Transcript ICE 6 Review

ICE 6 Review
Marty and Aaron
November 23, 2014
A quick review of kinases
•
A kinase is an enzyme that utilizes ATP to transfer a phosphate onto a serine, threonine,
or tyrosine amino acid.
•
This reaction is thermodynamically favorable, but slow.
•
Kinases are specific – only certain amino acids are phosphorylated.
•
Phosphorylation changes protein shape and function.
•
Kinases are often used in signaling.
ATP
Protein
substrate
Kinase regulation: The activation loop
Src-family kinases have another level of
regulation
A particular phosphatase (an enzyme that
dephosphorylates an amino acid side
chain) acts on Src and increases its activity.
On which tyrosine does this phosphatase
exert its enzymatic activity, Y527 or Y416?
Briefly explain your answer.
Closed,
inactive
Y527
Y416
Phosphatase
Kinase
Kinase
Open,
inactive
Phosphatase
Open,
active
This phosphatase dephosphorylates Y527. Dephosphorylation of
Y527 would destabilize the closed conformation since binding of
Y527 to the SH2 domain is dependent upon the phosphorylation
of Y527.
Abl is a Src-family kinase
A particular mutation decreases the Kd for
binding of the kinase C-lobe to the myristate.
How would this mutation affect the activity of
Abl?
Mutation lowers the
energy of closed, inactive
Lower Kd,
stronger interaction
Phosphorylation
of Y412
Myristate detaches
from C-lobe
Closed,
inactive
Myristate attaches
to C-lobe
Open,
inactive
This equilibrium
constant decreases
Dephosphorylation
of Y412
Open,
active
The amount of active
enzyme decreases
This equilibrium
shifts left
Abl activity would decrease. The lower Kd would stabilize the
energy of the “closed” conformation of Abl, which would lead to
a lower concentration of active enzyme.
CML is caused by a misregulated kinase:
Bcr-Abl
Bcr-Abl leads to increased Abl activity
Myristate detaches
from C-lobe
Closed,
inactive
Myristate attaches
to C-lobe
Open,
inactive
There is no myristate!
Keq increases
Open,
active
This equilibrium
shifts right
The amount of active
enzyme increases
Gleevec decreases the concentration of active
Bcr-Abl
Which decreases
closed, inactive…
Which decreases
open, inactive…
Which shifts this
equilibrium left…
Gleevec
Kd = 10 nM
+
Closed,
inactive
Which shifts this
equilibrium left…
Open,
inactive
This equilibrium heavily
favors bound state…
Closed, inactive,
bound to Gleevec
Which decreases
open, active…
Open,
active
Dasatinib and Gleevec bind to the same
location, but to different Abl conformations
Dasatinib
• Both drugs bind to the ATP
binding site.
• The drugs bind to different
Abl conformations. Gleevec
binds closed, inactive, while
Dasatinib binds open,
active.
Gleevec
Dasatinib vs. Gleevec
Binds the
open, active
conformation
Treats some
Drug-resistant
mutants
Inhibit Bcr-Abl
Bind to the ATP
binding site
Gleevec
Closed,
inactive
Binds the
closed, inactive
conformation
Dasatinib
Open,
inactive
Open,
active
Lots of terms…
Latch
Dasatinib
Clamp
SH2
Src
Closed
Myristate
Abl
Gleevec
Kinase
Myr
Phosphorylation
Bcr-Abl
CML
Open
Inactive
Linker
SH3
Activation loop
Active
Y412/Y416
Y527
An example concept map…
Src kinases have an
additional level of
regulation in which they
can be either open or
Open
closed.
