Download The relationship between amino acid sequences and protein folds.

Protein Structure and Function
Instructor: !
!
James Omichinski!
!
Contact Information:!
!
E-mail: !
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[email protected]!
Section #1:
The relationship between amino acid
sequences and protein folds.
Correlation between sequence and protein fold.
There are two competing models:
Model 1: !The Local Model"
•! fold specificity is defined by a few critical residues.
•! this model is supported by by misfolding mutations associated with
certain diseases such as cystic fibrosis.
Model 2: !The Global Model"
•! the entire sequence of the protein contributes equally to the fold.
•! this model is supported by mutation studies that show most
mutations at any position have no measurable impact on protein
function.
The case of the IgFF fold.
If one examines the members of the immunoglobulin fold family
(IgFF):
•! Many members of the IgFF are distally related proteins or
evolutionarily unrelated proteins
•! Despite their common fold there is low sequence identity between
subgroups suggesting both convergent and divergent evolution.
•! It seems the common connection for all IgFF members are
residues that form a common hydrophobic core.
•! This supports the global model.
Reference: Halby et al, Prot. Engineering 12, 563-571 (1999)
The Common Core for IgFF Proteins.
The conserved core region
is sheet I (green) and sheet
II (blue) of 52 IgFF
members with less than
55% sequence identify. !
!
There are wide variations
in the loop (red) regions of
IgFF members. These
regions have developed as
part of the evolutionary
process.!
!
Relation Between IgFF Sequence and Structure
Zone I!
Zone II!
Zone I: High identity and similar structures.!
Zone II: Low identity and similar structures.!
Sequence Identity, Homology and Fold
•! Amino acid homology is a better criteria then amino acid
identities.
•! General rule is that sequences that share 30% sequence homology
will fold similarly.
•! In the immunoglobulin fold family (IgFF) there can be <10%
identity.
•! In IgFF the residues constituting the core are concentrated in a
small number of conserved positions that define the fold.
Reference: Wood and Pearson, J. Mol. Biol.291, 977-995 (1999)
Protein G as a Model for Studying Cores
•! Protein G contains two types of domains (GA and GB).
•! GA binds to human serum albumin and has a 3-! fold .
•! GB binds to Fc region of IgG and has a 4! + " fold.
•! It is possible to make two proteins, a GA like domain and a GB like
domain that share 77% identity in 56 amino acids (GA77 and
GB77).
•! GA77 and GB77 have different folds and different functions despite
having 77% identity.
Reference: P.A. Alexander et al., Proc Natl Acad Sci (2009), V106:21149-54
GA77 versus GB77
GB77
GA77
Reference: P.A. Alexander et al., Proc Natl Acad Sci (2009), V106:21149-54
Interconverting GA77 and GB77
•! The goal was to interconvert the two proteins with the fewest number of
mutations.
•! The conversion should change both the structure and the function to be
considered successful.
•! It turns out that it is possible to do this with a single point mutation.
Reference: P.A. Alexander et al., Proc Natl Acad Sci (2009), V106:21149-54
A point mutant converts GA77 to a GB77
Reference: P.A. Alexander et al., Proc Natl Acad Sci (2009), V106:21149-54
Section #2:
Intrinsically disordered proteins.
The case of acidic activation domains
Intrinsically disordered domains in proteins
•! Many disordered segments fold on binding (coupled folding and binding).
•! It is now clear that the occurrence of disordered regions is surprisingly
common in functional proteins.
•! These regions can be highly conserved between species in both amino acid
composition and sequence.
•! These regions are often characterized by a low content of hydrophobic
amino acids and a high concentration of polar or charged amino acids.
•! They are particularly abundant in transcription factors, signaling
proteins, autoinhibitory domains and viral proteins.
•! The classic example of functional unstructured region is the
transactivation domain (TAD) of transcription factors.
Liu et al., PNAS USA 106: 19819-23 (2009).
PIC, HATs, remodeling and mediator
Role of activators in transcription
•! Activators function through the recruitment of general transcription
factors, the mediator complex, histone acetyl transferases and the
chromatin remodeling proteins.
•! Activators function by participating in a series of protein/protein
interactions through their transactivation domain (TAD).
•! TADs are characterized by a high percentage of a particular amino acid
and are generally thought to be unstructured in the unbound state.
