenance during the cell cycle, by promoting cell cycle
recovery from replication arrest and limiting the amount of single-stranded DNA
(11). CDK-9 has two regulatory subunits, cyclin T (PBD ID: 3BLQ) and cyclin K. CDK9/cyclin-T has several functions as a component of the positive transcription elongation factor
b (P-TEFb). Researchers at the Department of Radiation Oncology at Emory University
School of Medicine and the Department of Biochemistry at Vanderbilt University
Medical determined, however, that only when bound to cyclin K can CDK-9 perform
its genome integrity functions. The removal of either the CDK-9 or cyclin K
impaired cell cycle recovery in response to replication stress, thus inducing
spontaneous DNA damage. The same is not true for the removal of cyclin T. Researchers,
therefore, concluded that only the CDK9/cyclin-K complex, not the CDK9/cyclin-T
complex, can interact with ATR, a checkpoint kinase that responds to
single-stranded DNA formed by processing double-strand breaks or at stalled
replication forks, and other DNA damage response and DNA repair proteins (1,11).
Additionally,
cyclin K is transcriptionally activated in response to p53-dependent cellular
stresses. Using cDNA microarray technology, researchers at the Human
Genome Center Institute of Medical Science at the University of Tokyo found higher
levels of cyclin K mRNA in cells deficient in wild-type p53 when in the
presence of exogenous p53. Using electrophoretic mobility-shift assay,
researchers showed that a possible p53-binding site could bind to the p53
protein in intron 1 of the cyclin K gene. In addition, a heterologous reporter
assay showed that the possible p53-binding site had transcriptional activity
dependent on p53. Colony-formation assays revealed that glioblastoma cell
growth was suppressed by overexpression of cyclin K. Researchers, therefore,
concluded that cyclin K plays a role in regulating the cell cycle or apoptosis
after being targeted for transcription by p53 (3).
Cyclin K plays a key role in the eukaryotic cell cycle. Although the structure of cyclin K is identified, much of cyclin K’s functionality is still unknown. Within the cyclin family, cyclin K’s function is relatively underexplored. Further research into cyclin K’s response to p-53 dependent stresses may help scientists study cancerous cell proliferation and explore responses to deficiencies in p53.