CELL0010 – Integrative Cell Biology
First part is molecular, then practicals and then second part is cellular.
Molecular – protein structure/chemistry
Keywords
GEF – Protein domains which that activate monomeric GTPases by stimulating the release of
of GDP to allow GTP binding.
GAP – Bind to activated G proteins to stimulate their GTPase activity and turn them off.
From GTP to GDP.
Ras – single subunit small GTPase related in structure to the G alpha subunit. Has intrinsic
GTPase activity and will hydrolyse bound GTP to GDP. However too slow by itself for
function, so bound to GAP to make RasGAP complex that supply additional catalytic residues
so that a water molecule is optimally positioned for nucleophilic attack of the gamma-
phosphate of GTP.
Sos – Bind to Ras-GTPases and force them to release their bound nucleotide.
Lecture 1: Protein kinase structure-function and reversible post-translational protein
modification by phosphorylation
Models made out of X-ray diffraction data so best guesses, but not exactly right. Only
have structure for things they get in the state. Many proteins are partially stable and
unwound, and take up thick structures transiently, so don’t have much data with
changing structures.
Much of cell signalling and signal transduction is for moving info across the
membrane. Changes to the environment or transmitted messenger molecule which
causes a signal transduction to occur. Molecules which cannot pass through have
receptors on the outside of the cell.
There is a receptor and then there are effectors inside which stimulate activity
internally. Look at protein kinases and GTPases.
Transduction events lead to signal in the nucleus, or something gets secreted out of
the cell.
How the input turns into the output.
Proteins which respond to phosphorylation signal.
What is phosphorylation?
Kinase places phosphate onto other proteins such as other kinases.
Only 5 accept phosphate group: serine, threonine, tyrosine, histidine (transient – only holds
it briefly) and aspartate.
Prokaryotes can use phosphorylated aspartate but little evidence seen in eukaryotes.
Commonly share an -OH group.
Thinking as a chemist, works as a condensation reaction. Esterify phosphate. H 2O as a by-
product.
Phosphatase inject a molecule of water to break the bond and release the phosphate. So
hydrolysis in the backwards direction.
Source of phosphate from ATP. Some kinases use GTP.
,All phosphorylated in the tissue are attached to serine and threonine. Antibodies recognise
phosphotyrosine and lead to bias in literature, with phosphorylation of tyrosine residues.
Plenty of oncogenes that are mutated that give rise to cancers are tyrosine kinases. Tiny
changes in signal. Levels of tyrosine phosphorylated proteins are held low because they are
such a strong signal so change in 1% is a 10-fold change in signal.
Importance of archetypes
Once a molecular machine exists with particular function, if function can develop in different
ways they develop families. Different members of family have specific individual functions.
Map principles from one protein kinase and apply it to the other members of the family.
Protein kinase A is the first protein discovered. First signal transduction mechanism was the
signal for transmitting adrenaline. cAMP then has its effect in cells through the activation of
PKA. cAMP is a second messenger that sets off this enzyme.
Heterotetramer – 2 regulatory subunits (blue) and catalytic subunits (green).
On regulatory subunit, they have binding sites for cAMP. If one molecule binds into binding
site, then the second binding site is more avid for binding cAMP, so a non-linear response
curve is produced. Diffuses through the cell and binds to regulatory subunits. Once the
cAMP is bound, there is a conformational change in regulatory subunits and change the
shape to no longer hold onto catalytic subunit. Exchange ADP for ATP to phosphorylate
things.
Protein kinases have a bilobal structure. Small lobe on top and larger lobe on bottom.
Small lobe – ATP binding
Large lobe – peptide binding
ATP binds at the ATP binding site which has a crevice.
Proteins that start as phosphorylating kinases to phosphorylating other proteins has the
same structure.
Has a gap in the middle of the two lobes.
Beta strand sheets on the top and mostly alpha helices on bottom. Articulated spine keeps
two lobes in proximity.
Kinases have sites where they themselves can be phosphorylated.
Cleft as the catalytic site has to be close to target protein, recognise and clasp it.
Glycine rich loop and catalytic loop (moving electrons).
P+1 – activation loop
Mg2+ loop – aspartate, phenylalanine, glycine
4 kinases picked at random. Protein kinase A has an identical structure to the other protein
kinases. All four kinases have similar structures as seen when laid one on top of the other,
with tiny differences in structure that contribute to different functions.
Taken the main chain backbone of structure and superimposed them. Entirely different
kinases with different structures. Helices superimpose on helices and beta strands
superimpose on beta strands, so chemically look the same and function based on core
enzyme activity with small elaborations to do particular things at particular times.
