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Domain Structures
Branden & Tooze, Chapter 3
Alpha Domain: The Lone Helix
• There are a number of examples of small
proteins (or peptides), which consist of little
more than a single helix.
• A striking example is glucagon, a hormone
involved in regulating sugar metabolism in
mammals (as is insulin).
Alpha-Domain Structures
Coil-coil a Helices
• A coiled-coil a helix has a
repetitive heptad amino acid
sequence (transcription factor
GCN4). Within each heptad the
amino acids are labeled a-g.
• The a helices in the coiled-coil are
slightly distorted so that the helical
repeat is 3.5 residues rather than
3.6, as in a regular helix. There is
an integral repeat of seven
residues along each helix.
Packing of a Coil-coil
• Every seventh residue in both a
helices is a leucine, labeled “d”.
• Due to the heptad repeat, the dresidues pack against each
other along the coiled-coil.
• Residues labeled “a” are also
usually hydrophobic and
participate in forming the
hydrophobic core along the
coiled-coil.
Heptad Repeats.
• Salt bridges can stabilize coiled-coil structures and are
sometimes important for the formation of heterodimeric
coiled-coil structures. The residues labeled “e” and “g” in the
heptad sequence are close to the hydrophobic core and can
form salt bridges between the two a helices of a coiled-coil
structure, the e-residue in one helix with the g-residue in the
second and vice versa.
Helix-helix Packing
• The side chains of an a helix
are arranged in a helix row
along the surface of the helix
• They form ridges separated by
shallow furrows or grooves on
the surface.
a helices pack with the ridges
with one helix packing into the
grooves of the other and vice
visa.
• Ridges can be formed by the
sidechains separated by four
residues with an angle of 25 º
(i +4) and by 3 residues with
an angle of 45 º (i +3) to the
helical axis.
Helix-helix
Packing
• By fitting the ridges of side
chains from one helix into the
grooves between side chains of
the other helix and vice versa, a
helices pack against each
other. (a) Two a helices, I and
II, with ridges from side chains
separated by four residues
marked in red and blue,
respectively. In panel 4, the
orientation of the helices has
been rotated 50° in order to
pack the ridges of one a helix
into the grooves of the other. (b)
In the red a helix, the ridges are
formed by side chains
separated by four residues and
in the blue a helix by three
residues. The a helices are
rotated 20° in order to pack
ridges into grooves, in a
direction opposite that in (a).
Knobs in Holes Model
• One way to visualize how the helices fit together is using the
Knobs in Holes Model.
• The positions of the side chains along the surface of the
cylindrical a helix is projected onto a plane parallel with the
helical axis for both a helices of the coiled-coil.
• The side-chain positions of the first helix, the "knobs,"
superimpose between the side-chain positions in the second
helix, the "holes."
Alpha-Domain Structures
Four-helix Bundles
• Four-helix bundles
frequently occur as
domains in a proteins.
• The arrangement of the a
helices is such that
adjacent helices in the
amino acid sequence are
also adjacent in the threedimensional structure.
• Some side chains from all
four helices are buried in
the middle of the bundle,
where they form a
hydrophobic core.
Four-helix
Bundles
• In cytochrome
b562 (a)
adjacent helices
are antiparallel,
whereas the
human growth
hormone (b) has
two pairs of
parallel a helices
Dimeric RNA-binding Protein Rop
• The four helix bundle may be formed by
more than a single protein sequence.
• Each subunit of Rop comprises two a
helices arranged in a coiled-coil
structure with side chains packed into
the hydrophobic core according to the
"knobs in holes" model.
• The two subunits are arranged in such a
way (i.e., “ridges in grooves”) that a
bundle of four a helices is formed.
Some Bioinformatics Tools for Structure
Visualization
• Pymol http://www.pymol.org/
– Software for viewing/manipulating protein structure
• Protein Data Bank: http://www.rcsb.org/pdb/
– Database of proteins. Includes downloadable structure and
sequence files.
Ex. ROP Protein (1ROP)
Alpha-Domain
Structures
The Globin Fold
• This fold has been found in a large group of related proteins (i.e.,
conserved) including myoglobin and hemoglobin.
• The globin fold usually consists of 8 α helices (A-H). The two helices
at the end of the chain are antiparallel, forming a helix-turn-helix motif,
but the remainder of the fold does not include any characterized
supersecondary structures.
• These helices pack against each other with larger angles, around 50 °
between them than occurs between antiparallel helices
(approximately 20°) so that the helices form a hydrophobic pocket for
the heme active site.
