Protein Structure and Function
CHAPTER3.
Control of Protein Function
3-0 Overview : Mechanisms of Regulation

Protein function in living cells is precisely regulated
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Cells are densely packed (250,000 proteins/ a bacteria); regulation of protein function is
essential to avoid chaos. (by localization, interaction with effector molecules and by the
amount and lifetime of the active protein)

Proteins can be targeted to specific compartments and
complexes
-
by signal sequences (specific amino-acid sequence), lipid tails and interaction domains
When the protein is not in the location where it is needed, very often it is maintained in an
inactive conformation.
3-0 Overview : Mechanisms of Regulation

Protein activity can be regulated by binding of an effector
and by covalent modification
-
Binding of effector induces conformational changes that produce inactive or active
forms of the protein.
-
Post-translational covalent modification may either activate or inactivate the
proteins. (phosphorylation, methyltation, acetylation, carbohydration, proteolytic
cleavage…)
-
Signal amplification is an essential for the control of cell function and
covalent modification is the way such amplification is usually achieved; kinase
cascade, blood clotting
3-0 Overview : Mechanisms of
Regulation

Protein activity may be regulated by protein quantity and
lifetime
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Amount of protein can be set by the level of transcription
(promoter strength or transcription factor)
-
Amount of mRNA can be regulated by RNA degradation.
-
Amount of protein can by regulated by Ubiquitinproteosome degradation.
3-0. Overview : Mechanisms of Regulation

