Computational analysis of membrane proteins
implicated in metal transport
in Arabidopsis thaliana
CIAVVLCLVFMSVEVVGGIKANSLAILTDAAHLLSDVAAFAISLFSLWAAGWEATPRQTYGFFRIEILGALVSIQLIWLLT
ALFLLINTAYMVVEFVAGFMSNSLGLISDACHMLFDCAALAIGLYASYISRLPANHQYNYGRGRFEVLSGYVNAVFLVLVG
CFVVVLCLLFMSIEVVCGIKANSLAILADAAHLLTDVGAFAISMLSLWASSWEANPRQSYGFFRIEILGTLVSIQLIWLLT
LIAVLLCAIFIVVEVVGGIKANSLAILTDAAHLLSDVAAFAISLFSLWASGWKANPQQSYGFFRIEILGALVSIQMIWLLA
---IFLYLIVMSVQIVGGFKANSLAVMTDAAHLLSDVAGLCVSLLAIKVSSWEANPRNSFGFKRLEVLAAFLSVQLIWLVS
Stefanie Hartmann
Max Planck Institute for Molecular Plant Physiology
Supervisors: Joachim Selbig, Ute Krämer
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Q ui ck Ti m e ™ an d a T I FF ( U nc om p r es se d) de co m pr e ss or ar e n ee de d t o se e t hi s p i ct ur e .
Q ui ck Ti m e ™ an d a T I FF ( U nc om p r es se d) de co m pr e ss or ar e n ee de d t o se e t hi s p i ct ur e .
Q ui ck Ti m e ™ an d a T I FF ( U nc om p r es se d) de co m pr e ss or ar e n ee de d t o se e t hi s p i ct u re .
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12 membrane proteins
involved in metal transport
in Arabidopsis
Q ui ck Ti m e ™ an d a T I FF ( U nc om p r es se d) de co m pr e ss or ar e n ee de d t o se e t hi s p i ct ur e .
Q ui ck Ti m e ™ an d a T I FF ( U nc om p r es se d) de co m pr e ss or ar e n ee de d t o se e t hi s p i ct ur e .
Q ui ck Ti m e ™ an d a T I FF ( U nc om p r es se d) de co m pr e ss or ar e n ee de d t o se e t hi s p i ct u re .
QuickTi me™ and a T IFF (Uncom pressed) decom pressor are needed to see t his pict ure.
Metal transporters are of great importance because…
…they provide an adequate supply of essential trace metals
…they prevent an excess of these potentially toxic ions
in silico analyses may help design further experiments on
• basic research on metal homeostasis
• development of new ways of phytoremediation
Cation Diffusion Facilitator (CDF) proteins
also referred to as cation efflux (CE) proteins
• occur in archaea, bacteria, eukaryotes
• are involved in transporting heavy metals (Co2+, Cd2+, Zn2+, Ni2+)
• the CDF family of proteins had 13 members in 1997
• the CE Pfam family today has 348 members (July 2003)
426
(Jan 2004)
CDF signature sequence:
S X (ASG) (LIVMT)2 (SAT) (DA) (SGAL) (LIVFYA) (HDN) X3 D X2 (AS)
The Arabidopsis thaliana CDF protein family
CDF1: At2g46800
S LAILTDAAHLLS D VAA
CDF2: At3g61940
CDF3: At3g58810
CDF4: At2g29410
CDF5: At2g04620
CDF6: At2g47830
CDF7: At2g39450
CDF8: At1g16310
CDF9: At1g79520
CDF10: At3g58060
CDF11: At3g12100
CDF12: At1g51610
S LAILADAAHLLT D VGA
exact match
S LAILTDAAHLLS D VAA
S LAVMTDAAHLLS D VAG
S LGLISDACHMLF D CAA
1 mismatch
S TAIIADAAHSVS D VVL
S LAIIASTLDSLL D LLS
S MAVIASTLDSLL D LLS
2 mismatches
S MAVIASTLDSLL D LLS
S IAIAASTLDSLL D LMA
R VGLVSDAFHLTF G CGL
S HVIMAEVVHSVA D FAN
3 mismatches
4 mismatches
Research questions:
Can all 12 proteins be classified as CDF
proteins? i.e., are there predicted structural and
functional similarities of these 12 Arabidopsis
proteins?
secondary structure
prediction, inclusion in
membrane- and
transporter
databases, evaluation
of common motifs, etc
Research questions:
Can all 12 proteins be classified as CDF
proteins? i.e., are there predicted structural and
functional similarities of these 12 Arabidopsis
proteins?
secondary structure
prediction, inclusion in
membrane- and
transporter
databases, evaluation
of common motifs, etc
What are the relationships of the 12 Arabidopsis
proteins among each other and to other
published sequences?
intron/exon structure,
phylogenetic
reconstructions
Research questions:
Can all 12 proteins be classified as CDF
proteins? i.e., are there predicted structural and
functional similarities of these 12 Arabidopsis
proteins?
secondary structure
prediction, inclusion in
membrane- and
transporter
databases, evaluation
of common motifs, etc
What are the relationships of the 12 Arabidopsis
proteins among each other and to other
published sequences?
intron/exon structure,
phylogenetic
reconstructions
Is it possible to predict the 3D structure of these
proteins?
fold recognition by
threading
Sequence retrieval - four ambiguous sequences
 TIGR Arabidopsis thaliana database
 TAIR: The Arabidopsis Information Resource
 MIPS Arabidopsis thaliana genome database
• different assignment of introns, use of alternative start
codons
Sequence analysis - three additional ambiguous sequences
 SWALL
 Pfam vs. TIGR/TAIR/MIPS
• insertions and deletions, different amino acid sequence
Cloning and RT-PCR revealed correct sequences for six of the
seven ambiguous CDFs
Inclusion in membrane and transport databases
cation efflux,
Pfam entry
PF01545
Membrane Protein
Library (AMPL)
CDF1