•! The tumour suppressor protein p53 and the Herpes Virion Simplex
protein 16 (VP16) are two of the most potent activators known and they are
acidic-rich.
PIC, HATs, remodeling and mediator
TFIIH and TADs
TFIIH
Core TFIIH
CAK Complex
•! TFIIH is recruited to the PIC through interactions with p62 /Tfb1
(human/yeast) and the acidic carboxyl-terminal domain of TFIIE!.
•! Several acidic TADs interacts directly with the p62/Tfb1 subunit of
TFIIH and this interaction correlates with their ability to activate
initiation and elongation.
•! The interaction of p62/Tfb1 with p53 and VP16 requires the aminoterminal Pleckstrin Homology (PH) domain of p62/Tfb1.
p53
•! p53 is a potent transcriptional activator.
•! It induces expression of many genes whose products mediate cellgrowth arrest and apoptosis, thus blocking cell transformation and
tumor formation.
•! Mutations in p53 leading to inactivation of its tumor suppressor
function leads to the development of about 50% of human cancers.
•! A large percentage of the critical mutations occur within the DNA
binding domain of p53.
•! Amino terminal 100 amino acids contains a extremely potent
activation domain that is highly acidic and unstructured.
Schematic of the functional domains of p53
Proline
N-terminal
Transactivation Rich
Domain
Domain
TAD
PP
DNA Binding Domain
Tetradimerization
domain
DBD
TET
REG
C-terminal
Regulatory Domain
P P
P
P
P
P
S6 S8 S15 T18 S20
TAD1
MDM2
P
P
S33 S37
P
S46 T55
P
T81
TAD2
Tfb1/p62
•! The transcriptional activation function of
p53 is associated with its highly acidic TAD,
which can be divided into two independent
subdomains, TAD1 (residues 1-40) and TAD2
(residues 40-83).
Structure of the Tfb1/p53 complex
The structure of Tfb1 in the complex presents a typical PH fold.
The p53 TAD2 in the complex is unstructured except for a short
amphipathic !-helix involving residues 47-55.
Di Lello et al., Molecular Cell, V22, pg731, 2006.
Details of the Tfb1/p53 interface
N
C
Three hydrophobic residues I50, W53 and F54 of p53 form the
interface with Tfb1.
(Di Lello et al., Mol. Cell, 2006)
Schematic of the functional domains of VP16
Protein/protein
Interaction Domain
C-terminal
Transactivation
Domain
TAD
Core
•! VP16 is composed of two functional
domains: A core regulatory domain and a
TAD (residues 412-490).
•! The acidic TAD can be divided into two
independent subdomains, VP16N (residues
412-455) and VP16C (residues 456-490). (pI=
3.43)
VP16N
VP16C
Structure of Tfb1/VP16C
VP16C
N
"7#
"6#
C
"5#
N
C
Tfb1
Like p53 TAD2, VP16C in complex with Tfb1 is unstructured except
for a 9-residue ! helix between residues 472-480.
Langlois et al., J. Am. Chem. Soc., 130: 10596, 2008
Comparison of Tfb1/p53 and Tfb1/VP16 c
p53
C
M478!
C
W53
VP16
F54
I50
F479!
N
F475!
N
- F475, M478 and F479 of VP16 in
the Tfb1/VP16 complex participate
in many of the same interactions as
I50, W53 and F54 of p53 in the
Tfb1/p53 complex.
Langlois et al., J. Am. Chem. Soc., 130: 10596, 2008
Comparison of Tfb1/p53 and Tfb1/VP16
VP16
p53
T480!
T55
E51
E476!
D472!
S46
•! E476 and T480 of VP16 in the
Tfb1/VP16 complex participate in
the same interactions as E51 and
T55 of p53 in the Tfb1/p53
complex. However, S46 of p53
corresponds to A471 of VP16.
Langlois et al., J. Am. Chem. Soc., 130: 10596, 2008
Summary
•! The viral activator VP16 can mimic many of the properties of the
mammalian activator p53 in complex with Tfb1/p62.!
•! Hydrophobic and acidic residues play key roles in defining the
interface between acidic TADs and Tfb1/p62.!
!
•! Select activators like p53 have evolved so that phosphorylation
events play a key role in their regulation. This is not the case for
the viral activator VP16.
Why disordered regions?
•! Allow protein to bind to multiple partners using lower affinity
interactions!