Phosphorylation
Enables…
Catalyzes…
Active
Inactive
Determines
whether a
kinase is…
Activation loop
Kinase
Src
Regulated by
the position of
the…
Determines the
position of the…
Y412/Y416
Closed
Stabilized
by the…
Abl
Is regulated
by…
Misregulated by a
translocation that creates a
fusion protein called…
Gleevec
Is a particular
type of…
Interacts with the Clobe to form a…
Myristate (“Myr”)
Inhibits by binding to
its closed, inactive
form…
Is missing
its…
Latch
Interact to
form the…
Clamp
Interact to
form the…
Bcr-Abl
Causes…
Dasatinib
Inhibits Bcr-Abl and is capable
of binding to Gleevec-resistant
mutants
CML
Y527
+
SH2
Linker
+
SH3
The HIV Viral Replication Cycle
(Membrane Fusion)
HIV viral replication cycle
The Viral Life Cycle
Arts EJ and Hazuda DJ. “HIV-1 antiretroviral drug therapy.” Cold Spring Harb Perspect Med. 2012 Apr; 2 (4): 1-23.
Membrane Fusion
We’ve learned two examples, which work analogously to one another
• gp41
– gp41 extends into the pre-hairpin conformation after gp120 recognizes
CD4 and CCR5 or CXCR4
– N- and C-terminal domains of gp41 convert from a pre-hairpin to a
hairpin conformation
– N-terminal domain begins as an alpha helix, C-terminal domain gets
ordered into a helix as the two domains favorably wind together
• SNAREs
– v-SNARE and t-SNARE are both pre-formed alpha helices
– Cognate v-SNAREs and t-SNAREs recognize one another
– Cognate v-SNARE and t-SNARE favorably wind around another
• In both, as alpha helical domains wind together, the two
membranes that the alpha helices are embedded in are brought
closer together
gp41 facilitates
membrane fusion
between the viral
envelope and the host
cell plasma membrane
SNAREs facilitate
membrane fusion between
vesicles and their target
membranes (secretory
pathway)
SNAREs
gp41
fusion peptide
cytosol
t-SNARE TM
target organelle
membrane
cell membrane
N-terminal
gp41
extracellular
space
gp41 pre-hairpin
C-terminal
gp41
target organelle lumen
t-SNARE
v-SNARE
vesicle membrane
viral envelope
v-SNARE TM
gp41 TM domain
viral particle interior
vesicle lumen
gp41
SNAREs
Hemifusion
Hemifusion
gp41
SNAREs
target organelle lumen
fusion peptide
t-SNARE TM
cytosol
cell membrane
target organelle
membrane
t-SNARE
N-terminal gp41
extracellular
space
C-terminal gp41
v-SNARE
vesicle membrane
gp41 TM domain
viral envelope
viral particle interior
v-SNARE TM
vesicle lumen
Structure of the N- and C-domains of gp41
(same structure, one image rotate 90
degrees from the other)
Fusion peptide
C-terminal
N-terminal
• Which conformation is this structure
of gp41: pre-hairpin or hairpin?
Hairpin
• Which set of helices are the Nterminus of gp41? Which are the Cterminus?
• Where would the fusion peptide be?
• Which set of helices would Fuzeon
replace?
C-terminal, cyan
Viral Protein Trafficking
After integration into the host genome, an HIV-infected cell will start to
synthesize gp120, which travels through the endomembrane system until it
reaches the plasma membrane. Once the virus starts synthesizing gp120, it
has to begin degrading the cell’s supply of CD4. In addition to decreasing the
expression of CD4 at the plasma membrane to prevent other strains of HIV
from entering the cell, why else must the cell begin decreasing its cellular
concentration of CD4 once synthesis of gp120 begins? Why is it less necessary
for the cell to also decrease its cellular concentration of CCR5 or CXCR4?
Newly synthesized cellular CD4 would bind newly synthesized gp120 in the
endomembrane system and render it nonfunctional (it actually stalls it in
the ER). In order for gp120 to be expressed on the viral envelope and
available to bind with the next cell’s CD4, it must be free of CD4 to begin
with. It is less necessary to downregulated CCR5/CXCR4 since gp120 can
only interact with them AFTER having interacted with CD4, first.