Post on 11-Jan-2016
description
Laura Guerrero
Ainhoa Plaza
Nira Suárez
Rosa Turbau
WW domainthe smallest natural
protein domain known
Structure
• only 35-40 aa• Monomeric• stable in
the absence of disulfide bonds
• three-strandedantiparallel beta-sheet
• 2 loops• WWP = 2 tryptophans (20-23aa) + 1 proline.
WW domain. Mitotic rotamase PIN1. (Homo sapiens)
Trp (W)
Pro (P)
Loop 1
Loop 2
WW domain. PIN1. (Human)
Function
• adaptor module• functional similarity
with SH3 domains• attach the enzyme enhancing
the catalytic activity
• found in many different proteins often localized in the cytoplasm as well as in the cell nucleus.
• Proteins participating in signalling paths and development• binds proline-motifs, [X]-P-P-[X]-Y, and/or phosphoserine-
phosphothreonine-containing motifs.
P IN1. (Homo sapiens)
Dystrophin. (Homo sapiens)
Group Proteins Ligand Sequence (ligand)
I YAP65, Need4, Dystrophin PEBP2 transc.activ.
ENaC sodium channel
beta-dystroglycan
PPxY
II Formin Binding Proteins, FE65 Formin, Mena, Bat2 PPPPPPL/RP
III Formin Binding proteins Splicing factors: SmB, SmB’, U1C
(PxxGMxPP)N
IV Ess1 / Pin1 RNApol II, tau
Cdc25C
(Phospho-S/T)P
V Npw38/PQBP-1 NpwBP Rx(x)PPGPPPxR
Classification
Liddle’s sindrome
Muscular distrophies
Alzheimer disease
Hierarchy
Hierarchy
WW domain family
Sequence Alignment (Clustal W)
COMANDS
•‘ww_totes.fasta’ file:
1PIN.fa, 1f8aB.fa, 1I8H1.fa, 1i6c1.fa, 1nmv1.fa, 1e0l1.fa, 1eg3A.fa, 1eg4A.fa, 1i5h1.fa, 1e0n1.fa, 1jnq1.fa, 1k9r1.fa, 1k9q1.fa, 1k5r1.fa, 1o6w1.fa, 1tk71.fa
• $ clustalw ww_totes.fasta
•Outputs are: ww_totes.aln
ww_totes.dnd
Group I. Clustal alignment
Group II-III. Clustal alignment
Group IV. Clustal alignment
Comandes: ClustalW alignments. WW domain Groups:
Group I
fileGroupI.fasta {1eg3A.fa, 1eg4A.fa, 1i5h1.fa, 1jmq1.fa, 1k5r1.fa, 1k9q1.fa, 1k9r1.fa}
$clustalw
Input: fileGroupI.fasta
Output: fileGroupI.aln, fileGroupI.dnd
Group II-III
fileGroupII-III.fasta {1e0l1.fa, 1e0n1.fa, 1o6w1.fa, 1tk71.fa}
$clustalw
Input: fileGroupII-III.fasta
Output: fileGroupII-III.aln, fileGroupII-III.dnd
Group IV
fileGroupIV.fasta {1f8aB.fa, 1i6c1.fa, 1i8g1.fa, 1I8H1.fa, 1nmv1.fa, 1pinA.fa}
$clustalw
Input: fileGroupIV.fasta
Output: fileGroupIV.aln, fileGroupIV.dnd
WW domain family
Sequence Alignment (Clustal W)
1 2 3
WW domain family
Sequence Alignment (Clustal W)
Sequence Alignment
Native proteins
COMANDS
•‘nativas.fasta’ file:
1nmv1.fa, 1e0l1.fa, 1e0n1.fa, 1o6w1.fa, 1tk71.fa
• $ clustalw nativas.fasta
•Outputs are: nativas.aln
nativas.dnd
Structure Alignment
normal STAMP
Cluster: 4 ( 1nmv & 1tk7 1o6w 1e0l 1e0n )
Sc 1.43
RMS 1.62
3
COMANDS
•‘nativas_stamp.domains’ file:
$ pdbc -d 1nmv1.pdb >nativas_stamp.domains $ pdbc -d 1o6w.pdb >> nativas_stamp.domains $ pdbc -d 1tk7.pdb >> nativas_stamp.domains $ pdbc -d 1e0l1.pdb >> nativas_stamp.domains $ pdbc -d 1e0n1.pdb >> nativas_stamp.domains
•$ stamp -l nativas_stamp.domains -rough -n 2 -prefix nativas_stamp > nativas_stamp.out
•$ aconvert -in b -out c <nativas_stamp.4> nativas_stamp.aln
Structure Alignment
advanced STAMP
Cluster: 4 ( 1nmv & 1tk7 1o6w 1e0l 1e0n )
Sc 1.11
RMS 1.72
3
COMANDS
•aconvert -in c -out b <nativas.aln> nativas.align
•alignfit -f nativas.align -d nativas_stamp.domains -out nativas_stamp_a.trans