•! Enhance probability of posttranslational modifications such as
phosphorylation, acetylation and methylation.!
!
•! Enhance turn over rates of proteins, turn on and turn off theory.
Section #3:
Membrane Proteins.
Types of transmembrane receptors
•! 7-transmembrane segment (7-TMS) receptors- contain a seven
transmembrane (helical) segment, an extracellular ligand binding
site and and intracellular recognition site for a GTP-binding
protein.
•! Single-transmembrane (1-TMS) catalytic receptors- contain a
single transmembrane segment, an extracellular ligand domain
and intracellular catalytic domain (tyrosine kinase or guanylyl
cyclase)
•! Oligomeric ion channels- contain more than one subunit each of
which contains transmembrane segments, they are ion-ligated so
binding of ligand opens the ion channel.
Structures of transmembrane proteins
•! The ones solved to date are minimal since it is very hard to mimic
the membrane using NMR or X-ray techniques.
•! Two types of structures have been observed so far, either all !
helical or " barrels.
•! You need 20-25 residues in an ! helical sequence to span the
thickness (30Å) of the lipid bilayer.
•! You need only 7-9 residues in a " conformation to span the lipid
bilayer since it is a more extended conformation.
•! Hydrophathic plots can be used to predict ! helical
transmembrane domains but not " barrels.
Bicontinuous lipidic cubic phase
•! This system was developed to help obtain better crystals of
membrane proteins.
•! Once inserted into this continuous 3D curved lipid bilayer,
membrane proteins diffuse laterally to nucleate and eventually to
form well-ordered crystals.
•! Used to crystallize bacteriorhodopsin and rhodopsin.
Rhodopsin
•! It is a G-protein-coupled receptor (GPCR)
•! Rhodopsins are largest subfamily (90%)
•! They are light activated and turn on a signaling pathway that
leads to vision.
•! Composed of protein opsin and 11-cis retinal through K296.
•! Absorption of photon changes 11-cis form to all-trans-retinal.
•! The process is transient as trans-retinal is hydrolyzed and
dissociates from the opsin to be replaced by newly synthesized 11cis retinal.
Schematic of a 7-TMS receptor
Structure of Rhodopsin
Crystals were generated in micelles!
!
Helices are arranged differently then!
in Bacteriorhodpsin (bR).!
!
The extramembrane regions are larger!
and more ordered in Rhodopsin then in !
bR which demonstrates the functional !
difference.!
!
Reference: Palczewski et al, Science 289 739-745!
(2000)!
The retinal binding pocket
A269 and F261 are responsible for absorption differences!
between red and green pigments.!
"2 Adrenergic Receptor ("2AR)
•! It is a G-protein-coupled receptor (GPCR)
•! The ! and " andrenergic receptors differ in tissue localization,
ligand specificity, G-protein coupling and downstream effector
mechanisms
•! Genetic modifications of andrenergic receptors are associated with
a variety of human diseases including asthma, hypertension and
heart failure.
•! "2AR#s are found in smooth muscles throughout the body.
•! "2AR agonists are used in treatment of asthma and they are of
considerable interest to the pharmaceutical industry.
•! It differs from rhodopsin in that the ligand is freely diffusible and
not covalently bound.
"2 Adrenergic Receptor ("2AR)
"2AR Crystal Structure
•! There have been several crystal structures GPCR over the last five
years that are based on some interesting tricks to help cyrstallize
membrane proteins.
•! The first one solved was the structure of the "2AR (Cherezov et al,
Science V318, pg 1258, 2007) and two different techniques were
used.
•! In the higher resolution (2.4 Å) structure, the third intracellular
loop was replaced by inserting the sequence for T4-lysozyme).
•! In the lower resolution (3.4/3.7 Å) structure, the "2AR was
crystallized in complex with a Fab that binds the third
intracellular loop.
•! In both structures, the "2AR was crystallized in the presence of
the partial inverse agonist carazolol (Inactive conformation).
•! These structures have led to several other structures of GPCRs
using variations of the T4-lysozyme strategy.
The two "2AR structures:T4L vs FAB5
2.4Å resolution
3.4/3.7Å resolution
Rosenbaum et al, Science, 2007.
The T4L-"2AR structure
Cherezov et al, Science, 2007.