CDF2
Arabidopsis
ARAMEMNON
Transport
Protein
Database
PlantsT









CDF3





CDF4





CDF5

()

-
CDF6



CDF7


-
CDF8

-
-
CDF9

-
-
CDF10


-

CDF11




CDF12


-

Inclusion in membrane and transport databases
cation efflux,
Pfam entry
PF01545
Membrane Protein
Library (AMPL)
CDF1

CDF2
Arabidopsis
ARAMEMNON
Transport
Protein
Database
PlantsT









CDF3





CDF4





CDF5

()
()

-
CDF6

()
()


CDF7

()
()

-
CDF8

()
()
-
-
CDF9

()
()
-
-
CDF10

()

-

CDF11

()



CDF12

()

-

Inclusion in membrane and transport databases
cation efflux,
Pfam entry
PF01545
Membrane Protein
Library (AMPL)
CDF1

CDF2
Arabidopsis
ARAMEMNON
Transport
Protein
Database
PlantsT









CDF3





CDF4





CDF5

()
()

–
CDF6

()
()


CDF7

()
()

–
CDF8

()
()
–
–
CDF9

()
()
–
–
CDF10

()

–

CDF11

()



CDF12

()

–

Hidden Markov models used for secondary structure prediction
cytoplasmic side
membrane
non-cytoplasmic side
• states (loops, transmembrane domains, etc) are defined
• states are connected in a biologically reasonable way (transitions)
• each state has a specific probability distribution over the 20 amino acids
• each transition has a specific transition probability
• amino acid probabilities and transition probabilities are learned
• models are first taught using a training set, the trained model is then
used for the prediction
Results of secondary structure predictions
number of TMD
N-terminus within
cytoplasm
CDF1
6
2/3
CDF2
6
3/3
CDF3
6
2/3
CDF4
5-6
2/3
CDF5
6
CDF6
0-6
1/3
CDF7
4-6
2/3
CDF8
5-6
3/3
CDF9
5-6
3/3
CDF10
4-6
2/3
CDF11
6
3/3
CDF12
4-6
3/3
TMHMM v2
HMMTOP v2
Memsat2
(14)
(Tusnady and Simon, 1998, 2001)
(Sonnhammer et al. 1998)
(Jones et al. 1994, McGuffin et al. 2000)
3/3
Results of secondary structure predictions
number of TMD
N-terminus within
cytoplasm
CDF1
6
2/3
CDF2
6
3/3
CDF3
6
2/3
CDF4
5-6
2/3
CDF5
6
CDF6
0-6
1/3
CDF7
4-6
2/3
CDF8
5-6
3/3
CDF9
5-6
3/3
CDF10
4-6
2/3
CDF11
6
3/3
CDF12
4-6
3/3
TMHMM v2
HMMTOP v2
Memsat2
(14)
(Tusnady and Simon, 1998, 2001)
(Sonnhammer et al. 1998)
(Jones et al. 1994, McGuffin et al. 2000)
3/3
CDF signature
CE signature
Prediction of subcellular localization
mTP: mitochondrial
targeting peptide
cTP: chloroplast
transit peptide
SP: signal peptide
(ER/secretory pathway)
Prediction of subcellular localization - methods
• N-terminal sorting signals display characteristic amino acid compositions
• sequence-based methods predicting N-terminal sorting signals are based
on this observation
 TargetP
mTP, cTP, SP
neural network-based
 iPSORT
mTP, cTP, SP
decision list
 Predotar
mTP, cTP
neural network-based
 SignalP NN
 SignalP HMM
mTP: mitochondrial
targeting peptide
SP neural network-based
SP based on hidden Markov models
cTP: chloroplast
transit peptide
SP: signal peptide
(ER/secretory pathway)
Prediction of subcellular localization - results
TargetP
iPSORT
Predotar
SignalP
NN
HMM
CDF1
CDF2
3/4
CDF3
CDF4
CDF5
CDF6
mTP
cTP
cTP
cTP
mTP
cTP*
mTP*
1/4
CDF7
CDF8
2/4*
Y*
CDF9
CDF10
CDF11
CDF12
mTP: mitochondrial
targeting peptide
mTP
mTP
cTP: chloroplast
transit peptide
SP: signal peptide
(ER/secretory pathway)
Exon structure of the CDF proteins
# of exons
CDF1
1
CDF2
1
CDF3
1
CDF4
1
CDF5
1
CDF11
9
CDF6
12
CDF12
13
CDF7
6
CDF8
6
CDF9
7
CDF10
5
Gene organization of the CDF proteins
CDF1
CDF1
CDF2
CDF2
CDF3
CDF3
CDF4
CDF4
CDF5
CDF5
CDF11
CDF10
CDF6
CDF6
CDF12
CDF12
CDF7
CDF7
CDF8
CDF8
CDF9
CDF9
CDF11
CDF10
Phylogenetic Relationships within Cation Transporter Families of Arabidopsis