•stamp -l nativas_stamp_a.trans -prefix nativas_stamp_a > nativas_stamp_a.out
RMSD table
1 2 3 4 5
1 1.08 2.43 0.98 1.75
2 0.00 2.78 1.30 1.51
3 0.00 0.00 2.05 3.27
4 0.00 0.00 0.00 2.02
5 0.00 0.00 0.00 0.00
Structure Alignment
XAM
Fragment 1 Fragment 2
1nmv 9 16 20 38
1o6w 2 9 11 29
1tk7 15 22 24 42
1e0l 6 13 15 33
1e0n 1 8 9 27
COMANDS:
• $ /disc9/Superposition/xam/xam
• Input files: 1nmv.pdb, 1o6w.pdb, 1tk7.pdb, 1e0l1.pdb, 1e0n1.pdb
Outputs are: nativas_xam.out
nativas_xam.pdb
Structure Alignment
XAM
Structure Alignment
XAM
Structure Alignment
XAM
TYR (Y)
TRP (W)
Loop II
Loop I
3
2
1
X-P groove
PRO (P)
TRP (W)
Loop I
Loop II
Structure Alignment
XAM
TRP (W)ASN (N)
3
2
1
Loop I
Loop II
Structure Alignment
XAM
ASN (N)
PRO (P)
TRP (W)
Loop II3
2
1
2
Loop I
Structure Alignment
XAM
GROUP IV
PIN1
Phosphorilation-dependent regulation enzime.
Implicated in multiple aspects of cell cycle regulation.
p53, Myt1, Wee1, Cdc25 have been identified as Pin1
interacting proteins.
tau, in neuronal CKS, hyper-phosphorylated in
Alzheimer's disease.
Conserved from yeast to human.
Its WW domain is a IV class domain: bind peptides
containing a proline residue preceded by a phosphoserine
or a phosphothreonine (pSer/pThr –Pro motif)
PIN1 > Introduction
Structure
N-terminal WW domain
Structure > WW domain
Three stranded β-sheet
Loop I
Loop II
Structure > WW domain > interacting residues
Trp29
Tyr18
These two residues form an hydrophobic area on the molecular surface of the WW domain
Structure > WW domain > interacting residues
Pin1’s WWdomain binding site implicates the conserved residues Tyr18 and Trp29 which constitute an
hydrophobic groove.
This hydrophobic binding site alone is not likely to explain the phospho-dependent
character of the ligand binding to the Pin1 WW domain.
But...
Structure > WW domain > interacting residues
1st: Phosphate binding pocket:
- side chains of Ser11and Arg12
- the backbone amide of Arg12
Phosphorilated residue
Hydrogen bonds
Ser11
Arg12
Structure > WW domain > interacting residues
Located in loop I
2nd: Hibrophobic groove:
- Tyr18
- Trp29
Structure > WW domain > interacting residues
constrain the proline at position +1 of the interacting phosphoserine
Structure > WW domain > interacting residues
Trp29
Tyr18
Structure > WW domain > interacting residues
Arg12Ser11
Tyr18 Trp29
Important residues in ligand interaction
Structure > WW domain > Structural residues
Trp6
Pro32
Pro32
Trp6
Distance = 2.80 Å
Structure > WW domain > Structural residues
Trp29
Tyr18 The hydrophobic groove formed by Trp29 and Tyr18 has a dual role
Distance = 3.04 Å
Interaction between Pin1 and its ligands
Microtubule-associated tau (τ) protein is a Pin1 WW domain ligand
pThr7
Pro8
Pro9
Interaction between Pin1 and its ligands
Interaction between Pin1 and its ligands
Pro8
Pro9
pThr7
Ser11
Arg12
Arg12
Ser11
pThr7
Interaction between Pin1 and its ligands
Arg12
pThr7
Distance = 2.77 Å
Interaction between Pin1 and its ligands
Ser11
pThr7
Distance = 7.74 Å H bond via phosphate!
Trp29
Tyr18
Interaction between Pin1 and its ligands
Tyr18Trp29
Structure > WW domain > interacting residues
Arg12 and Ser11 side chains (loop I) anchor the
interacting phosphate moiety via several hydrogen bonds. Form the phosphate binding pocket.
