1.
STUDYING THE STRUCTURE OF THE MITOTIC CHECKPOINT COMPLEX USING COMPUTATIONAL ANALYSIS
AND TEMPERATURE-SENSITIVE YEAST MUTANTS
Trevor Van Eeuwen*^, James Luginsland^, Patricia Melloy*, Gloria Anderle^
Fairleigh Dickinson University, Madison, New Jersey 07940
* Department of Biological and Allied Health Sciences, ^ Department of Chemistry and Pharmaceutical Science
Cellular Studies of cdc20-1
References
Acknowledgements
Hypothesis
The cdc20-1 mutation, the result of a glycine to arginine substitution at amino acid 544 (G544R), causes
cells to arrest in metaphase.2 The mechanism by which this occurs is not understood, though we
hypothesize that one of the following occurs:
The mutation affects binding domains of Mad2 or Mad3 and restricts APC substrate
recognition.
The mutation affects thermodynamic stability at non-permissive temperatures, disrupting the
hydrophobic core causing the protein to denature.
The mutation affects protein folding kinetics, causing the unfolded protein to be disposed of
by the unfolded protein response (UPR).
Research was funded in part by Pfizer PURE Grant, the Novo Nordisk Summer Research Fellowship, the Novartis
Research Scholarship and an NSF Equipment grant number 0721251 for which we are immeasurably thankful.
Funding also provided Becton College Grant–in-Aid.
25oC 27oC 30oC 32oC 37oC
Wildtype +++ +++ +++ +++ +++
cdc20-1 +++ +++ ++ +/- -25oC 27oC
30oC 32oC 37oC
wt
cdc20-1
wt
cdc20-1
wt
cdc20-1
wt
cdc20-1
We wanted to use molecular dynamic simulations of protein models to observe protein behavior and
compare this with cellular studies.
Figure 5A. Analyzing the temperature-sensitive phenotype
of cdc20-1. Shown are serial dilutions of the wildtype control (top
row) and cdc20-1 (second row) budding yeast strains grown at
permissive temperature (25oC), non-permissive temperature (37oC)
and three intermediate temperatures for 7 days on YPD media. The
temperature-sensitive phenotype is first visible at 32oC (red arrow)
and confirmed with no growth seen at 37oC (yellow arrow).
Figure 5B.
Cdc20-1-GFP is visible in the
nucleus and then the vacuole
depending on growth
temperature. Shown are
images of the candidate
cdc20-1-GFP strain at 25oC
and 37oC. At 25oC, the GFP
signal (orange arrow)
localizes to bud neck of
mitotic cells and nucleus
and is distinct from the
central vacuole (blue
arrow)5. At 37oC, the GFP
signal (yellow arrow)
colocalizes with the central
vacuole (purple arrow).6
Images were taken using the
Leica DM5500 microscopy
system and an ORCA ER
camera(Hamamatsu). Expos
ure times were 5ms (DIC) ,
2s (GFP) and 2s (RHO) 2x2
binning.
1. Hartwell LH, Mortimer RK, Culotti J, Culotti M (1973). Genetic control of the cell division cycle in yeast. V. Genetic analysis of cdc mutants. Genetics, 74:267-286.
2. Schott EJ, Hoyt MA (1998). Dominant alleles of Sacchromyces cerevisiae CDC20 reveal its role in promoting anaphase. Genetics 148(2):599-610.
3. Chao WCH, Kulkarni K, Zhang Z, Kong EH, Barford D (2012). Structure of the mitotic checkpoint complex. Nature, 484: 208-214
4. Chen et al. (2010). MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallographica D66:12-21.
5. Melloy PM, Holloway S (2004). Changes in the Localization of the Saccharomyces cerevisiae Anaphase-Promoting Complex Upon Microtubule Depolymerization and Spindle
Checkpoint Activation. Genetics, 167:1079-1094.
6. Bertolotti, A., et al., Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat Cell Biol, 2000. 2(6): p. 326-32.
7. Chaudhury S, Lyskov S, Gray JJ (2010). PyRosetta: a script-based interface for implementing molecular modeling algorithms using Rosetta. Bioinformatics., 26:689–691
8. Tan KP, Khares S, Varadarajan R, Madhusudhan MS (2014). Tspred: a web server for the rational design of temperature sensitive mutants. Nucelic Acids Research. doi:
10.1093/nar.gku319.
Results and Discussion
Mitotic Checkpoint Complex (MCC)
Figure 1A. The MCC helps to regulate the cell division cycle by working with the anaphase-promoting
complex (an E3 ubiquitin ligase). In the event of unattached kinetochores or other spindle damage, the MCC forms
and inhibits the APC to block cell cycle progression until damage is repaired. Mad1 and Mad3 are recruited to
unattached kinetochores, facilitating MCC formation. Mad2, Mad3 and Bub3 bind to Cdc20 to form MCC which in turn
binds APC thereby blocking substrate recognition. The inability of APC to ubiquitinate securins and cyclins maintains
cells in metaphase until kinetochores are properly attached.