A single protein may be subject to many regulatory influences
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is achieved largely through signal transduction networks .
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Ex)The cyclin-dependent protein kinases (CDK) to control the cell cycle are
regulated by a number of different mechanisms.
3-0. Overview
: Mechanisms of Regulation
- Ckd and cyclin binding
→ conformational change
→ phosphorylation by CAK
→ activation
further phosphorylation or
dephosphotylation on other
tyrosines finely regulate the
activities
Figure3-1.The cyclin-dependent protein kinases that control progression
through the cell cycle are regulated by a number of different mechanisms
3-1. Protein Interaction Domains
Different small domains → distinct binding specificities and funtions
Phosphoserine and phosphothreonine motif
Toll-like receptors
- Interaction domains can be divided into distinct families whose members
are related by sequence structure, and ligand binding properties.
Figure3-2. Interaction domains
3-1. Protein Interaction Domains
b-catenin
Vesicle fusion
Figure3-2. Interaction domains
3-1. Protein Interaction Domains
IκBs mask NLS of NF-kB proteins;
Figure3-2. Interaction domains
3-1. Protein Interaction Domains
Figure3-2. Interaction domains
3-1. Protein Interaction Domains
Figure3-2. Interaction domains
3-1. Protein Interaction Domains
Figure3-2. Interaction domains
3-2. Regulation by Location
Protein function in the cell is contextdependent
-Temporal and spatial control over a
protein’s activity must be exercised.
-Temporal control: regulating gene
expression and protein lifetime.
-Spatial control: location control.
- Translocated by a specific organelle,
a cargo vesicle.
-Precise localization of protein is
essential for proper protein function
Figure3-3.The internal structure of cells
Various Kinases, Tem1
3-2. Regulation by Location
There are several ways of targeting proteins in cells
(a). By sequences in the protein
itself
(b-c). Covalent chemical
modification of the protein
(d). Binding to scaffold proteins
Figure3-4. Mechanisms for targeting proteins
3-3. Control by pH and Redox Environment
- Protein function is modulated by the
environment in which the protein operates
-Changes in redox environment can greatly
affect protein structure and function
: cysteine residues in proteins are usually fully
reduced to –SH groups inside the cell but are readily
oxidized to disulfide bond when secreted.
-Changes in pH drastically alter protein
structure and function
Active site open
: Modulation of the surface charge of a protein by
pH change could influence binding strength .
: charged group in their active sites can be
changed by pH alteration (two Asp in active site).
Figure3-5. Cathepsin D, endopeptidase conformational switching in
3-3. Control by pH and Redox Environment
Proton pump
Diphtheria toxin : B, T, A domain
B domain : bind to a receptor in the target-cell membrane.
A domain : kills cells by catalyzing the ADP-ribosylation of elongation factor 2 on the
ribosome.
T domain : deliver the catalytic domain into the cytoplasm of the target cell.
Exposure to the reducing environment inside the endocytic vesicle breaks the disulfide bond
between the A and B domains, releasing the toxic A domain.
In low pH, the exposure of hydrophobic residues of the diphtheria toxin T domain results in its
insertion into the endosomal membrane →As a channel, T domain transfers the A domain
Figure3-6. Schematic representation of the mechanism
by which diphtheria toxin kills a cell
Glutathione serves as a sulfhydryl buffer and an antioxidant
Structure of glutathione peroxidase
2GSH + RO-OH  GSSG +
H2O + ROH
3-4. Effector Ligands
: Competitive Binding and Cooperativity
Protein function can be controlled by effector ligands that bind competitively to
ligand-binding or active sites
- FEED BACK INHIBITION : the end product of the pathway acts as a
competitive inhibitor of the first enzyme.
Figure3-7. Competitive feedback inhibition
3-4. Effector Ligands
: Competitive Binding and Cooperativity
Cooperative binding by effector ligands amplifies their
effects
-Amplification: covalent modification,
cooperativity
-Cooperativity : positive cooperativity
negative cooperativity
-Positive cooperativity : binding of one molecule of a ligand
to a protein makes it easier for a second molecule of that
ligand to bind.
-Nagative cooperativity : binding of the second molecule is
more difficult.
Figure3-8. Cooperative ligand binding
3-5. Effector Ligands
: Conformational Change and Allostery
Effector molecules can cause conformational changes at distant sites
(a) The binding of successive effector molecules causes a
Sequential model Concerted model
sequential series of conformational changes form the initial
state to the final state by induced fit.
-subunits need not exist in the same conformation
-substrate-binding causes increased substrate affinity in
adjacent subunits
-conformational changes are not propagated to all subunits
(b). The effector can only bind to one of these forms, and its
binding shifts the equilibrium in a concerted manner on favor of
the bound form.
-Allostery : from the Greek for “another structure”
-Allostery activator
-Allostery inhibitor
Figure3-9.Two models of allosteric regulation
3-5. Effector Ligands
: Conformational Change and Allostery
ATCase is an allosteric enzyme with regulatory
and active sites on different subunits
Carbamoyl phosphate + Aspartate  N-carbamoyl
Aspartate,
CTP
-ATCase is a hetero-oligomer. Six catalytic and six regulatory
subunit.
-Allosterically inhibited by cytidine triphosphate (end product,
feedback inhibitor).
-Allosterically activated by ATP(= activator).
-Mutation of Y77F, the site away from active site stabilized
the T state resulting in the inhibition of enzyme.
Figure3-10. Ligand-induced conformational change activates aspartate transcarbamoylase
3-5. Effector Ligands
: Conformational Change and Allostery
Binding of gene regulatory proteins to DNA is often controlled by ligandinduced conformational changes.
- Co-activators and Co-repressors: small molecules, metal ions or proteins
control binding of the activator or repressor to DNA.
DtxR: specific repressor of
Diphtheria toxin .
Binding of DtxR to its operator
sequence is controlled by the
concentration of Fe2+ in the
bacterial cell.
Figure3-11. Iron binding regulates the repressor of the diphtheria toxin gene
3-6. Protein Switches Based on Nucleotide Hydrolysis
Most protein switches are enzymes that catalyze the hydrolysis of a nucleoside
triphosphate to the diphosphate
- GTPase : major class of switch protein (G protein)
- ATPase : usually associated with motor protein complexes or transporters
GTPase
ATPase
-two-component response regulator: histidine kinase, response regulator proteins
Why ATP or GTP are used for trigger of switch?
Figure3-12. Structure of the core domains of a typical GTPase and an ATPase
3-6. Protein Switches Based on Nucleotide Hydrolysis
- Triphosphate-bound state = “on”, spring-loaded
- Loss of gamma phosphate group → conformational change.
- Two hydrogen bonds in the each switch (Ⅰ and Ⅱ).
a- and b- phosphates are bound to Ploop (GXXXXGKS/T)
g-phospate is bound to both switch I and
II (DXnT and DXXG respectively)
Although common structural and functional
features in switch proteins, many insertions
of other domains in individual GTPases
present various functions.
Figure3-13. Schematic diagram of the universal switch mechanism of GTPases
3-7. GTPase Switches
: Small Signaling G Proteins
The switching cycle of nucleotide hydrolysis and exchange in G proteins is
modulated by the binding of other proteins
GTP hydrolysis rate is very low
→ GAP(GTPase-activating protein)
increase the rate by 105 fold
GDP release is conducted by
GEF(guanidine-nucleotide exchange factors)
Opening up the binding site
Figure3-14.The switching cycle of the GTPase involves interactions with
proteins that facilitate binding of GTP and stimulation of GTPase activity