Plant Physiology 2001; 126 (4): 1646–1667
omitted:
CDFs
5, 7, 8, 9
CDF6
CDF11
CDF4
CDF3
CDF2
CDF10
CDF12
CDF1
Phylogenetic analysis of the Arabidopsis CDF proteins
AtCDF4
100
AtCDF3
group I
100
AtCDF1
98/94/99
AtCDF2
AtCDF12
100
AtCDF6
AtCDF10
67/–/69
100
AtCDF7
group II
86/100/95
–/9479
AtCDF9
100
AtCDF8
AtCDF5
100/
73/68
8
AtCDF11
RmCzcD
Phylogenetic analysis of sequences containing the CE signature
Escherischia coli
ZITB
Ralstonia metallidurans
CZCD
Ralstonia metallidurans
CZCD
Mus musculus
ZNT4
Rattus norvegigus
ZNT2
Mus musculus
ZNT3
Arabidopsis thaliana
CDF4
Arabidopsis thaliana
CDF2
Eucalyptus grandis
Arabidopsis thaliana
CDF3
Arabidopsis thaliana
CDF1
Thlaspi caerulescens
ZTP1
Thlaspi goesingense
MTP1
Thlaspi goesingense
MTP1
Thlaspi goesingense
MTP1
Oryza sativa
Zea mays
Lotus japonicus
Medicago trunculata
Oryza sativa
Triticum aestivum
Caenorhabditis elegans
CDF1
Rattus norvegicus
ZNT1
Schizosachharomyces pombe
ZHF
Saccharomyces cerevisiae
COT1
Saccharomyces cerevisiae
ZRC1
Oryza sativa
Stylosanthes hamata
MTP1
Oryza sativa
Arabidopsis thaliana
CDF10
Arabidopsis thaliana
CDF7
Stylosanthes hamata
MTP4
Stylosanthes hamata
MTP2
Stylosanthes hamata
MTP3
Oryza sativa
Arabidopsis thaliana
CDF8
Arabidopsis thaliana
CDF9
Arabidopsis thaliana
CDF6
Saccharomyces cerevisiae
Mmt2
Saccharomyces cerevisiae
Mmt1
T. thermophilus
czrB
Oryza sativa
Arabidopsis thaliana
CDF12
Homo sapiens
ZNT5
Homo sapiens
ZTL1
Homo sapiens
ZNT7
Homo sapiens
ZNT6
Arabidopsis thaliana
CDF11
S. cerevisiae
MSC2
Oryza sativa
Arabidopsis thaliana
CDF5
Staphylococcus aureus
Staphylococcus aureus
Bacillus stearothermophilus
Bacillus stearothermophilus
Arabidopsis group I sequences,
monocot and dicot sequences,
mammalian metal transporters
Arabidopsis group II sequences,
monocot and dicot sequences,
prokaryotic and eukaryotic seqs
several two-domain proteins
outgroup
working model:
topology of Arabidopsis CDF proteins
CDF
signature
sequence
cell exterior/organelle
cytoplasm
N
C
Information derived from the 3D structure of a protein
assignment
of function
guide mutagenesisexperiments
ligand and
functional sites
evolutionary
relationships
residue solvent
exposure
putative
interaction sites
Structure determination
1. Classical approaches
• X-ray crystallography
• NMR spectroscopy
2. Computational approaches
• comparative (“homology”) modeling
• fold recognition (“threading”)
• ab initio methods
The basis of fold recognition (“threading”)
The number of folds occurring
in nature is limited:
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
PDB statistics: http://www.rcsb.org/pdb/holdings.html
There are many sequences with no significant sequence identity
but with the same or similar folds
…HEAIDHKPKLTGMKTGRVVSSMKSNFFADLP…
…HDGRSSMTRFSRYFRKTGRVSEYYKKQERLLE…
Fold recognition methods
aim: to find an optimal sequence-structure alignment
1. “threading” of an unknown target sequence into the backbone
structure of template proteins of known structure
………CLVFMSVEVVGGIKANSLAILTD………
Fold recognition methods
2. evaluation of the compatibility between target sequence and
proposed 3D structure
using environment-based mean force potentials
or
using knowledge-based mean force potentials
3. Output:
4.99 Å
a list of folds (sorted or unsorted),
their “compatibility score”,
sometimes other information such as SCOP descriptors,
alignment, rudimentary 3D model of the query protein, raw
scores, solvation energy for the model, links