Mad1
Bub3
MCCCdc20
binding
event
Cdc20 APC
Unattached
Kinetochore
Inactivated
APC
Mad3
Bub3
Mad2
Figure 1B. The MCC (left) is
responsible for maintaining chromosome and
spindle integrity during the cell cycle. Mutations
in these genes can cause errors in response to
spindle damage. The Cdc20-1 mutant (obtained
from the ATCC) causes cells to arrest in
metaphase.1
Major Results of our study are:
Fluorescence Microscopy indicates cdc20-1-GFP is localized to the nucleus at room temperature and
the vacuole at 37 oC. Comparative studies of Cdc20-GFP localization at 25 oC and 37 oC are in progress.
We have created Saccharomyces cerevisiae homology models of Cdc20p, Mad2p, and Mad3p using an
existing S. pombe crystal structure (4AEZ) as a template. Structures have been validated using the
MolProbity structural validation server.
Using a molecular dynamics in-silico simulation, we have shown that a folded cdc20-1 protein is
predicted to be stable at 37 oC.
Our current data suggests that cdc20-1 causes defects in protein folding.
 Localization to central vacuole suggests involvement of Unfolded Protein Response (UPR) in removing
misfolded cdc20-1.
 Current simulations at permissive and non-permissive temperatures indicate no major changes in
thermodynamic stability. Recent studies found that centrally located hydrophobic amino acids in
protein structure were the most successful at producing a temperature sensitive phenotype by
protein destabilization8. Based on our model and the sequence, residues surrounding G544 were
identified as potential disrupters of the hydrophobic core.
Bub3
Mad3
Cdc20
Mad2
Cdc20
Mad3
Bub3
Mad2
Cdc20
Mad2
Mad3
Cell-division cycle protein 20 (Cdc20)
Figure 4. RMS plot of molecular dynamic simulations in AMBER indicate wild type and cdc20-1 have relatively similar
thermodynamic stability after running simulations at permissive and non-permissive temperatures for 1000 picoseconds. This
suggests all models reside in a potential well and are not experiencing short-timeframe structural changes.
Molecular Dynamics of Cdc20p and cdc20-1p
Figure 2A. Cdc20 is an Anaphase Promoting Complex
(APC) activator characterized by the beta sheets arranged in
seven beta propellers, known as a WD40 repeat motif.2
Saccharomyces cerevisiae Cdc20p is 640 amino acids long.3 We
generated the 313 residue long Cdc20 model pictured, that
focuses on the functional regions near the N-terminus. Residues
in green indicate the location of the D box receptor (a motif
used to bind APC substrates), red residues indicates the KEN
box receptor residues (another substrate binding motif) and
blue indicates location of cdc20-1 (GR).3
Cdc20 wild type
(Glycine)
cdc20-1
(Arginine)
Figure 2C. The cdc20-1 mutation is
characterized by a glycine to arginine point mutation at
amino acid 544.1,2 This single change renders the cdc20-1
protein temperature-sensitive, meaning that the protein
is stable at permissive temperature, but non-functional at
higher temperatures. We are attempting to understand
the nature of the temperature-sensitivity on the protein
level with this study. Pictured here is a computational
model of the wild type protein with the small, achiral
glycine (blue, left) at the top of a beta sheet. The mutant
cdc20-1 has a larger, sterically hindered and charged
arginine (blue, right) in the same position.
Mitotic Checkpoint Complex Docking
Figure 6A. Areas of
Mad3p and Cdc20p interface are of
great interest as they include the
KEN Box of Mad3 (K30 E31 N32)
and the KEN Box Receptor (D260
D261 F262 Y263) on Cdc20.3 The
KEN Box Receptor has been
identified, along with the D Box, as
sites involved in ubiquitination of
cyclin and securin. Amino acids
typically act by hydrogen bonding
though side chains and with the
protein backbone.
Homology Modeling Cdc20
Figure 3B. The S. cerevisiae homology
model of Cdc20p (purple) exhibits an extremely high
degree of structural similarity with the template
structure (textured). The S. pombe (fission yeast)
structure (4AEZ chains A, D and G) and the S.
cerevisiae homology model are both characterized
by the WD40 repeats and beta sheet secondary
structure.3
DIC GFP VACUOLE MERGE
Cdc20-1at25oCCdc20-1at37oC
Figure 6B. Docking
the homology models of Mad3
(orange), Mad2 (green) and
Cdc20 (purple) with PyRosetta
will help us with further
computational analysis of the
MCC and associated proteins.7
Future research will focus on
modeling the Cdc20-Mad2
interaction points, an area out
of the scope of our current
model.
Figure 3A.
Structural analysis via
Ramachandran plots and
MolProbity structural
validation server
indicate a stable and
realistic model. Analysis
shows all rotamers in
favored position, five
Ramachandran outliers,
no bad backbone angles
and ranks in the 83rd
percentile of structures
as ranked by MolProbity
score.4
Figure 2B. A linear representation of S. cerevisiae
(budding yeast) Cdc20 protein. The functional features relative
to sequence including Beta-propellers (blue), D box receptor
(green star) and KEN box receptor (red star).