,Protein kinases undergo concerted molecular movements
Whether catalytic cycle turns depends on protein it wants to phosphorylate. Once ATP
binding site is filled, suck in the protein that it wants to phosphorylate. Phosphopeptide
leaves and kick out ADP to repeat cycle. No metabolic pathway runs faster than its slowest
step which is removing ADP.
ADP and ATP binding is slightly different because they are differently shaped molecules.
Small flexing and changes in the shape of the kinase as the different steps happen.
Small lobe
Key residues – target to make a manipulated sort of biology. Handful of residues are critical.
Glycine rich loop makes interactions with ATP through backbone
Valine – aliphatic short chain
Important targets that target the specific residue.
All lie in around the binding cleft of ATP.
No bonds are made with gamma phosphate to ATP.
Recreating new bond between phosphate and peptide substrate which is catalytic loop.
Most ATP found in cells are in complex with magnesium, so it is a critical part of any enzyme
that binds ATP. ATP and magnesium must bind at the same time.
ATP is sitting in the cleft holding the two parts of the enzyme together. Bonds are made and
unmade as part of the catalytic cycle.
Having ADP and ATP there comes to the light of structural spines such as hydrophobic
spines. Cyan amino acids are hydrophobic to stack up like a spine except for the gap that fills
up with ADP. Only when ATP is bound is it in the correct structural conformation, where the
hydrophobic spine is stabilised by the binding of ADP/ATP. Key residues are valine 57,
alanine 70 and lysine 173. Enzyme will not take up the correct shape of what it needs to do
as a key structural part of the enzyme. The conformation becomes more closed up when
ADP/ATP bind.
Appreciation of cell signalling from atoms to organisms
The importance of sequence consensus or context
Kinases can be highly specific or have a broader range of acceptable substrates.
Proteins take up stable structures due to water hating core.
Polar amino acids enriched on the surface. Not all accept a phosphate.
Phosphorylation sites have a few characteristics that are needed:
1. Physcial accessibility: Surface outside that cleft which sucks in the protein substrate.
Whatever needs to be phosphorylated has to be readily accessible.
2. Cryptic sites: change in protein structure required to reveal phosphorylation site. Not
all are immediately accessible normally but can become exposed.
3. Consensus sequence – which amino acids flank the S, T and Y. Any amino acids will
have 2 neighbours on either side so is embedded in particular sequence flanking the
amino acids.
, Serine/threonine kinases will recognise serine and/or threonine sufficiently to
phosphorylate them. Swapping S or T for a Y will inhibit or abolish activity.
Kinases only add phosphate to some surface-accessible residues but not all. Besides the S, T
or Y residue, the kinase protein targets other parts of the molecule as well. Across the active
site there is a place for the target to dock.
Protein-protein interactions favour some substrates. Adaptors and interaction domains
improve association between kinase and target substrate protein and even sub-optimal
sequences can be phosphorylated because the kinase and target are held in close proximity
for long periods of time.
Surfaces of the kinase structure which the incoming substrates are coming to dock to are
highly conserved and regulated. The binding substrate has a conserved surface that matches
it. Preference for particular amino acids flanking the sequence will also be conserved, giving
rise to the consensus sequence.
Lock and key model
Can have multiple indentations created by the kinase. Positive against negative,
hydrophobic with hydrophobic. Exactly what protein fits into the kinase will be
phosphorylated.
Kinase can read a word of sequence of amino acid. Sensitive to surrounding amino acids.
Consensus sequences can be identified in two ways:
1. Multiple sequence alignment
2. Degenerate peptide libraries
Multiple sequence alignment
Analysis amongst known targets of kinases.
Can be driven by computers – what does the kinase phosphorylate in cells? What do
they all have in common?
Take 5 sequences and do alignment along target site.
Serine as phosphorylation site in the middle. Each of the flanking positions have
something in common such as a pair of lysines always at -4/-5 amino acids from the
phosphorylation site. Methionine at -2, and glutamate at +4 and add them to
emerging consensus sequence.
Alanine is a softer kind of constraint than what it would go for. Coding the particular
bit of the target substrate to the particular conformation that the kinase
phosphorylates. Some parts of the consensus sequence can vary in multiple
substrates because it is a looser part of binding.
With the amino acid sequence, can search the database and see what other proteins
have this similar amino acid sequence, which means it can be a potential substrate.
Degenerate peptide libraries
If not all targets of a kinase is known, can use the above technique.
