Transcript TIM BARREL

TIM BARREL
Teresa Páramo
Miguel Hernández
Diana Garzón
Index

Introduction





Super families
Conserved regions




Comparison & prediction
Evolution




Examples
Residues
Super families & functions
Active sites


Structure
Composition
Loop regions
Evolution of sequences
Super imposition
Gene fusion
Fold change
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
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Insertion, Deletion & Substitution
Circular permutations
Strand Invasion
Beta Hairpin Flip Swap
Introduction

The TIM barrel is an extremely common
protein fold (which constitutes nearly 10%
of all known enzymes) consisting of eight
α-helices and eight parallel β-strands that
alternate along the peptide backbone.
Introduction

Lineage
 Class:
α/β Proteins
 Fold: TIM α/β Barrel
 Super families:

32 super families
Structure

The α-helices and β-strands form a
solenoid that curves around to close on
itself in a doughnut shape, topologically
known as a toroid.
Structure

The parallel β-strands form the inner wall
of the doughnut (hence, a β-barrel),
whereas the α-helices form the outer wall
of the doughnut.
Composition
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The protein's core is tightly
packed with bulky hydrophobic
amino acid residues.
The packing interactions
between the sheets and helices
are also dominated by
hydrophobicity.
The branched aliphatic
residues valine, leucine, and
isoleucine comprise about 40%
of the total residues in the βstrands.
Loop regions

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Out of the 200 residues required to form a TIM barrel,
only 160 are considered structurally equivalent between
different proteins sharing this fold.
The remaining residues are located on the loop regions
that link the helices and sheets; the loops at the Cterminal end of the sheets tend to contain the active site,
which is one reason this fold is so common.
The residues required to maintain the structure and the
residues that effect enzymatic catalysis are for the most
part distinct subsets. The linking loops can, in fact, be so
long that they contain other protein domains.
Super families
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Triosephosphate isomerase (TIM)
Ribulose-phoshate binding barrel
Thiamin phosphate synthase
Pyridoxine 5'-phosphate synthase
FMN-linked oxidoreductases
Inosine monophosphate dehydrogenase
(IMPDH)
PLP-binding barrel
NAD(P)-linked oxidoreductase
(Trans)glycosidases
Metallo-dependent hydrolases
Aldolase
Enolase C-terminal domain-like
Phosphoenolpyruvate/pyruvate domain
Malate synthase G
RuBisCo, C-terminal domain
Xylose isomerase-like
Bacterial luciferase-like
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
Nicotinate/Quinolinate PRTase Cterminal domain-like
PLC-like phosphodiesterases
Cobalamin (vitamin B12)-dependent
enzymes
tRNA-guanine transglycosylase
Dihydropteroate synthetase-like
UROD/MetE-like
FAD-linked oxidoreductase
Monomethylamine methyltransferase
MtmB
Homocysteine S-methyltransferase
(2r)-phospho-3-sulfolactate synthase
ComA
Radical SAM enzymes
GlpP-like
CutC-like
ThiG-like
TM1631-like
Conserved regions

As structure in general is more conserved than sequence, thorough
structural comparisons and structure-based sequence alignments
can help, in cases where standard sequence-alignment tools fail, to
determine distant evolutionary relationships.

One of the most intriguing features among members of this class of proteins
is although they all exhibit the same tertiary fold there is very little sequence
homology between them.

Of the approximately 200 residues required to fully form a TIM
Barrel, about 160 are considered structurally equivalent between
different proteins sharing this fold.
Alignment of sequences or
structures
TRNA super family.
 TIM super family.
 Aldolase super family