The side chain of aromatic Trp29 makes an hydrophobic
interaction with the conserved Proline at position +1 of the
interacting phosphothreonine and with hydrophobic residue at +2
(Pro9 en tau peptide).
Indeed, Tyr18 makes no interaction with the phosphorilated
ligand.
Phosphopeptide ligands are principally fixed by a charge-
charge interaction and a proline-aromatic stacking.
Interaction between Pin1 and its ligands
Intermolecular stacking structure between Trp29 and Prolines at
positions +1 and +2 is analogous to the intramolecular stacking
between conserved residues Trp6 and Pro32 in the WWdomain.
By mutagenesis studies importance of the Trp6-Pro32
interaction for the folding and the stability of WW domains.
Similar strong intermolecular Trp-Pro interaction is used to
promote protein-protein interactions between the WW domain and
its substrates.
Interaction between Pin1 and its ligands
Pro9-Trp29 interaction
Pro32-Trp6 interaction
Interaction between Pin1 and its ligands
Ser11
Arg12 Trp29
Tyr18
Ser11
Arg12
Trp29Tyr18
Interaction between Pin1 and its ligands
Mutations in the loop I appeared to weaken the interaction
without destroying it completely. The binding affinity between
phospho-substrates and Pin1 WW domain seems thus generated by
a sum of at least two energetically favorable interactions:
- charge-charge interaction between the phosphate group
and the positively charged WW loop
- proline-aromatic interaction
with none of the individual interactions appearing as absolutely
essential.
Complexed - Non-complexed
Red chain: non-complexed protein
Blue chain: complexed protein
Complexed - Non-complexed
Sequence alignment revealed that, indeed, these two sequences were obtained from the same protein:
Complexed –Non-complexed tau (τ) Conserved Trp
Complexed – Non-complexed
Loop I
Complexed – Non-complexed
Arg – Arg
Distance = 5.06 Å
Ser - Ser
Distance = 3.98 Å
Complexed – Non-complexed
Pro – Pro
Distance = 0.91 Å
Trp – Trp
Distance = 1.40 Å
Complexed – Non-complexed
Complexed – Non-complexed
Tyr – Tyr
Distance = 1.78 Å
Trp – Trp
Distance = 1.63 Å
Complexed – Non-complexed
The non-phosphorylated variants of peptide ligands did not
induce modifications of the WW domain resonances, confirming
the phospho-dependence of the interaction.
Complexed – Non-complexed
Residue Distance (amstrongs)
Arg12 5.06 Å
Ser11 3.98 Å
Trp29 1.78 Å
Tyr18 1.63 Å
Pro32 0.91 Å
Trp6 1.40 Å
Fosfat-binding pocket
Hydrophobic groove
Structural residues
GROUP I
DYSTROPHIN
WW domains have highly diverse sequence preferences
• Pin1 binds:
pS-P or pT-P
• Yes-associated protein YAP-65 and Dystrophin prefer:
P-P-X-Y (Pro-Pro-X-Tyr)
It has been a challenge to clearly delineate any common pattern of recognition across the family, aside from the
general preference for proline
DYSTROPHIN
Dystrophin is a 427 KDa molecule containing:
• N-terminal actin-binding domain
• Central rod-like region
• C-terminal region that interacts with other proteins in the dystrophin-glycoprotein complex (DGC)
• EF hand domains
• WW domain
Important structural role as a part of a large complex in muscle fiber membranes
The C-terminal region of dystrophin:
• binds the cytoplasmic tail of -dystroglycan, in part through the interaction of its WW domain with a proline-rich motif in its tail
• deletions give rise to a severe distrophic phenotype (DGC fails to form)
WW domain
EF hand domain like
Dystroglycan binding region: X-Ray crystal structure of
residues 3046-3306 of human
dystrophin
WW domainEF hand like
domain
-dystroglican
All WW domains share a core X-P binding groove
Formed by a conserved Tyr and Trp
Proline residues are recognized by these
grooves
Specific recognition?
1.- Use of variable loops
2.- Neighbouring domains
WW domain binding motif: P-P-X-Y
-dystroglycan peptide sequence: KNMTPYRSPPPYVPP
Three consecutive proline residues
form a single turn of left-handed
polyproline type II helix
Polyproline helix
PRO9
PRO10
PRO11
P-P-P-Y sequence: The core of the interaction
AROMATIC CRADLE: concave hydrophobic surface
PRO10
PRO9
TYR72
TRP83
Shallow hydrophobic pocket
TYR12
GLN79
ILE74
HIS76
Distance TYR12 hydroxil group and HIS76 aromatic nitrogen: 2.46 A
Outside the motif: Hydrogen bond
ARG7 and TRP83
Distance between ARG7 carbonil oxygen and TRP83 indolic nitrogen: 2.71A
ARG7
TRP83
¿ EF-HAND DOMAIN LIKE ?