The T4L-"2AR structure
Carazolol
"2AR
Bound lipids
(Cholesterol and palmitic acid)
T4 Lysozyme
Cherezov et al, Science, 2007.
The T4L-"2AR structure:Contacts with T4L
There are only a limited number of intramolecular interactions
between the T4 Lysozyme (T4L-green) and the "2AR (gold).
Cherezov et al, Science, 2007.
The T4L-"2AR structure:Ligand binding site
Binding site
cleft
Negative charge
Positive charge
Cherezov et al, Science, 2007.
Ligand Binding:T4L-"2AR versus rhodopsin
"2AR
Rhodopsin
"2AR ECL2 (A) contains a short helix and two disulfide bonds (yellow). One disulfide bond connects
to ECL1 the second connects to helix III. Phe193 of ECL2 interacts with the ligand carazolol (purple).
In contrast, ECL2 in rhodopsin (B) does not have direct access to retinal (peach).
Cherezov et al, Science, 2007.
More Recent "2AR Crystal Structure
•! There has been three new crystal structures of the "2AR using
similar techniques and in two cases with a new twist.
•! There are two crystal structures of the "2AR with an agonist
bound in the active site (active confirmation). One was done with
the T4L in the third EC loop as before (Rosenbaum et al, Nature
V469, pg 236, 2011) and one was done with the T4L and a
nanobody (Rasmussen et al, Nature V469, pg 175, 2011).
•! There is also a crystal structure of the "2AR with an agonist
bound in the active site bound to a Gs protein. In this complex, the
T4L was placed at the amino-terminal end of the protein and a
nanobody was also used (Rasmussen et al, Nature V477, pg 549,
2011).
G Protein Cycle for the "2AR –Gs Complex
Rasmussen et al, Nature(2011), 477: 549-555.
"2AR –Gs Complex
Rasmussen et al, Nature(2011), 477: 549-555.
Other Crystal Structures of GPCRs
A2A Adenosine receptor- Jaakola et al, Science (2008), 322: 1211-1217.
CXCR4 Chemokine Receptor- Wu et al, Science (2010), 330: 1066-1071.
Sphingosine 1-phosphate Receptor- Hanson et al, Science (2012), 335: 851-855.
M2 Muscarinic receptor- Haga et al, Nature(2012), 482: 547-552.
M3 Muscarinic receptor- Kruse et al, Nature(2012), 482: 552-559.
Kappa-opioid receptor- Wu et al, Nature(2012), 485: 327-334.
FQ-opioid receptor- Thompson et al, Nature(2012), 485: 395-400.
Delta-opioid receptor- Granier et al, Nature(2012), 485: 400-405.
µ$opioid receptor- Manglik et al, Nature(2012), 485: 321-327.
The A2A$denosine Receptor
Inserted T4L into the 3rd intracellular loop and truncated c-terminal end of the protein.
Jaakola et al, Science (2008), 322: 1211-1217.
The A2A$denosine Receptor
ZM241385 is an antagonist of the A2A receptor
Jaakola et al, Science (2008), 322: 1211-1217.
The CXCR4 Receptor
Inserted T4L into the 3rd intracellular loop and a thermostable point mutant.
Wu et al, Science (2010), 330: 1066-1071.
The CXCR4 Receptor
IT1t is a small molecule and CVX15 is a cyclic peptide that bind to CXCR4.
Wu et al, Science (2010), 330: 1066-1071.
The M2 Muscarnic Receptor
Inserted T4L into the 3rd intracellular loop.
M2 Muscarinic receptor- Haga et al, Nature(2012), 482: 547-552.
The M2 Muscarinic Receptor
QNB is a small molecule that binds selectively to the M2 receptor.
M2 Muscarinic receptor- Haga et al, Nature(2012), 482: 547-552.
The M3 Muscarinic Receptor
Inserted T4L into the 3rd intracellular loop.
M3 Muscarinic receptor- Kruse et al, Nature(2012), 482: 552-559.
The M3 Muscarinic Receptor
Tiotropium is a small molecule that binds selectively to the M3 receptor.
M3 Muscarinic receptor- Kruse et al, Nature(2012), 482: 552-559.
The Delta Opioid Receptor
Inserted T4L into the 3rd intracellular loop.
Delta-opioid receptor- Granier et al, Nature(2012), 485: 400-405.
.