No new insights regarding the structure of CDF proteins
Membrane proteins are significantly under-represented
in structural databases – and therefore also in fold libraries
If there is no fold similar to the native fold of the target protein,
this approach cannot succed.
Threading methods cannot be used for modeling of
transmembrane proteins
Will the 3D structure of CDFs be available soon?
• for fold recognition methods to be used successfully:
significantly more 3D structures of membrane proteins are
needed
• fold recognition methods specifically for integral
membrane proteins may eventually be developed
• cyrystallization of bacterial homologs and subsequent
extraploation of structural features as an alternative?
• approach for globular proteins:
predicting a protein’s solubility and propensity to crystallize,
based on results from high-throughput structure determination
Can threading results be used as an independent way to verify
group assignment?
Were some structural hits specific for any of the CDF groups?
1. Which hits were common to
which of the CDF sequences?
1 • • • • • •
•
2 • • • • • •
3 • • • • • •
4
• • • • • •
5
• • • • • •
• •
• • •
1
• • •
•
• •
2
• •
•
3
• •
•
•
2. “Phylothreading”
•
• •
•
4
• •
5
Can threading results be used as an independent way to verify
group assignment?
Were some structural hits specific for any of the CDF groups?
1. Which hits were common to
which of the CDF sequences?
1 • • • • • •
•
2 • • • • • •
3 • • • • • •
4
• • • • • •
5
• • • • • •
• •
• • •
1
• • •
•
• •
2
• •
•
3
• •
•
•
2. “Phylothreading”
•
• •
•
4
• •
5
Which hits were common to which of the CDF sequences?
Structural hits predicted
• for most CDF sequences
• for group I sequences
• for group II sequences
• for CDF5 and CDF11
• for CDF6 and CDF12
1…
…170
1
• • • • • •
2
• • • • • •
…
• • • • • •
11
• • • • • •
12
• • • • • •
•
• •
• • •
• • •
•
• •
• •
•
•
Results were unable to provide evidence to verify group
assignments based on other methods
•
•
•
•
• •
•
•
•
“Phylothreading”
CDF1
CDF2
CDF3
CDF4
CDF5
CDF6
78
CDF7
99
68
CDF8l
CDF8s
CDF9
CDF10
CDF11
CDF12
Phylothreading results can
neither
verify nor refute
group assignments based
on other methods
Threading: non-transmembrane CDF fragments
cell exterior/organelle
cytoplasm
N
C
N-terminus
histidine-rich loop
between TMD 4 and 5
C-terminus
“Phylothreading”: CDF C-terminal fragments
CDF1
CDF2
group I
CDF3
“phylothreading” results
confirm the assignment of
CDF sequences to groups
that were based on
independent methods
CDF4
CDF5
67
CDF11
CDF6
69
CDF7
68
79
85
CDF8
group II
CDF9
CDF10
CDF12
Conclusions
•
The 12 Arabidopsis protein sequences reveal structural and
therefore probably functional conservation
•
My results support the classification of these proteins as CDF
metal transporters
•
I propose that the CDF protein family of A. thaliana contains
two groups, each containing at least four proteins that are
structurally and functionally closely related
•
Threading methods cannot be used for transmembrane
proteins or for their non-transmembrane domains (yet)
•
Threading results for multiple sequences may be used to
confirm (or find?) relationships among these sequences
(“phylothreading”)
•
I was able to evaluate and compare a number of online tools
that are available for the analysis of sequence data
Conclusions
1. Sequence retrieval revealed conflicting information for 7
of the 12 proteins
2. The 12 Arabidopsis protein sequences reveal striking