tRNA super family
(PDBSEQ vs FASTA, ClustalW)
TIM super family
(PDBSEQ vs FASTA, ClustalW)
Aldolase super family
(PDBSEQ vs FASTA, ClustalW)
Residues
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
The remaining residues that are not structural
form the loop regions that connect the β
strands with the α helices containing the amino
acids responsible for its catalytic chemistry.
The specific enzymatic activity is, in each case,
determined by the loop regions at the carboxy
end of the β strands (length and amino acid
sequences), which do not contribute to the
structural stability
Residues
The number of active site residues at the eight βα motifs. The active site
residues, which could be aligned using multiple structure alignment, are
indicated with asterisks (*).
Super families & functions
Super family
Functions
Triosephosphate isomerase (TIM)
Isomerase
Ribulose-phoshate binding barrel
Isomerase / Lyase / Synthase
Thiamin phosphate synthase
Transferase
Pyridoxine 5'-phosphate synthase
Oxidoreductase
FMN-linked oxidoreductases
Oxidoreductase / Dehydrogenase
Inosine monophosphate
dehydrogenase
Oxidoreductase
PLP-binding barrel
Isomerase / Lyase
NAD(P)-linked oxidoreductase
Oxidoreductase
(Trans) glycosidases
Hydrolase / Transferase / Glycosidase /
Isomerase / Oxidoreductase
Metallo-dependent hydrolases
Hydrolase / Phosphotriesterase
Super families & functions
Super family
Functions
Aldolase
Lyase / Hydrolase / Transferase / Oxidoreductase
Enolase C-terminal domain-like
Lyase / Hydrolase / Isomerase
Phosphoendpyruvate/ pyruvate
domain
Lyase / Kinase / Transferase / Isomerase / Synthase
Malate synthase G
Lyase
RubIsCo large subunit C-terminal
domain
Lyase
Xylose isomerase-like
Lyase / Hydrolase / Isomerase / Oxidoreductase
Bacterial luciferase-like
Oxidoreductase
Nicotinate/Quinolinate PRTase CTerminal domain like
Hydrolase / Transferase
PLC-like phosphodiesterases
Hydrolase
Cobalamin (Vitamin B12)dependent
enzymes
Lyase / Isomerase
Super families & functions
Super family
Functions
tRNA-guanine transglycosylase
Transferase
Dihydropteroate synthetase-like
Transferase / Shyntase
UROD/MetE-like
Transferase / Lyase
FAD-linked oxidoreductase
Oxidoreductase
Monomethylamine
methyltransferase MtmB
Transferase
Homocysteine S-methyltransferase
Transferase
(2r)-phospho-3-sulfolactate
synthase ComA
Lyase
Radical SAM enzymes
Transferase / Oxidoreductase / Ligase
GlpP-like
Transcription
CutC-like
Metal binding protein
ThiG-like
Biosyntetic protein
TM1631-like
Unknown
Super families & functions
(Nagano et al. 2002, Rison et al. 2000, Pujadas and Palau, 1999,
Wierenga, 2001).
Active sites (general)

We have described a general relationship between structure and
function for the βα barrel structures. They all have the active site at
the same position with respect to their common structure in spite of
having different functions as well as different amino acid sequences.

TIM barrels serve as scaffolds for active site residues in a diverse
array of enzymes. Residues that form the active site are always
located at the same end of the barrel, associated with the C-terminal
ends of b-strands and the loops connecting these to a-helices.

In almost every one of the more than 100 different known a/b
structures of open a/b sheet the active site is at the carboxy edge of
the B sheet. Functional residues are provided by the loop regions that
connect the carboxy end of the b strands with the amino end of the
alpha helices. This is similar therefore to the a/b barrel structures.
Active sites (comparison)

The general shapes of the active sites are quite different, however.
Open a/b structures cannot form funnel shaped active sites like the
barrel structures. Instead, they form crevices at the edge of the B
sheet. Such crevices occur when there are two adjacent
connections that are on opposites sides of the B sheet.

The position of such crevices is determined by the topology of the B
sheet and can be predicted from a topology diagram. The crevices
occur when the strand order is reversed and can be easily identified
in a topology diagram as the place where connections from the
carboxy ends of two adjacent B strands go in opposite directions
one to the left and one to the right.
Active sites (prediction)

Such positions in a topology diagram are called topological switch
points. It was postulated in 1980 by Carl Branden, in Uppsala,
Sweden that the position of active sites could be predicted from
such switch points. Since then at least one part of the active site has
been found in crevices defined by such switch points in almost all
new a/b structures that have been determined.