Small GTPase Ras family: H-, N-,and K-ras, 21kDa, lipid
attachment
Signal transduction by Ras is dependent on the GTP-bound
state. A prolonged on state are found in up to 30% of human
tumors. Reduction of GTP hydrolysis is caused by point
mutations at 12, 13 or 61 resulting in uncontrolled cell growth
and proliferation. Good target for anti-tumor therapy.
How the GAP facilitate GTP hydrolysis?
- GAP insert an arginine side chain into the nucleotide-binding
site of the GTPase. The positive charge on the side chain helps
to stabilize the negative charge in the transition state for
hydrolysis of the g-phosphate group of GTP
How
the GEF facilitate GDP release?
- GEF binding induces conformational changes in the P loop and
switch regions of the GTPase while the rest of the structure is largely
unchanged. The binding of the GEF sterically hinders the magnesiumbinding site and interferes with the phosphate-binding region by
insertion of an alpha helix into nucleotide binding site. When
the GEF binds the GTPase, the phosphate groups are released first and
the GEF is displaced upon binding of the entering GTP molecule.
- After GDP has disassociated from the GTPase, GTP generally
binds in its place, as the cytosolic ratio of GTP is much higher than
GDP at 10:1. The binding of GTP to the GTPase results in the release
of the GEF, which can then activate a new GTPase. Thus, GEFs both
destabilize the GTPase interaction with GDP and stabilize the
nucleotide free GTPase until a GTP molecule binds to it.
From wikipedia
3-8. GTPase Switches
: Signal Relay by Heterotrimeric GTPases

-
-
Heterotrimeric GTPase
α, β and γ subunit.
α subunit consist of the canonical G domain and an extra helical domain.
β and γ subunit are tightly associated with each other by coiled-coil
interaction.
G protein associated with G protein coupled receptor(GPCR).
GDP-bound G protein bind to GPCR = “off” state.
When activated by ligand, these receptors act as GEF for their partner
G protein.
When GDP is released and GTP binds, G protein dissociates from the
GPCR.
In the absence of β and γ, α does not bind to GPCR.






Regulator of G-protein signaling proteins (RGS proteins) are
responsible for the GTPase catalytic rate. How it increase the
rate?
a subunit of G-protein has a “built-in” arginine residue in the
extra helical domain that projects into the catalytic site. RGS
proteins bind to the switch regions, reducing the flexibility and
stabilization the transition state for hydrolysis.
Paticular RGS proteins regulate particular GPCRs; specificity
GPCRs are the most numerous receptors in all eukaryotic
genome (1-5% of the total number of genes)
various ligands such as light, orants, lipids, peptide hormones.
8 families
3-8. GTPase Switches
: Signal Relay by Heterotrimeric GTPases
GPCR =
“Off” state
WD40
Coiled-coil interaction
Figure3-15. Hypothetical model of a heterotrimeric G protein in a complex
with its G-protein-coupled receptor
1994 Nobel prize in Physiology & Medicine
3-9. GTPase Switches
: Protein Synthesis
EF-Tu(elongation factor)
– GTP bound form
Figure3-16.The switching cycle of the elongation factor EF-Tu delivers
aminoacyl-tRNAs to the ribosome
3-10. Motor Protein Switches
Myosin is ATP-dependent nucleotide switches
①
②
③
④
-① ADP and Pi are bound to the heads as a result
of hydrolysis of a bound ATP by the intrinsic
ATPase activity of the catalytic core(blue).
-② Myosin head docking onto a specific binding
site(green) on the actin thin filament(gray).
-③ On actin docking Pi is released from the active
site, and there is a conformational change in the
head that causes the lever arm to swing to its
“poststroke” ADP-bound position(red).
-④ ADP dissociates and ATP binds to the active
site and undergoes hydrolysis.
Figure3-17. Models for the motor actions of muscle myosin and kinesin
3-10. Motor Protein Switches
Kinesin is ATP-dependent nucleotide switches
①
②
③
④
-① The “trailing” head has ADP bound and the
“leading” head is empty and neither linker is
docked tightly to the micro-tubule.
-② When ATP binds to the leading head, its linker
adopts a conformation that as well as docking it
firmly to the microtubule reverses its position and
thus throws the trailing head forward by about 160Å
towards the next binding site on the microtubule.
-③ Binding also accelerates the release of ADP
from this head, and during this time the ATP on the
other head is hydrolyzed to ADP-Pi.
-④ After ADP dissociates from the new leading
head ATP binds in its turn, causing the linker to
zipper onto the core.
Figure3-17. Models for the motor actions of muscle myosin and kinesin
3-10. Motor Protein Switches
Switch Ⅱ region of the
motor protein kinesin
Switch Ⅱ region of the
motor protein Myosin
Switch Ⅱ region of the
G protein
ATPase domains of motors and the GTPase domains of G proteins are different.
Figure3-18. Structural and functional similarity between different families of molecular switches
The Inner Life of the Cell