structural and therefore probably functional conservation
3. My results support the classification of these proteins as
CDF metal transporters
4. I propose that the CDF protein family of A. thaliana
contains two groups, each containing four proteins that are
structurally and functionally closely related
5. I was able to evaluate and compare a variety of online
tools available for the analysis of sequence data
Conclusions
1. Sequence retrieval revealed conflicting information for 7 of the
12 proteins
2. The 12 Arabidopsis protein sequences reveal striking structural
and therefore probably functional conservation
3. My results support the classification of these proteins as CDF
metal transporters
4. I propose that the CDF protein family of A. thaliana contains two
groups, each containing four proteins that are structurally and
functionally closely related
5. I was able to evaluate and compare a variety of online tools
available for the analysis of sequence data
6. Threading methods cannot be used for transmembrane proteins
or for their non-transmembrane domains (yet)
7. Threading results for multiple sequences can be used to
confirm (or find?) relationships among these sequences
(“phylothreading”)
METHODS
Phylogenetic analysis: tree-building methods
• distance-based methods
overall distance between all pairs of sequences are calculated and
then used to calculate a tree
(Neighbor Joining)
• character-based methods
the individual substitutions among the sequences are used to
determine the most likely ancestral relationships
(Maximum Parsimony, Maximum Likelihood)
• Bayesian inference of phylogenies
...CLVFMSVEVVGGIKANSLAILTD...
...NTAYMVVEFVAGFMSNSLGLISD...
...CLLFMSIEVVCGIKANSLAILAD...
...CAIFIVVEVVGGIKANSLAILTD...
...YLIVMSVQIVGGFKANSLAVMTD...
Phylogenetic analysis: statistical evaluation of trees
• bootstrap analysis
how much support exists for particular branches in a phylogeny?
1.
2.
3.
4.
5.
6.
tree construction, determination of the “best” tree
bootstrap datasets (pseudosamples) are created from the original
dataset by random sampling with replacement
tree construction using the bootstrap datasets
comparison of the bootstrap tree with the inferred tree
this is repeated several hundred times
bootstrap value: percentage of times an interior branch in the
bootstrap tree was the same as the one in the inferred tree
...CLVFMSVEVVGGIKANSLAILTD...
...NTAYMVVEFVAGFMSNSLGLISD...
...CLLFMSIEVVCGIKANSLAILAD...
...CAIFIVVEVVGGIKANSLAILTD...
...YLIVMSVQIVGGFKANSLAVMTD...
Fold recognition methods
2. evaluation of the compatibility between target sequence and
proposed 3D structure
• using environment-based mean force potentials
(Bowie, Fischer, Eisenberg: 1991-1996)
- residue positions are categorized into environment classes
- the 3D protein structure is converted into a 1D sequence
- generate alignment of this 1D string to target sequence
• using knowledge-based mean force potentials
(Sippl: 1990-1995)
- information is automatically learned from databases of protein structures
- pairwise interactions between structurally adjacent
residues are calculated
- transformation of mean force potentials as a function of distance
Fold recognition methods
aim: to find an optimal sequence-structure alignment
1. “threading” of an unknown target sequence into the backbone
structure of template proteins of known structure
………CLVFMSVEVVGGIKANSLAILTD………
fold library
query sequence
Fold recognition methods
2. evaluation of the compatibility between target sequence and