The Globin Fold has been Preserved
During Evolution
• The 3D structures of globin proteins from
different organisms (mammals, plants, and
insects) are solved and they share the
same essential features of the globin fold.
• The sequence homology is from 99 % to
16 %, which is very low.
• How can amino acid sequences that are
very different for proteins be very similar in
their 3D structures?
Evolution of Globins
• Arthur Lesk & Cyrus Chothia in the UK examined residues that
are structurally equivalent to positions in 9 known globin
structures, that are involved in helix-heme contacts, and in the
packing of the helices against each other.
– There are a total of 59 positions preserved, 31 buried in the
middle of protein and 28 in contact with the heme group.
– There is no conserved sequences nor size-compensatory
mutations in the hydrophobic core formed by the 31 a.a.
– Conclusion: The evolutionary divergence of globins has
been constrained primarily by conservation of the
hydrophobicity of the residues buried in the helix-helix and
helix-heme contact.
How do Proteins Adopt to Changes
in the Size of Buried Residues?
• The mode of packing for the a helices are the same
in all the globin structures
• The same types of packing ridges into grooves occur
in corresponding a helices in all these structures.
– The relative positions and orientations of the a
helices change to accommodate changes in the
volume of sidechains involved in the packing.
– The structure of loop regions changes so that the
movement of one helix is not transmitted to the
rest of the structure to preserve the geometry of
the heme pocket.
Globin fold
Visualization
PDBfile 1mbn
•
“The Stereochemistry of the Protein Myoglobin.” Watson, H.C. (1969)
Prog. Stereochem. 4: 299.
Hemoglobin:
Quaternary
Structure
• The hemoglobin molecule
is built up of four
polypeptide chains: two a
chains and two b chains.
Each chain has a threedimensional structure
similar to that of
myoglobin: the globin
fold.
• In sickle-cell anemia, Glu
6 in the b chain of
hemoglobin is mutated to
Val, thereby creating a
hydrophobic patch on the
surface of the molecule.
Sickle-cell Hemoglobin
•
•
Sickle-cell
hemoglobin
molecules
polymerize due to
the hydrophobic
patch introduced
by the mutation
Glu 6 to Val in the
b chain.
Hemoglobin in
cells polymerize
into fiber that
deform
erythrocytes into
sickle shapes:
confers
resistance to
malaria for
heterozygote
carriers.
Alpha / Beta Structures
• Alpha/beta domains are found in many proteins.
• The most regular and common domain structures
consist of repeating beta-alpha-beta super-secondary
units, such that the outer layer of the structure is
composed of alpha helices packing against a central
core of parallel beta sheets. These folds are called
alpha/beta, or wound alpha beta.
• There are three main classes
of a/b proteins (b-a-b):
– TIM barrel
– Rossman Fold (open sheets)
– Horseshoe fold
TIM Barrel
• A core of twisted parallel b
strands arranged close
together, like the staves of a
barrel.
• Here shows a closed barrel
exemplified by schematic
and topological diagrams for
the enzyme triosephosphate
isomerase.
The b-a-b motifs are connected sequentially, and
a helices are on the same side of the b sheet.
Properties of TIM Barrel
 a/b-barrel has been found
in more than 15 proteins.
 Most of them are enzymes
with completely different
amino acid sequences and
different functions.
 Branched hydrophobic
side chains dominate the core
of this class of proteins.
• In most a/b-barrel structures the eight b strands of the barrel
enclose a tightly packed hydrophobic core formed entirely
by side chains from the b strands. The core is arranged in
three layers, with each layer containing four side chains
from alternate b strands. The schematic diagram shows this
packing arrangement in the a/b barrel of the enzyme
glycolate oxidase.
Methylmalonyl-coenzyme A Mutase
• One exception, the
inside of the barrel
methylmalonylcoenzyme A mutase
is lined by small
hydrophilic side
chains (serine and
threonine) from the b
strands, which
creates a hole in the
middle where one of
the substrate
molecules, coenzyme
A (green), binds
along the axis of the
barrel from one end
to the other.
Coenzyme A (CoA)
a/b Barrel Active
Site
• The active site in all a/b barrels
is a pocket formed by the loop
regions that connect the
carboxy ends of the b strands
with the adjacent a helices (a).
• A view from the top of
the barrel of the active site of
the enzyme RuBisCo (ribulose
bisphosphate carboxylase),
which is involved in CO2
fixation in plants (b).
Alpha / Beta Structures
Open a/b Sheet
• Open twisted b sheet is
surrounded by ahelices on both sides of
the b-sheet.