http://www.youtube.com/watch?v=wJyUtbn0O5Y
3-12. Control of Protein Function by Phosphorylation

Protein function can by controlled by covalent modification
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50~90% of the proteins in the human body are post-tranlationally modified.
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Phosphorylation, glycosylation, lipidation, and limited proteolysis……
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Most covalent modifications can change the location of the protein, or its activity, or its
interatcions with other proteins and macromolecules.
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Two phosphorylation effect : 1). Change the activity of the target protein
2). Provide a new recognition site for another protein to bind
3-12. Control of Protein Function by Phosphorylation
-Phosphorylation is reversible → be suited
as a regulatory mechanism
-Dimerization of two receptor by ligand
binding → phosphorylated at cytoplasmic
domain → creating binding sites for an
adaptor molecule(Grb2) → Ras activated →
MAPKKK phosphorylation → MAPKK
phosphorylation → MAPK phosphorylation
→ other substrate phosphorylation
Figure3-21. A kinase activation cascade in an intracellular signaling pathway
that regulates cell growth
3-12. Control of Protein Function by Phosphorylation
- Phosphorylation of Glycogen phosphorylase : rearrangement of the
amino-terminal residues about 50Å.
Figure3-22. Conformational change induced by phosphorylation in glycogen phosphorylase
3-12. Control of Protein Function by Phosphorylation
-TCA-cycle enzyme.
-There are no conformational changes
by phosphorylation
- attachment of a phosphoryl group
Substrate bound
inhibits binding of the negatively
charged substrate by steric exclusion
and elecrostatic repulsion.
Phosphotylated
Figure3-23. Inactivation of the active site of E.coli isocitrate dehydrogenase by phosphorylation
3-13. Regulation of Signaling Protein Kinases
: Activation Mechanism
Protein kinases are themselves controlled by phosphorylation
Protein kinases reponsible for
posphorylating proteins on
serine, threonine and tyrosine
residues all have the same fold
for the catalytic domain
Many of them also have other
subunits or other domains that
serve regulatory functions or
targeting to substrate
Figure3-24.The conserved protein kinase catalytic domain
3-13. Regulation of Signaling Protein Kinases
: Activation Mechanism
-Most kinases are normally inactive.
-Before they can phosphorylate other
proteins they must themselves be activated
by their activation loop .
D
T/Y
D
T/Y
The activation loop plays a central part
in regulating catalyric activity.
A conserved aspartate in the activation
loop is critical to the catalytic action of
the kinase.
Figure3-25. Conserved mechanism of kinase activation
3-13. Regulation of Signaling Protein Kinases
: Activation Mechanism
Src kinases both activate and inhibit themselves
SH2 bind to an inhibitory phosphate
on a tyrosine
SH2 releases the carboxyl tail of the
protein. New conformation for substrate
binding and auto-phosphorylation in the
activation loop
Figure3-26. Regulation of a Src-family protein kinase
3-14. Regulation of Signaling Protein Kinases
: Cdk Activation
Cyclin acts as an effector ligand for cyclin-dependent kinases
- The structure of Cdk2 alone.
- In unphosphorylated state, Cdk2 is autoinhibited by the activation loop.
Figure3-27. Regulation of Cdk2 activation
3-14. Regulation of Signaling Protein Kinases
: Cdk Activation
- The structure of the complex of unphosphorylated Cdk2 with cyclinA.
- Only 0.3% active, Show come significant conformational changes.
Figure3-27. Regulation of Cdk2 activation
3-14. Regulation of Signaling Protein Kinases
: Cdk Activation
- The structure of the phospho-Cdk2-CyclinA complex.
- Fully activated.
Figure3-27. Regulation of Cdk2 activation
3-15. Two-Component Signaling Systems in Bacteria
Two-component system in Bacteria
1). ATP-dependent histidine protein
kinase(HK)
2). Response regulator protein(RR)
-Dimerization of HK and dimerization of RR
→ HKs catalyze ATP-dependent
autophosphorylation of Histidine →
phosphoryl group transfer to aspartate of RR
→ Responses
Figure3-29.Two-component signaling mechanisms
3-15. Two-Component Signaling Systems in Bacteria
Blue : unphosphorylated
Purple : phosphorylated
-All RRs have the same general fold and share a set of conserved residues.
-A common mechanism appears to be involved in the structural changes
that propagate from the active site.
Figure3-30. Conserved feature of RR regulatory domains
