Good Vibrations?
Kettle Moraine SMART Team: Dams, Greg; Drachenberg, Disa; Goelz, Mike; Greene, Allie; Jastrow, Bronson; Krause, Kris; Laux, Jake; Merritt, Nick; Murray, Nate; Reese, Kara; Wilson, Bradley
Teacher: Beck, Kelly; Plum, Stephen
Kettle Moraine High School, 349 N. Oak Crest Drive, Wales, WI, 53183
Kovrigin, Evgenii Ph.D., Medical College of Wisconsin, Milwaukee, WI, 53226
Abstract
The Switch
Ras Movement
GEF + GTP
Active
Ras
- GDP
GDP
- pi
GDP
Inactive
Ras
Activation of the Ras protein is
controlled by the binding of GTP. The
structure of the GTP bound Ras
molecule (the active form) permits the
binding/activation binding of Ras to the downstream
of downstream effectors. Downstream effectors are
effectors
proteins that activate cellular processes
leading to the cell growth and division.
Hydrolysis of GTP renders Ras inactive
thus turning the signaling cascade 'off'.
Ras and Cancer
Cancer is manifested by uncontrolled cell growth, which occurs
due to mutations in signaling proteins. Ras GTPase (Ras) is one of
the most important signaling proteins that help regulate cell growth
and division. Mutations in Ras leading to its permanent activation
and uncontrolled cell growth are responsible for nearly 30% of
human cancers. When functioning normally, Ras binds Guanosine
triphosphate(GTP), and adopts active signaling conformation.
When activated, Ras interacts with other proteins and activates
them resulting in cell growth and cell division (proliferation). When
GTP is hydrolyzed and turned into Guanosine diphosphate(GDP),
Ras adopts its non-signaling conformation and can no longer bind
other proteins and activate them (signaling was 'turned off').
However, if Ras has a specific oncogenic mutation, it cannot
hydrolyze GTP and instead permanently signals for cell growth,
causing cancer. One of the keys to figuring out how Ras causes
cancer might be to better understand how the protein changes its
conformation while performing its signaling function. Structural and
dynamic studies of Ras using NMR techniques suggest that the
protein, when active and bound to GTP, has two majorly different
conformations it can take. Understanding how the protein changes
conformation and interacts with other proteins could shed light on
how it signals for cell growth. This information could further cancer
treatments and potentially lead to a cure for cancers caused by
Ras mutations.
X-Ray Crystallography
and NMR
5P21.pdb
The above picture shows a crystallographic model of RasGTP with most recent NMR data highlighted with color [1].
The nitrogen atoms that report significant motional
dynamics are shown in blue. The red area, the effectorbinding surface, experiences motion so extensive that NMR
signals from those atoms are not detected at all.
The key finding here is that this movement is spread
around most of the Ras-GTP structure, instead of just to
the effector-binding interface (red). This result indicates
that Ras-GTP experiences significant conformational
change in order to convert to the 'tight' conformer, only
suitable for binding its signaling partners.
Function of the 'weak' conformer is yet to be established
but likely important in regulation of Ras function. It may be
targeted by specific drugs to attenuate signaling of the
oncogenic mutated Ras. Thus 30% of people with cancer
may get additional hope for combating the disease, which
makes these studies worth the effort.
[1] O'Connor, C. and Kovrigin, E.L., Global conformational dynamics in Ras.
Biochemistry, 2008. 47(39): p. 10244-10246.
Ras Binding
http://www2.kumc.edu/urology/images/
photos/bladderCancer.jpg
Mutations in Ras cause 30% of human cancers,
including bladder, colon, and rectal cancer.
Because of this, if we were to understand
exactly how Ras works, it would be extremely
beneficial in discovering cures for those
cancers. Using NMR, a technique used to view
protein structures, to view Ras will help to
determine how Ras configures itself during cell
signaling, which, in turn, will help in
understanding why Ras sometimes constantly
signals for cell division and how to prevent it.
PDB ID: 5P21
Pai et al, (1990) Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A
resolution: implications for the mechanism of GTP hydrolysis. EMBO J. 9: 2351-2359
5P21.pdb
Ras GTP
The ‘weak’
conformer
Downstream
Effector
Ras GTP
The ‘tight’
conformer
Above, the X-Ray Crystallography-based model of RasGTP (shown here in ribbon form)
X-Ray Crystallography is a technique for determining
the structure of proteins in the protein crystal. It shows
a protein in its most compact, static form, meaning it is
difficult to study proteins with more than one
conformation. Nuclear Magnetic Resonance (NMR)
spectroscopy, however, can determine protein
structures and resolve conformational switching in
physiological solution conditions, not much different
from intracellular environment.
Downstream
Effector
GTP
NMR spectroscopy uncovered that active Ras-GTP is not a
single well-defined conformation ready to bind and activate
its signaling partners. Instead, Ras-GTP spends most of the
time in the 'weak binding', inefficient state, which must be
converted into the 'tight binding' conformation for activity.
A SMART Team project supported by the National Institutes of Health (NIH) –
National Center for Research Resources Science Education Partnership Award (NCRR‐SEPA)