proposed 3D structure
using environment-based mean force potentials
or
using knowledge-based mean force potentials
4.99 Å
Fold recognition methods
2. evaluation of the compatibility between target sequence and
proposed 3D structure
using environment-based mean force potentials*
or
using knowledge-based mean force potentials*
4.99 Å
* distant-dependent forces that act between atoms/residues
(electrostatic and van der Waals interactions, influences on the surrounding medium on these
interactions, contacts between two or three amino acids, angles between residue pairs, …)
Fold recognition methods
2. evaluation of the compatibility between target sequence and
proposed 3D structure
using environment-based mean force potentials
or
using knowledge-based mean force potentials
3. Output:
4.99 Å
a list of folds (sorted or unsorted),
their “compatibility score”,
sometimes other information such as SCOP descriptors,
alignment, rudimentary 3D model of the query protein, raw
scores, solvation energy for the model, links
Threading methods used
Qu i ck Ti me ™a nd a TIFF (Unc om pres se d) de co mp re ss or are n ee de d to s ee th is pi ctu re .
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
Quick Time™a nd a TIFF ( Uncomp res sed) deco mpre ssor are n eede d to s ee this picture .
Qu i ck Ti me ™a nd a TIFF (Unc om pres se d) de co mp re ss or are n ee de d to s ee th is pi ctu re .
UCLA-DOE Fold Server
P. Mallick et al., 2002
(BLAST, PSI-BLAST, SDP, DASEY)
Threader
D.T. Jones et al., 1992
mGenThreader
L.J. McGuffin & D.T. Jones 2003
3D-PSSM
L.A. Kelley et al., 2000
Arby
I. Sommer et al., unpublished
(PSI-BLAST, 123D, Jprop)
Selection of structural hits for further analysis
UCLA-DOE:
top 10 structural hits are returned, all were kept
Threader:
compatibility of target sequence and all 2000
available templates is evaluated; lists were sorted
by Z-value, approximately 10-20 best hits were
kept
mGenThreader: top 20 structural hits are returned, all were kept
3D-PSSM:
top 20 structural hits are returned, all were kept
Arby:
a list of the 10-20 best scores is returned; the
corresponding hits were extracted from a large
table
Evaluation of the top score for each CDF sequence
UCLA
350
Threader
300
0
very poor score
250
1
200
2
150
100
50
0
3
borderline significant
4
significant
very significant
scores: no guidelines
mGenThreader
3D-PSSM
1.0
0.8
guess
0.6
0.4
2.5
2.0
1.5
low confidence
0.2
0.0
poor score
1.0
worthy of attention
0.5
medium confidence
high confidence
certain
0.0
highly confident
There is no consensus of top fold predicted by different methods
example: top two structural hits for CDF1
Threader:
1ONE
1C3Q
phosphopyruvate hydrolase
thiazole kinase
mGenThreader:
1L8M
1QGR
his-rich protein (model)
importin beta
UCLA-DOE:
1B8F
1HFA
histidine ammonia-lyase
clathrin assembly protein
3D-PSSM:
1PW4
1KPW
glycerol-3-phosphate transporter
green cone pigment
Arby:
1HZX
1EZV
bovine rhodopsin
yeast cytochrome bc1
No new insights regarding the structure of CDF proteins
Membrane proteins are significantly under-represented
in structural databases – and therefore also in fold libraries
If there is no fold similar to the native fold of the target protein,
this approach cannot succed.
Threading methods cannot be used for modeling approaches
Threading results: C-termini
1. Structural information
no information of domains for metal transport available.
BUT: several of the returned hits are proteins in which bound