First part is molecular, then practicals and then second part is cellular.
Molecular – protein structure/chemistry
Keywords
GEF – Protein domains which that activate monomeric GTPases by stimulating the release of
of GDP to allow GTP binding.
GAP – Bind to activated G proteins to stimulate their GTPase activity and turn them off.
From GTP to GDP.
Ras – single subunit small GTPase related in structure to the G alpha subunit. Has intrinsic
GTPase activity and will hydrolyse bound GTP to GDP. However too slow by itself for
function, so bound to GAP to make RasGAP complex that supply additional catalytic residues
so that a water molecule is optimally positioned for nucleophilic attack of the gamma-
phosphate of GTP.
Sos – Bind to Ras-GTPases and force them to release their bound nucleotide.
Lecture 1: Protein kinase structure-function and reversible post-translational protein
modification by phosphorylation
Models made out of X-ray diffraction data so best guesses, but not exactly right. Only
have structure for things they get in the state. Many proteins are partially stable and
unwound, and take up thick structures transiently, so don’t have much data with
changing structures.
Much of cell signalling and signal transduction is for moving info across the
membrane. Changes to the environment or transmitted messenger molecule which
causes a signal transduction to occur. Molecules which cannot pass through have
receptors on the outside of the cell.
There is a receptor and then there are effectors inside which stimulate activity
internally. Look at protein kinases and GTPases.
Transduction events lead to signal in the nucleus, or something gets secreted out of
the cell.
How the input turns into the output.
Proteins which respond to phosphorylation signal.
What is phosphorylation?
Kinase places phosphate onto other proteins such as other kinases.
Only 5 accept phosphate group: serine, threonine, tyrosine, histidine (transient – only holds
it briefly) and aspartate.
Prokaryotes can use phosphorylated aspartate but little evidence seen in eukaryotes.
Commonly share an -OH group.
Thinking as a chemist, works as a condensation reaction. Esterify phosphate. H 2O as a by-
product.
Phosphatase inject a molecule of water to break the bond and release the phosphate. So
hydrolysis in the backwards direction.
Source of phosphate from ATP. Some kinases use GTP.
,All phosphorylated in the tissue are attached to serine and threonine. Antibodies recognise
phosphotyrosine and lead to bias in literature, with phosphorylation of tyrosine residues.
Plenty of oncogenes that are mutated that give rise to cancers are tyrosine kinases. Tiny
changes in signal. Levels of tyrosine phosphorylated proteins are held low because they are
such a strong signal so change in 1% is a 10-fold change in signal.
Importance of archetypes
Once a molecular machine exists with particular function, if function can develop in different
ways they develop families. Different members of family have specific individual functions.
Map principles from one protein kinase and apply it to the other members of the family.
Protein kinase A is the first protein discovered. First signal transduction mechanism was the
signal for transmitting adrenaline. cAMP then has its effect in cells through the activation of
PKA. cAMP is a second messenger that sets off this enzyme.
Heterotetramer – 2 regulatory subunits (blue) and catalytic subunits (green).
On regulatory subunit, they have binding sites for cAMP. If one molecule binds into binding
site, then the second binding site is more avid for binding cAMP, so a non-linear response
curve is produced. Diffuses through the cell and binds to regulatory subunits. Once the
cAMP is bound, there is a conformational change in regulatory subunits and change the
shape to no longer hold onto catalytic subunit. Exchange ADP for ATP to phosphorylate
things.
Protein kinases have a bilobal structure. Small lobe on top and larger lobe on bottom.
Small lobe – ATP binding
Large lobe – peptide binding
ATP binds at the ATP binding site which has a crevice.
Proteins that start as phosphorylating kinases to phosphorylating other proteins has the
same structure.
Has a gap in the middle of the two lobes.
Beta strand sheets on the top and mostly alpha helices on bottom. Articulated spine keeps
two lobes in proximity.
Kinases have sites where they themselves can be phosphorylated.
Cleft as the catalytic site has to be close to target protein, recognise and clasp it.
Glycine rich loop and catalytic loop (moving electrons).
P+1 – activation loop
Mg2+ loop – aspartate, phenylalanine, glycine
4 kinases picked at random. Protein kinase A has an identical structure to the other protein
kinases. All four kinases have similar structures as seen when laid one on top of the other,
with tiny differences in structure that contribute to different functions.
Taken the main chain backbone of structure and superimposed them. Entirely different
kinases with different structures. Helices superimpose on helices and beta strands
superimpose on beta strands, so chemically look the same and function based on core
enzyme activity with small elaborations to do particular things at particular times.