Thus we can predict the approximate position of the active site and
possible loop regions that form this site in a/b proteins. This is
contrast to proteins of the other two main classes a helical proteins
and ant parallel B proteins, where no such predictive rules have
been found.
Evolution

Because the members of this family of
protein catalyze a wide range of reactions
and the lack of strong sequence
homology, The evolutionary history of this
family has been the subject of vigorous
debate.
Evolution
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
If members of a protein family show common
function as well as a common structure, it
has generally been assumed that all
members have diverged from a common
ancestor.
If members of a protein family has no
common function, It is possible that these
proteins are related by convergent evolution
to a stable fold.
Evolution

The evolutionary history of this family
of enzymes is still not completely
certain, but divergent evolution from a
common ancestor explains more of the
available data than does convergent
evolution to a stable fold. (Reardon and
Farber 1995; Petsko 2000; Gerlt and
Babbitt 2001)

Notwithstanding the diversity of their catalytic reactions, the active site is
always found at the C-terminal end of the barrel sheets, suggesting divergence
from an ancestral TIM barrel (Bränden & Tooze 1991).

Fifteen years ago, Farber & Petsko (1991) classified 17 TIM barrel structures
into four families on the basis of their different geometries, and suggested
divergence from a common ancestor.

Bränden also analyzed 19 TIM barrel structures, considering their domain
organization, the metal and phosphate binding sites as well as catalytic
centres. It was suggested that the presence of the common phosphate-binding
site, formed by loop-7 loop-8 and a small helix (helix-8), is the strongest
evidence obtained for the divergent evolutionary history of TIM barrels (Bräden
1991, Wilmanns et al 1991).

Using 30 TIM barrel structures and their sequence families, the evolution of TIM
barrels was discussed by Reardon & Farber (1995), and they also agree with
the divergent evolution.

More recently Copley & Bork (2000) re-analyzed a subset of the TIM barrels,
including those which bind phosphate. They suggest that five of these families
(two classes of aldolase; dihydropteroate synthetase; pyruvate kinase and
enolase) are distantly related to the enzymes with a common phosphatebinding motif.
Evolution


Because 3-dimentional structure evolves more slowly
than primary structure, the lack of sequence homology
could be due to the age of the ancestral enzyme.
The similar location of the active site in all of the
members of the family, combined with geometric
arguments concerning the barrel structure and the clear
indications of gene duplication, followed by specialization
whiting the various families, let the argued of these
proteins are related by divergent evolution from a
common ancestor.
The yellow markers indicate
the glycosidase subgroups or
family. The green color shows
the enzyme families with the
SPB motif, whilst the blue one
shows those with phosphatemoiety of the ligands in the
same position as the SPB
motif. Filled markers indicate
the metal-binding families,
whilst open markers indicate
the remainder. The triangles
show seven-stranded TIM
barrel family.
Nagano et al 2002
Evolution of the sequence

Are new enzymes formed from random sequences generated by
recombination and other genetic rearrangement or do they arise by
divergent evolution from a preexisting set of enzymes.

There is debate over whether the many different TIM barrel enzymes
are evolutionarily related, since in spite of the structural similarities
there is tremendous diversity in catalytic functions of these enzymes
and little sequence homology.

Greg Petsko provided strong evidence for the latter case from
studies of a/B barrel enzymes in a rare metabolic pathway,
conversion of mandelate to benzoate. This rare metabolic pathway
is thought to be of recent evolutionary origin, since it’s present in
only a few pseudomonad species.
Evolution of the sequence

Petsko found that the three dimensional structure of mandelate,
including it’s a/b barrel, is very similar to that of a quite different
enzyme, muconate lactonizing enzyme, which catalyzes a different
chemical reaction, but which also involves the formation of an
intermediate by proton abstraction.

The amino acid sequences of the 350 residues of these enzymes
showed 26% sequence identity, which clearly demonstrates that
they are evolutionary related. By comparing these two structures in
detail Petsko found significant similarities in the region of the active
site that catalyzes proton abstraction and intermediate formation but
substantial difference in those regions of the active site that confer
substrate specifity.
Evolution of the sequence

These results are compatible with an evolutionary history in
which the new enzyme activity of mandelate racemase has
evolved from a pre existing enzyme that catalyzes the basic
chemical reaction of proton abstraction and formation of an
intermediate.