• Examples of different
types of open twisted
a/b structures (a) the
FMN-binding redox
protein Flavodoxin and
(b) the enzyme
adenylate kinase,
which catalyzes the
reaction AMP + ATP
<=> 2ADP.
Active Site of Open a/b Sheet
• There are always two adjacent bstrands on opposite sides of a bsheet. One of the loops from one
of these two b-strands goes
above the b-sheet, whereas the
other loop goes below, which
creates a crevice outside the
edge of the b-sheet between two
loops.
• Almost all binding sites in this
class of proteins are located in
crevices at the carboxy end of
the b sheet.
•A schematic view of the active site of tyrosyl-tRNA synthetase. Tyrosyl adenylate,
the product of the first reaction catalyzed by the enzyme, is bound to two loop
regions: residues 38 - 47, which form the loop after b strand 2, and residues 190 - 193,
which form the loop after b strand 5. The tyrosine and adenylate moieties are bound
on opposite sides of the b sheet outside the carboxy ends of b strands 2 and 5.
a/b–horseshoe
Fold
What would the consensus sequence be ?
a/b –horseshoe Fold is formed by amino acid
sequences that contain repetitive regions of a
specific pattern of a helices and b-strands. The b
strands form a curved parallel b sheet with all the
a helices on the outside.
Schematic diagram of the structure of the
ribonuclease inhibitor. The molecule, which is built
up by repetitive b-loop-a motifs, resembles a
horseshoe with a 17-stranded parallel b sheet on
the inside and 16 a helices on the outside.
Leucine-rich Motifs
• Consensus amino acid sequence and
secondary structure of the leucine-rich
motifs of type A and type B. “X”
denotes any amino acid; “a” denotes
an aliphatic amino acid. Conserved
residues are shown in bold in type B.
• In the ribonuclease inhibitor, leucine
residues 2, 5, and 7 from the b strand
pack against leucine residues 17, 20,
and 24 from the a helix as well as
leucine residue 12 from the loop to
form a hydrophobic core between the
b strand and the a helix.
Alpha / Beta Structures
Visualization using Chimera
PDBfile 2fcr
•
“Crystal Structure of Oxidized Flavodoxin from a red algae Chondrus
crispus refined at 1.8A resolustion. Description of the flavin mononucleotide
binding site.” Fukuyama, K. (1992) J. Mol. Biol. 225: 775-789. PubMed
1602481.
PDBfile 1fue
Flavodoxin from Helicobacter pylori. Freigang, J. 2002.
PDBfile 4h2d
NDOR1 (NADPH-dependent diflavin oxidoreductase 1. Banci, L.
2013.
Beta Structures
• Anti-parallel b strands are usually
arranged in two b-sheets that
pack against each other and form
a distorted barrel structure, the
core of the structure.
• Depending on the way the bstrands around the barrel are
connected along the polypeptide
chain, they can be divided into
four major groups:
Up-and-down barrel
superoxide dismutase (SOD)
Greek Key barrel
Jelly roll barrel
b-helix
Up-and-down
Barrel
• Schematic and
topological diagrams of
an up-and-down b
barrel.
• The eight b strands are
all antiparallel to each
other and are connected
by hairpin loops.
• Beta strands that are
adjacent in the amino
acid sequence are also
adjacent in the threedimensional structure of
up-and-down barrels.
Retinol-binding Protein (RBP)
• The structure of human plasma retinol-binding protein
(RBP) is an up-and-down b barrel. A retinol molecule,
vitamin A (yellow), is bound inside the barrel,
between the two b sheets, such that its only
hydrophilic part (an OH tail) is at the surface of the
molecule.
Green Fluorescent Protein
 from Aequorea victoria (jellyfish)
 11-stranded b-barrel with central ahelix
 chromophore formed during folding
(from residues S65, Y66, G67) is
contained in the middle of the barrel
 contains 1 Trp, 10 Tyr
Excitation 395 nm and 475 nm
Emission 506 nm
http://www.plantsci.cam.ac.uk/Haseloff/GFP/GFPbackgrnd.html
Amino Acid Sequence Reflects b
Structure
• Amino acid sequence of b strands 2, 3, and 4 in
human plasma retinol-binding protein.
• The sequences are listed in such a way that residues
which point into the barrel are aligned.
• These hydrophobic residues are shown by arrows
and are colored green. The remaining residues are
exposed to the solvent.
Beta Structures
Greek Key Motifs•
This motif is formed when
one of the connections of
four antiparallel b strands is
not a hairpin connection.