metals have structural or catalytic roles
2. Verification of group assignment
i. Hits predicted for more than one C-terminus:
specific for group I:
specific for group II:
specific for CDF5 and CDF11:
ii. “Phylothreading”
48 folds
3
2
2
Positions of conserved domains and signature sequences
1
2
3
4
5
11
6
12
7
8
9
10
TMD
I II III
IV
V VI
CDF signature
Pfam CE signature
BLOCKS
(eMOTIF)
10, 11
11, 12
6-12
Arabidopsis CDF proteins
AtCDF1
AtCDF2
AtCDF3
AtCDF4
AtCDF5
AtCDF11
AtCDF6
AtCDF12
AtCDF9
AtCDF8
AtCDF10
AtCDF7
outgroup
group I:
- contain his-rich region between TMD 4 and 5
- one member is confirmed to transport Zn ions
- genome structure conserved (no introns)
no group assignment:
- CDF6, CDF12: possibly distant common ancestry
and mitochondrial localization
- CDF5, CDF11: close relationship also in PFAM tree
group II:
- lack the his-rich region between TMD 4 and 5
- proteins may transport Mn ions
- C-terminal regions differ from group I sequences
working model:
topology of Arabidopsis CDF proteins
CDF
signature
sequence
cell exterior/organelle
cytoplasm
N
C
Gene organization of the CDF proteins
CDF1
CDF2
CDF3
CDF4
CDF5
CDF10
CDF6
CDF12
CDF7
CDF8
CDF9
CDF11
Phylogenetic analysis of sequences containing the CE signature
100/52/83
100/100/70
100/98/97
100/100/69
100/
99/82
100/99/88
99/
94/
72
100/100/63
100/100/74
100/100/–
71/55
100/99/95
99/–/61
-/75
1000/100/94
100/
100/
53
62
100/100/82
100/100
/95
97/–/–
68/–/83
100/–/63
83/–/67
100/100/71
99/65/–
100/100/–
100/99/75
96/96/78
100/100/63
100/100/69
74/–/–
100/100/92
100
/100
/85
5
100
/100
/84
Escherischia coli
ZITB
Ralstonia metallidurans
CZCD
Ralstonia metallidurans
CZCD
Mus musculus
ZNT4
Rattus norvegigus
ZNT2
Mus musculus
ZNT3
Arabidopsis thaliana
CDF4
Arabidopsis thaliana
CDF2
Eucalyptus grandis
Arabidopsis thaliana
CDF3
Arabidopsis thaliana
CDF1
Thlaspi caerulescens
ZTP1
Thlaspi goesingense
MTP1
Thlaspi goesingense
MTP1
Thlaspi goesingense
MTP1
Oryza sativa
Zea mays
Lotus japonicus
Medicago trunculata
Oryza sativa
Triticum aestivum
Caenorhabditis elegans
CDF1
Rattus norvegicus
ZNT1
Schizosachharomyces pombe
ZHF
Saccharomyces cerevisiae
COT1
Saccharomyces cerevisiae
ZRC1
Oryza sativa
Stylosanthes hamata
MTP1
Oryza sativa
Arabidopsis thaliana
CDF10
Arabidopsis thaliana
CDF7
Stylosanthes hamata
MTP4
Stylosanthes hamata
MTP2
Stylosanthes hamata
MTP3
Oryza sativa
Arabidopsis thaliana
CDF8
Arabidopsis thaliana
CDF9
Arabidopsis thaliana
CDF6
Saccharomyces cerevisiae
Mmt2
Saccharomyces cerevisiae
Mmt1
T. thermophilus
czrB
Oryza sativa
Arabidopsis thaliana
CDF12
Homo sapiens
ZNT5
Homo sapiens
ZTL1
Homo sapiens
ZNT7
Homo sapiens
ZNT6
Arabidopsis thaliana
CDF11
S. cerevisiae
MSC2
Oryza sativa
Arabidopsis thaliana
CDF5
Staphylococcus aureus
Staphylococcus aureus
Bacillus stearothermophilus
Bacillus stearothermophilus
IV
I
V
II
III
Phylogenetic analysis: tree-building methods
• maximum parsimony methods
the best tree topology minimizes the total amount of evolutionary
change that has occurred
• distance methods
the best tree topology minimizes the the total distance among taxa
• maximum likelihood methods
given a particular substitution model and given a particular tree, how
likely is the observed data?
...CLVFMSVEVVGGIKANSLAILTD...
...NTAYMVVEFVAGFMSNSLGLISD...
...CLLFMSIEVVCGIKANSLAILAD...
...CAIFIVVEVVGGIKANSLAILTD...
...YLIVMSVQIVGGFKANSLAVMTD...
Inclusion in membrane and transport databases
cation efflux,
Pfam entry
PF01545
Membrane Protein
Library (AMPL)
CDF1