,Protein kinases undergo concerted molecular movements
Whether catalytic cycle turns depends on protein it wants to phosphorylate. Once ATP
binding site is filled, suck in the protein that it wants to phosphorylate. Phosphopeptide
leaves and kick out ADP to repeat cycle. No metabolic pathway runs faster than its slowest
step which is removing ADP.
ADP and ATP binding is slightly different because they are differently shaped molecules.
Small flexing and changes in the shape of the kinase as the different steps happen.
Small lobe
Key residues – target to make a manipulated sort of biology. Handful of residues are critical.
Glycine rich loop makes interactions with ATP through backbone
Valine – aliphatic short chain
Important targets that target the specific residue.
All lie in around the binding cleft of ATP.
No bonds are made with gamma phosphate to ATP.
Recreating new bond between phosphate and peptide substrate which is catalytic loop.
Most ATP found in cells are in complex with magnesium, so it is a critical part of any enzyme
that binds ATP. ATP and magnesium must bind at the same time.
ATP is sitting in the cleft holding the two parts of the enzyme together. Bonds are made and
unmade as part of the catalytic cycle.
Having ADP and ATP there comes to the light of structural spines such as hydrophobic
spines. Cyan amino acids are hydrophobic to stack up like a spine except for the gap that fills
up with ADP. Only when ATP is bound is it in the correct structural conformation, where the
hydrophobic spine is stabilised by the binding of ADP/ATP. Key residues are valine 57,
alanine 70 and lysine 173. Enzyme will not take up the correct shape of what it needs to do
as a key structural part of the enzyme. The conformation becomes more closed up when
ADP/ATP bind.
Appreciation of cell signalling from atoms to organisms
The importance of sequence consensus or context
Kinases can be highly specific or have a broader range of acceptable substrates.
Proteins take up stable structures due to water hating core.
Polar amino acids enriched on the surface. Not all accept a phosphate.
Phosphorylation sites have a few characteristics that are needed:
1. Physcial accessibility: Surface outside that cleft which sucks in the protein substrate.
Whatever needs to be phosphorylated has to be readily accessible.
2. Cryptic sites: change in protein structure required to reveal phosphorylation site. Not
all are immediately accessible normally but can become exposed.
3. Consensus sequence – which amino acids flank the S, T and Y. Any amino acids will
have 2 neighbours on either side so is embedded in particular sequence flanking the
amino acids.
, Serine/threonine kinases will recognise serine and/or threonine sufficiently to
phosphorylate them. Swapping S or T for a Y will inhibit or abolish activity.
Kinases only add phosphate to some surface-accessible residues but not all. Besides the S, T
or Y residue, the kinase protein targets other parts of the molecule as well. Across the active
site there is a place for the target to dock.
Protein-protein interactions favour some substrates. Adaptors and interaction domains
improve association between kinase and target substrate protein and even sub-optimal
sequences can be phosphorylated because the kinase and target are held in close proximity
for long periods of time.
Surfaces of the kinase structure which the incoming substrates are coming to dock to are
highly conserved and regulated. The binding substrate has a conserved surface that matches
it. Preference for particular amino acids flanking the sequence will also be conserved, giving
rise to the consensus sequence.
Lock and key model
Can have multiple indentations created by the kinase. Positive against negative,
hydrophobic with hydrophobic. Exactly what protein fits into the kinase will be
phosphorylated.
Kinase can read a word of sequence of amino acid. Sensitive to surrounding amino acids.
Consensus sequences can be identified in two ways:
1. Multiple sequence alignment
2. Degenerate peptide libraries
Multiple sequence alignment
Analysis amongst known targets of kinases.
Can be driven by computers – what does the kinase phosphorylate in cells? What do
they all have in common?
Take 5 sequences and do alignment along target site.
Serine as phosphorylation site in the middle. Each of the flanking positions have
something in common such as a pair of lysines always at -4/-5 amino acids from the
phosphorylation site. Methionine at -2, and glutamate at +4 and add them to
emerging consensus sequence.
Alanine is a softer kind of constraint than what it would go for. Coding the particular
bit of the target substrate to the particular conformation that the kinase
phosphorylates. Some parts of the consensus sequence can vary in multiple
substrates because it is a looser part of binding.
With the amino acid sequence, can search the database and see what other proteins
have this similar amino acid sequence, which means it can be a potential substrate.
Degenerate peptide libraries
If not all targets of a kinase is known, can use the above technique.
