Transcript Back to where we began: Protein structure combining

Ras and Cancer
Back to where we began:
Protein structure
combining Bioinformatics, protein
structure with Kinemage, and the
Map kinase path
page 776-780
Ras was the first human oncogene
found
• Take DNA from Human bladder cancer
cells
• Put DNA into mouse cells (these cells
already had other helpful mutations)
• Mouse cells become transformed=
cancerous
• Human Ras was found to be the oncogene
found in the human bladder cancer cells
We have a ClustalW analysis
showing that the number 12 amino
acid G (glycine) can be replaced
with C (cysteine) and this causes
human lung cancer.
The normal protooncogene Ras
becomes an oncogene
K-Ras can have a point mutation- a
single change in the sequence
OncogenesCommonly
Are from
Genes
That act in
Cell
signaling
Paths-Like the Ras
Map Kinase
path
Our Clustal W analysis of the amino acid
change in K Ras
From our lab: The number 12 amino acid is different. The
First listing is normal, second from cancer patient
Change from normal G to cancerous C-symbolized
by G12C. Note no other weird animal has a change (only
mutant human protein)
No change here in h
Why is there a different amino acid at position 12?
DNA coding strand sequence (you do not report the
template strand sequence) from a person with cancer due
to K Ras mutation…from our prior work
•
The Sequence Manipulation Suite: Show Translation
Results for 5312 residue sequence starting "GGCCGCGGCG".
1 A A A A E A A A A A A V A A A K V A A A
1 GGCCGCGGCGGCGGAGGCAGCAGCGGCGGCGGCAGTGGCGGCGGCGAAGGTGGCGGCGGC
Met is
21 R P V L P A P A I S D W E R A R R R H *
st
1 Amino 61 TCGGCCAGTACTCCCGGCCCCCGCCATTTCGGACTGGGAGCGAGCGCGGCGCAGGCACTG
41 R R R R G Q R L S G S Q V R E R G L L K
Acid
121 AAGGCGGCGGCGGGGCCAGAGGCTCAGCGGCTCCCAGGTGCGGGAGAGAGGCCTGCTGAA
12th amino acid is C not G
•
61 M T E Y K L V V V G A C G V G K S A L T
181 AATGACTGAATATAAACTTGTGGTAGTTGGAGCTTGTGGCGTAGGCAAGAGTGCCTTGAC
The normal individual with a protooncogene has GGT
not the dangerous TGT
Use the Genetic Code
from our text, for mRNA
and an
Amino acid
For Ras induced cancer,
change G (glycine or gly)
for a C (cysteine or cys)
by changing
GGU in mRNA to UGU –
only 1 change
Fig. 21-8, p 660 in 6th Ed
In the normal cancer free individual with normal
K Ras, the K Ras mRNA with Glycine would
have….(using the Genetic Code)
GGU or GGC
(also GGA or GGG code for glycine)
For Cysteine (C) in the mutant oncogene K Ras –the cancer
patient would have this sequence in their mRNA:
UGU or UGC
So, the first nucleotide base of the codon on mRNA
changes from a G to a U. This is due to a prior change in
the DNA of the cancer patient (a point mutation).
Follow change all the way through…note
that the coding strand is always reported
(same as mRNA except replace U with T)
Coding strand ---------G changes to T -G-T-------
DNA
Template strand---------C changes to A -C-A-------
---------G changes to U -G-U------
mRNA
Protein
------------#12 Glycine changes to Cysteine --------And K Ras protein cannot be shut off, causes cancer
A POINT MUTATION IN THE DNA; FROM G TO T
Normal protooncogene
coding
---GGT-----CCA---
Oncogene
---TGT-----ACA---
DNA
template
---GGU--- mRNA
---UGU---
AMINO
ACID
---GLYCINE---
--CYSTEINE--
Let’s look at Ras structure and see where this
number 12 amino acid is located…and why it is
so importantRas is “on” and will cause cell division if the
Ras has GTP bound to it (ras is a G Protein)
Ras is off when GTP is broken down to GDP.
Ras itself breaks down GTP (a GTPase
reaction) but Mutant Ras cannot be activated to
breakdown GTP.
So, mutant Ras stays on…causing cancer
(unregulated cell division)
Put another way, the mutation inhibits the
GTPase activity of Ras
In general, Ras a central Beta
sheet
and 5 alpha helices located on both
sides of the Beta sheet.
GTP binds in the end of the Beta
sheet
#1 Amino Acid
here
#12
Glycine
In P/G1
loop and
helps
bind GTP
Gly12 here
• Use the kinemage Ras file on web site, along with
Kinemage, to view Ras structure
• View1: the backbone of the amino acid chain is in
white, the bound GTP analogue in pink, and the
Mg++ in yellow
• View2 is from one edge of the twisted beta sheet of
this alpha/beta protein where GTP binds. Glycine 12
(where the most common mutation occurs) and
Glycine 13 are labeled in green and are found on
what is called the G1 or P loop.
• These 2 Glycines are located in a critical part of one
of the main GTP-binding loops (the G1 or P loop).
They are the two major sites of mutations that
convert this enzyme into an oncogene - when these
Gly's mutate to Cys, the GTP cannot be broken down
down (GTPase activity of ras is reduced) so Ras
stays in the "on" state more of the time- causing
cancer.
• To see details of interactions at the binding site, choose View3
and turn on "interact.", which shows sidechains in cyan and
weak H bonds in purple.