Subsequent mutations have modified the substrate specifity
while preserving the ability to catalyze the basic chemical
reaction. Chemistry is the important factor to preserve during
evolution of new enzymes, while specifity can be modified. It
would therefore seem that relatively nonspecific enzymes
which may have existed earlier in evolution or which may arise
occasionally through random genetic rearrangements are the
clay from which nature sculpts new enzymes.
Superimposition
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tRNA (PDB -> STAMP -> RasMol, 2 sets)
Score: 9.87
Superimposition
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Aldolase (PDB -> STAMP -> RasMol, 2 sets)
Score: 9.87
Gene Fusion

A fusion gene is a hybrid gene formed
from two previously separate genes. It can
occur as the result of a translocation,
interstitial deletion, or chromosomal
inversion
Gene Fusion

Gene duplication plays an important role in
enzyme evolution. It has been estimated
that 50% of all genes in microorganisms
are the result of duplication events, which
are followed by diversification of the twin
genes (Fani et al 1998, Lynch & Conery
2000).
Gene Fusion
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Gene duplication and fusion events that
multiply and link functional protein domains
are crucial mechanisms of enzyme evolution.
The analysis of amino acid sequences and
three-dimensional structures suggested that
the (αβ)8-barrel, has evolved by the
duplication, fusion, and mixing of (αβ)4-halfbarrel domains (Höcker et al 2004).
Gene fusion
It has been postulated that HisA
and HisF evolved from a common
(βα)4-half-barrel by a series of
gene duplication and diversification
events (Höcker et al 2004).
(βα)8-barrels were derived from
‘half-barrels’ will motivate
the search for ancestral domains
within other apparent
single-domain protein folds
(Petsko, 2000; Gerlt
and Babbitt, 2001, Jürgens et al
2000).
Birte et al. (2004)
Fold Change in Evolution of Protein
Structures
Mechanisms for evolutionary fold change:
1. Insertion, deletion, or substitution of
structure elements
2. Circular permutation
3. Strand invasion/withdrawal
4. Beta hairpin flip/swap
Insertion, Deletion, Substitution
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Luciferase and NFP
NFP deletes a 90-residue section that contains
an αβαβα segment of TIM barrel
Missing segment is compensated for by a single
anti-parallel strand
Shared regions are 30% identical, they are part
of same operon, and homology was detected
before Luciferase structure was solved
Other TIM-like proteins have less dramatic
changes of similar nature
Insertion, Deletion, Substitution
Circular Permutation
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Circular permutations can occur because the N- and Ctermini of proteins often end near each other
As a result, elements can be substituted into structure
from N- and C- termini
Though structure is barely changed, topology is different,
and so is different fold
C2 domains have simple permutation of only one strand
Have clear sequence homology to each other, as well as
very similar structures
Circular Permutation
We can observe circular permutations
between families of the TIM barrel super
family FAD-linked oxidoreductase and also
in the proteins of the super family PLPbinding barrel.
 The last ones perform a circular
permutation of the canonical fold.

Circular Permutation
Triosephosphate isomerase us Alanine racemase
(ClustalW)
Strand Invasion / Withdrawal

Is defined as on
both sides of the
invading strand
disruption of Hbonds in internal βstrands, which
requires changes in
H-bonding patterns
Beta-Hairpin Flip / Swap
• Β-hairpin
flip/swap shifts
location of two
strands so that
they have new
partners on one
side, and new
H-bonding on
the other
Implications

Two scenarios for homology in cases of a small region of similarity:

Local: segments of clear homology are inserted into different structural
frameworks, producing local regions of homology in the middle of nonhomologous proteins
 Global: shared segments of similarity are leftovers from a once completely
shared structure. Gradual change of rest of structure through sequence changes
and indels

All homology is local to a certain extent

Structures that are mostly similar are probably mostly homologous, though alien
segments could fill some sections without being detectable
 Structures that are very plastic and have changed considerably may only have
small sections of true homology


Homology modeling could give erroneous results in some cases
Understanding the natural changes in folds could help with protein design