• The motif occurs when
strand number n is
connected to strand n + 3 (a)
or n - 3 (b) instead of n + 1 or
n - 1 in an eight-stranded
antiparallel b sheet or barrel.
The two different possible
connections give two
different hands of the Greek
key motif.
• In all protein structures
known so far, only the hand
shown in (a) has been
observed.
The Fold of IgG
Domains
• Beta strands labeled A-G of the constant and variable domains
of immunoglobulins have the same topology and similar
structures. There are two extra b strands, C' and C'' (red) in the
variable domain. The loop between these strands contains the
hyper-variable region CDR2. The remaining CDR regions are at
the same end of the barrel in the loops connecting b strands B
and C and strands F and G.
Gamma Crystallin Domain
• The domain structure of g-crystallin is built up from
two b sheets of four antiparallel b strands, sheet 1
from b strands 1, 2, 4, and 7 and sheet 2 from
strands 3, 5, 6, and 8.
• It is obvious that the b strands are arranged in two
Greek key motifs, one (red) formed by strands 1 - 4
and the other (green) by strands 5 - 8.
Complete g-crystallin Molecule
• The two domains of the
complete molecule have the
same topology; each is
composed of two Greek key
motifs that are joined by a
short loop region.
• There is a greater amino
acid sequence homology
between the domains than
the motifs within each
domain, suggesting that the
four Greek Key motifs in gcrystallin are evolutionarily
related by gene duplication
and fusion.
Jelly Roll Motifs
• The eight b strands
are drawn as
arrows along two
edges of a strip of
paper. The strands
are arranged such
that strand 1 is
opposite strand 8,
etc..
• The b strands follow
the surface of the
barrel and the loop
regions provide the
connections at both
ends of the barrel.
Two-sheet b helix.
• The two parallel b sheets are
colored green and red, the loop
regions that connect the b
strands are yellow.
• Each structural unit is composed
of 18 residues with 9 consensus
sequence Gly-Gly-X-Gly-X-AspX-U-X forming a b-loop-b-loop
structure, where U is a large
hydrophobic residue, often Leu.
• Each loop region contains six
residues of sequence Gly-Gly-XGly-X-Asp where X is any
residue. Calcium ions are bound
to both loop regions.
What are the role of the Glycines?
What is the role of the Aspartic acid?
Extracellular bacterial proteinase
Three-sheet b Helix
• As shown in (a), two of the b sheets (blue and yellow) are
parallel to each other and are perpendicular to the third
(green). In (b), each structural unit is composed of three b
strands connected by three loop regions (labeled a, b and c).
• Loop a (red) is invariably composed of only two residues,
whereas the other two loop regions vary in length.
Beta Structures
Visualization using Chimera
PDBfile 2kfb
•
“Structure of the cataract causing P23T mutant of human gamma-D
Crystallin.” Jung, J. (2009) Biochemistry 48: 2597-2609.
PDBfile 4jgf, 1h4a.
PDB
Species
WT / mutant Method
1h4a
Human
WT
Xray 1.15A
2kfb
Human
P23T
NMR
4jgf
Human
P23T
Xray
2.5 A
Schematic Structural Diagrams of
Myoglobin
Richardson Diagrams
Myoglobin
Triosephosphate isomerase
Cylinder for a helices; arrows for b strands, which gives the
direction of the strand from N to C; and the ribbons for the
remaining part.
Calcium-binding proteins (CaBPs)
Binding affinities
Kd (M)
10-3CaR
Triggering
Ca
10-4
Thermolysin
10-5
Calmodulin
Ca2+-free
Calmodulin
(1cfc)
Ca2+-binding
Calmodulin
(3cln)
10-7
Protease K
Buffering
Stabilizing
10-8
Calbindin D9k
10-9
α-Lactalbumin
Parvalbumin
Calbindin D9K
(1b1g)
Thermolysin
(1tlx)
http://bioinformatics.org/firstglance/fgij/1000px-Amino_Acids.svg.png
Common Features of Globular Proteins
The non-polar residues V, L, I, M and F largely occur in the interior of a
protein, out of contact with the aqueous solvent.
The charged polar residues R, H, K, D and E are largely located on the surface
of a protein in contact with the aqueous solvent. If they are located inside of a
protein, they often have specific chemical function, such as promoting catalysis
or participating in metal binding.
The uncharged polar groups S, T, N, Q, Y and W are usually on the surface but
frequently occur in the interior of the protein, forming hydrogen bonds with
other groups.
Globular protein cores are efficiently arranged with their side chains in relaxed
conformations. The protein interior is efficiently packed.