3-16. Control by Proteolysis
: Activation of Precursors
Limited proteolysis can activate enzymes
-Limited proteolysis involves the
cleavage of a target protein at no
more than a few specific sites
(commonly one, by specific
protease).
-Maturation of the inactive
precursor chymotyrpsinogen to
active alpha-chymotrypsin.
- Cleavage between residues 15 and 16 results in a rearrangement of part of
the polypeptide chain.
Figure3-31. Activation of chymotrypsinogen
3-16. Control by Proteolysis
: Activation of Precursors
Red : Plasmonogen(inactive)
Blue : Plasmin(active)
- Plasminogen is activated by a proteolytic cleavage.
Figure3-32. Comparison of the active sites of plasminogen and plasmin
3-16. Control by Proteolysis
: Activation of Precursors
Polypeptide hormones are produced by limited proteolysis
- Limited proteolysis can also
produce polypeptides with new
functions.
- A number of other cleavages,
which are tissue-specific, occur
in different cell types to produce
different ensembles of hormones.
Figure3-33. Schematic diagram of prepro-opiomelanocortin and its processing
3-16. Control by Proteolysis
: Activation of Precursors
- Several such activations may follow
one another to generate a proteolytic
cascade in which the initial activation
of a single molecule of an inactive
proenzyme produces a huge final
output.
Figure3-34.The blood coagulation cascade
3-17. Protein Splicing
: Autoproteolysis by inteins
Some proteins contain self-excising intein
-Intein : A protein intron. An internal portion
of a protein sequence that is posttranslationally excised in an auto-catalytic
reaction.
-More than 100 inteins have been
discovered.
-In the protein splicing process, segment of
peptide chain excises itself from the protein
in which it is embedded.
Figure3-35. Protein splicing
3-17. Protein Splicing
: Autoproteolysis by inteins
-Intein : A to G
-Site A, B and G represent conserved sequences important for
self-splicing
-A : Ser1
-B : ThrXXHis
-G : HisAsn
Figure3-36. Schematic of the organization of intein-containing proteins
3-17. Protein Splicing
: Autoproteolysis by inteins
- Example of interin : From the gyrase A subunit of Mycobacterium xenopi
Figure3-37. Structure of an intein
3-17. Protein Splicing
: Autoproteolysis by inteins
Step 1 : A side chain oxygen or sulfur(X) of
the first intein residue attacks the carbonyl
group of the peptide bond that it makes
with the preceding amino acid
Step 2 : Carbonyl is attacked by the first
residue of the carboxyl-terminal extein
segment.
Figure3-38. Four-step mechanism for protein splicing
3-17. Protein Splicing
: Autoproteolysis by inteins
Step 3 : The last residue of the intein,
which is most commonly an asparagine,
then cyclizes internally through its own
peptide carbonyl group
Step 4 : Releasing both the intein and the
extien, in which the amino-terminal and
carboxy-terminal segments are connected
via side chain of the first residue of the
carboxy-terminal segment
Figure3-38. Four-step mechanism for protein splicing
3-17. Protein Splicing
: Autoproteolysis by inteins
The carboxy-terminal domain
of Drosophila Hedgehog
protein possesses an autoprocessing activity that
results in an intra-molecular
cleavage of full-lenth protein
Figure3-39. Structure of part of the Hedgehog carboxy-terminal autoprocessing domain
3-18. Glycosylation
Glycosylation can change the properties of a protein and provide recognition sites
-Glycosylaion : Attachment of carbohydrate chains
-Almost all secreted and membrane-associated proteins
of eukaryotic cells are glycosylated.
-Function
1). Specific oligosaccharides provide recognition sites
2). Shield large areas of the protein surface, providing
protection from proteases
Figure3-40. Immunoglobulin A protects mucosal surfaces from pathogenic organisms
3-18. Glycosylation
-Simple eukaryote attach only a simple
set of sugars.
-Mammals modify their proteins with
highly branched oligosaccharides.
-The commonest modifications
Figure3-41. Schematic representation of the core N-linked oligosaccharide
and a representative O-linked core oligosaccharide
3-18. Glycosylation
-As the protein is being synthesized in the endoplasmic reticulum(ER) the
precursor is transferred to asparagine residues in the signal sequence(NXS)
-To produce the mature oligosaccharides, the N-glycosylation core is first
trimmed in the ER.
-And additional processing occur in the Golgi complex
Figure3-42. Oligosaccharide processing
3-18. Glycosylation
- Structure determined by NMR
Figure3-43.The structure of Glc3Man9GlcNac2
3-19. Protein Targeting by Lipid Modifications