CDF2
Arabidopsis
ARAMEMNON
Transport
Protein
Database
PlantsT
CDF
zinc transporter
CDF
CDF

CDF
putative MTP
CDF
CDF
CDF3

CDF
putative MTP
CDF
CDF
CDF4

CDF
putative MTP
CDF
CDF
CDF5

singleton
(CDF related)
putative cation
transporter
CDF
-
CDF6

singleton
unknown protein
CDF
CDF
CDF7

family
unknown protein
CDF
-
CDF8

family
hypothetical protein
-
-
CDF9

family
unknown protein
-
-
CDF10

family
putative MTP
-
CDF
CDF11

singleton
putative MTP
CDF
CDF
CDF12

singleton
putative MTP
-
CDF
Inclusion in membrane and transport databases
cation efflux,
Pfam entry
PF01545
Membrane Protein
Library (AMPL)
CDF1

CDF2
Arabidopsis
ARAMEMNON
Transport
Protein
Database
PlantsT
CDF
zinc transporter
CDF
CDF

CDF
putative MTP
CDF
CDF
CDF3

CDF
putative MTP
CDF
CDF
CDF4

CDF
putative MTP
CDF
CDF
CDF5

singleton
(CDF related)
putative cation
transporter
CDF
-
CDF6

singleton
unknown protein
CDF
CDF
CDF7

family
unknown protein
CDF
-
CDF8

family
hypothetical protein
-
-
CDF9

family
unknown protein
-
-
CDF10

family
putative MTP
-
CDF
CDF11

singleton
putative MTP
CDF
CDF
CDF12

singleton
putative MTP
-
CDF
Inclusion in membrane and transport databases
cation efflux,
Pfam entry
PF01545
Membrane Protein
Library (AMPL)
CDF1

CDF2
Arabidopsis
ARAMEMNON
Transport
Protein
Database
PlantsT
CDF
zinc transporter
CDF
CDF

CDF
putative MTP
CDF
CDF
CDF3

CDF
putative MTP
CDF
CDF
CDF4

CDF
putative MTP
CDF
CDF
CDF5

singleton
(CDF related)
putative cation
transporter
CDF
-
CDF6

singleton
unknown protein
CDF
CDF
CDF7

family
unknown protein
CDF
-
CDF8

family
hypothetical protein
-
-
CDF9

family
unknown protein
-
-
CDF10

family
putative MTP
-
CDF
CDF11

singleton
putative MTP
CDF
CDF
CDF12

singleton
putative MTP
-
CDF
Inclusion in membrane and transport databases
cation efflux,
Pfam entry
PF01545
Membrane Protein
Library (AMPL)
CDF1

CDF2
Arabidopsis
ARAMEMNON
Transport
Protein
Database
PlantsT
CDF
zinc transporter
CDF
CDF

CDF
putative MTP
CDF
CDF
CDF3

CDF
putative MTP
CDF
CDF
CDF4

CDF
putative MTP
CDF
CDF
CDF5

singleton
(CDF related)
putative cation
transporter
CDF
-
CDF6

singleton
unknown protein
CDF
CDF
CDF7

family
unknown protein
CDF
-
CDF8

family
hypothetical protein
-
-
CDF9

family
unknown protein
-
-
CDF10

family
putative MTP
-
CDF
CDF11

singleton
putative MTP
CDF
CDF
CDF12

singleton
putative MTP
-
CDF