Download Glucose/Galactose Binding Protein (GGBP)

Molecular modeling of casein kinase-1 to determine relevance of conserved potential
phosphorylation sites
Paige R. Pritchett1, Troy C. Messina1, and Cynthia J. Brame2
1Centenary College of Louisiana, Department of Physics, 2911 Centenary Boulevard, Shreveport, LA 71104
2Center
for Teaching, Vanderbilt University, Nashville, TN 37212
Eukaryotic protein kinases transfer a phosphate from a nucleoside triphosphate to a protein substrate. The eukaryotic protein kinases are homologous and therefore exhibit significant structural similarity but can be
divided into eight subfamilies with closer structural and functional relationships. The CK1 subfamily is ubiquitous and highly conserved within Eukarya, and has members that are relevant to regulating cell proliferation and
differentiation, chromosomal segregation, and circadian rhythm. In humans, CK1s have been linked to neurodegenerative diseases and cancer. We are investigating regulation of CK1 activity by phosphorylation, using
yeast CK1 protein kinases as models. We have previously identified phosphorylation sites that negatively regulate activity through in vitro and in vivo studies of phosphorylation-mimicking and -preventing mutations. In
this study, we have used NAMD to investigate the effect of mutations on protein structure in silico. We have identified structural changes large enough to modify kinase activity, for example S179 shifts towards a positively
charged RD pocket by 3-4 Å upon phosphorylation or mutation to a glutamic acid. The phosphorylated amino acid is also subject to a much more restrictive potential of mean force over the coordinate between S179 and
the RD pocket. The simulation results will be further contextualized in terms of the laboratory work.
Overview
Protein kinases are highly conserved in form and
function within the domain Eukarya. The Casein
kinase-1 family of protein kinases has been implicated
in controlling neural processes and in the normal
development of yeast buds. This second
consequence suggests CK1 may be important in the
proliferation and development of cells in eukaryotes.
In yeast cells, phosphorylation leads to deactivation of
the protein. Blast alignments show conserved regions
across many CK1 orthologs. Experiments mutating
phosphorylatable amino acids in the conserved
regions have been performed and implicate
a
particular serine as important to the activity of CK1.
Mutations to this amino acid was performed using
molecular modeling software VMD and NAMD. We
then performed molecular dynamics simulations and
steered molecular dynamics to better understand the
structural relation to CK1 activity.
Methodology/Procedure
Conclusion and Ongoing Work
The crystal structure of the wildtype, non-phosphorylated
(active) version of casein kinase-1 was obtained from the
protein data bank (PDB ID: 1CSN). NAMD 2.9 (Win32
version) and VMD 1.9.1 (Windows OpenGL version) were
used to mutate, solvate, visualize, and simulate the
wildtype and phosphorylated versions of the protein.
The current results show that there is a movement of the
lowest energy conformation for the phosphorylated
version that holds the phosphorylated S179 closer to the
RD pocket at R130.
The graph at left displays the movement of the
phosphorylated S179 in relation to the wildtype.
The serine at position 179 (S179) was mutated or
phosphorylated to see what structural changes resulted.
Molecular dynamics (MD) simulations were run on the
solvated enzyme to provide structural information for
determining change in structure as a result of the
changes to S179.
The structures are both tightly held at those points and are
unlikely to cross any energy barriers that involve a force of
more than ~3 kcal/mol due to thermodynamic hindrance.
Adaptive biasing force simulations are still being run to
obtain a complete graph of the potential of mean force.
The distance between S179 and a conserved, arginine
(R130) was calculated from the simulation. R130 is a
putative charge interaction partner for pS179. A distance
histogram was generated from the distance data and
used to support the investigation of the phosphorylated
S179 and its possible role in activation/deactivation of
CK1.
Acknowledgements
• Funding
–
–
Centenary College Biophysics Department
Centenary College Gus S. Wortham Endowed Chair of
Engineering
• Dr. Lucy Robinson
References
The above left image shows the relation of protein
kinase families. Casein kinase-1 is located at the top
center of the image.
The above right image displays the location of labeled
CK1 during the budding of a yeast cell. The
highlighted portions correspond to the position of the
CK1 and make it clear that it is consistently
associated with the site where the bud forms.
Serine
Phosphoserine
Because MD at physiological temperatures only allows for
low energy structures that are near the original starting
structure to be observed, another way of determining
lowest energy structures that may be separated by large
barriers had to be used. To determine any other
differences in conformation as a result of phosphorylation
adaptive biasing force simulations were run. This
simulation directed the S179 to move closer and further
from the conserved RD pocket at residue 130 and
calculated the force it took to hold the protein at each
position from 0-20 Å.
The above center image shows the protein
solvated in water for simulation.
The above lower image displays the protein
with S179 and R130 highlighted in blue and
yellow, respectively.
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