Lipid attachment is one of the most common posttranslational modifications in eukaryotic cells.

The process is sequence specific, always involves
residues either at or near either the carboxyl terminus or
the amino terminus of the protein.
3-19. Protein Targeting by Lipid Modifications
- Myristoylation : 14-carbon fatty acid chain
is attacked via a stable amide linkage to an
amino-terminal glycine residue
- Palmitoylation : 16-carbon fatty acid chain
is attacked via a labile thioester linkage to a
cysteine residue
- Prenylation : a prenyl group is attacked via
a labile thioester linkage to a cysteine
residue initially four positions from the
carboxyl terminus that becomes carboxylterminal after proteolytic trimming and
methylation of the new caboxyl terminus
Figure3-44. Membrane targeting by lipidation
3-19. Protein Targeting by Lipid Modifications
Glycosylphosphoatidylinositol (GPI) anchor
-The anchor consists of an
oligosaccharide chain.
-The protein is connected
through an amide linkage to a
phosphoethanolamine molecule
Figure3-45. Glycosylphosphatidylinositol anchoring
3-19. Protein Targeting by Lipid Modifications
-ARF is myristoylated at its amino
terminus and when GDP is bound this
hydrophobic tail is sequestered within
the protein.
- ARF is itself recruited to the Golgi
membrane by a GTP-exchange
protein, and on binding GTP, ARF
undergoes a conformational changes.
-Membrane-bound ARF then recruits
coat proteins necessary for vesicle
budding and transport.
Figure3-46. Working model for vesicular transport between Golgi compartments
3-20. Methylation, N-acetylation,
Sumoylation and Nitrosylation
Figure3-47. Structures of methylated arginine and lysine residues
3-20. Methylation, N-acetylation,
Sumoylation and Nitrosylation
Figure3-48. N-acetylation
3-20. Methylation, N-acetylation,
Sumoylation and Nitrosylation
-SUMO(Small Ubiquitin-related modifier)
-The consensus sequence for sumoylation is ψKXE(ψ = hydrophobic aa.)
-A SUMO precusor is processed to SUMO by Ulp proteins.
Figure3-49. Sumoylation
3-20. Methylation, N-acetylation,
Sumoylation and Nitrosylation
Nitrosylation : reversible modification of proteins by NO groups
Figure3-50. Cysteine nitrosylation