The Delta Opioid Receptor
Naltrindole is a small molecule that binds selectively to the Delta opiod receptor.
Delta-opioid receptor- Granier et al, Nature(2012), 485: 400-405.
.
The Kappa Opioid Receptor
Inserted T4L into the
3rd intracellular loop.
Kappa-opioid receptor- Wu et al, Nature(2012), 485: 327-334.
The Kappa Opioid Receptor
JDTic is a small molecule that binds selectively to the Kappa opioid receptor.
Kappa-opioid receptor- Wu et al, Nature(2012), 485: 327-334.
The FQ Opioid Receptor
Inserted the thermostabilize apocytrochrome b562RIL (BRIL) in place of 43 amino
acids at the n-terminus and deleted 31 residues as the c-terminus.
FQ-opioid receptor- Thompson et al, Nature(2012), 485: 395-400.
The FQ Opioid Receptor
C24 is a small molecule that binds selectively to the FQ receptor.
FQ-opioid receptor- Thompson et al, Nature(2012), 485: 395-400.
The µ Opioid Receptor
Inserted T4L into the 3rd intracellular loop.
µ$opioid receptor- Manglik et al, Nature(2012), 485: 321-327.
The µ Opioid Receptor
"-funaltrexamine ("-FNA)is a small molecule that binds selectively to the µ opioid receptor.
µ$opioid receptor- Manglik et al, Nature(2012), 485: 321-327.
Section #4:
DNA as an allosteric ligand of proteins.
Reference: S.H Meijsing et al., Science 324, 407-410 (2009).
Glucocorticoid Receptor
•! The glucocorticoid receptor (GR) is a potent transcriptional
activator that contains a ligand-binding domain for
dexamethasone.
•! Ligand binding is required prior to binding DNA and
inducing gene expression.
•! It induces expression of many target genes whose by
associating with specific DNA-binding sites, the sequences of
which differ dramatically between genes.
•! GR binds to palindromic and non-palindromic sequences as a
dimer and binding to DNA induces dimerization.
Schematic of the functional domains of GR
•! GR is composed of three functional domains: Activation
Function 1 (AF1: residues 1-406), DBD (residues 440-525) and
LBD/AF2 (residues 550-770).
•! The DNA-binding domain (DBD) contains two zinc fingers.
Binding to DNA occurs subsequent to hormone binding.
•!The DBD binding site consists of two hexameric half sites
separated by 3- or 4-base pairs (imperfect palindromic
sequences).
GR target DNA sites (GBS) differ in sequence
•! All sequences are derived from endogenous target genes.
•! All sequences have comparable basal activity but differ
considerably when induced with dexamethasone (dex).
•! There is not a good correlation between in vitro binding and
in vivo transcriptional activity.
Mutations in GR domains effect GBS activity
•! Mutations were introduced into the dimerization motif (red),
the AF1 (yellow) and in the AF2 (green).
•! The mutations had different effects on different GBS sites.
•! This suggested that binding to different targets were causing
different interactions at regions distant from the DNA-binding
residues.
The role of the lever arm in GR function
•! In 13 crystal structures of GR-DBD:GBS complex, the
structures were virtually identical except the lever arm.
•! In one chain H472 was flipped in and in the other chain
H472 was flipped out.
•! In the flipped out chain, the conformation of the lever arm
was heterogeneous and this suggests that the DNA sequence
directs distinct changes in the conformation of the lever arm.
The role of the lever arm in GR function
Chain A versus chain B
Chain B: 3 bp versus 4 bp spacer
The role of the lever arm in GR function
•! The GR% splice variant differs from GR! in the lever arm.
•! The two isoforms display similar abilities to repress the
osteocalcin gene .
•! The two isoforms display varying abilities to activate genes
depending on the target GBS sequence.
The role of the lever arm in GR function
•! The lever arm and interactions with residues in the core help
to define the specificity of the steroid hormone receptors.
DNA as an allosteric ligand of proteins
Summary:
•! The binding interface of the 13 GR:GBS complexes are very
similar and suggest the different roles is not due to the
specific interactions with DNA.
•! The structures demonstrate the importance of the lever arm
to the function of the GR and potentially to other steroid
hormone receptor.
•! The results suggest that binding DNA can alter the properties
of the GR. Thus, DNA is acting as an allosteric ligand for the
proteins functions.