additional interactions
The dystrophin WW domain cannot bind the dystroglycan ligand; the WW domain must be paired with the adjacent helical EF hand-like domain
The two domains form a composite recognition surface
It is possible that the primary function of WW domains is to act as an auxiliary recognition motif in tandem with other domains
Neighbouring domains are critical for specificity
PRO5 TYR6
THR188
SEQUENCE AND STRUCTURAL ALIGNMENTS FOR SUPERPOSITION
Sequence alignment (ClustalW)
Structural alignment (Stamp)
CONFORMATIONAL CHANGE
TYR72
ILE74
HIS76 (0.83A)
GLN79 (1.27 A)
Complexed
Not complexed
COMMANDS FOR SEQUENCE ALIGNMENT with CLUSTALW:
$ clustalw distrofina.fasta
1. Input sequence for alignment (fasta format)
2. Multiple alignment
1. Do complete multiple alignment now (Slow/Accurate)
Output: clustalw format (distrofina.aln and distrofina.dnd)
COMMANDS FOR STRUCTURAL ALIGNMENT with STAMP:
$ stamp –l distrofina.domains –rough –n 2 –prefix distrofina > distrofina.out
$ aconvert –in b –out c <distrofina.1> distrofina.aln
$ transform –f distrofina.1 –g –o distrofina1.pdb
• Structural basis for phosphoserine-proline recognition by group IV WW domains.
Verdecia, M.A., Bowman, M.E., Lu, K.P., Hunter, T., Noel, J.P.
• Converging on proline: the mechanism of WW domain peptide recognition.
Zarrinpar A, Lim WA.
• Structure of a WW domain containing fragment of dystrophin in complex with beta-dystroglycan.
Huang X, Poy F, Zhang R, Joachimiak A, Sudol M, Eck MJ.
• 1H NMR study on the binding of Pin1 Trp-Trp domain with phosphothreonine peptides.
Wintjens R, Wieruszeski JM, Drobecq H, Rousselot-Pailley P, Buee L, Lippens G, Landrieu I.
Bibliography
PREGUNTES PEM
1. El X-P groove del ww domain quin ambient proporciona?
a) acid
b) hidrofobic
c) basic
d) polar
e) cap de les anteriors
2. Els triptofans que donen nom al ww domain en que estan implicats?
a) en mantenir estable l’estructura
b) en mantenir estable l’interacció amb el lligant
c) les dues anteriors
d) en mantenir estable l’interacció amb l’EF-hand
e) b i d son correctes
3. Quina estructura té el domini WW?
a) TIM barril
b) Beta-meander
c) 3 cadenes beta antiparal·leles
d) 4 - helix bundle
e) 3 cadenes beta paral·leles
4. A quines malalties es pensa que pot estar associat el WW domain?
a) Distròfies musculars
b) Alzheimer
c) Les dues anteriors
d) Parkinson
e) Totes les anteriors
5. A què s’uneix principalment un domini WW?a)Prolinesb)Fosfoserines/fosfotreoninesc)Les dues anteriorsd)Tirosines fosforiladese)Totes les anteriors
6. A quin domini és funcionalment semblant el domini WW?a)SH3b)SH2c)Els dos anteriorsd)PHe)Cap dels anteriors
7. Quins residus formen el solc hidrofòbic a la proteïna Pin1?
a)Tyr18
b)Trp29
c)Els dos anteriors
d)Arg12
e)Tots els anteriors
8. Quin loop està implicat en la unió al lligand fosforilat a la Pin1?
a)El loop I
b)El loop II
c)Els loops I i II
d)El lligand no s’uneix quan està fosforilat
e)El lligand s’uneix quan està fosforilat però no està implicat cap loop.
9.- Tots els dominis WW, tot i tenir el mateix solc d’unió per seqüències amb patró X-P s’uneixen especificament als seus lligands. Com aconsegueixen aquest reconeixement específic?a) a) Utiliçant loops variables pel reconeixement del lligand.b) b) Mitjançant interaccions amb dominis veïns.c) c) a i b son correctesd) d) El reconeixement es depenent del reconeixement de l’aminoàcid X e) Totes són certes.
10.- La distrofina, indica la resposta incorrecta:a) a) És una proteïna amb funció estructural.b) b) Té un WW domain.c) c) Té dominis EF hand-like.d) d) Desfosforilada és inactiva.e) e) S’uneix a motius del tipus P-P-X-Y mitjançant el seu domini WW.
PREGUNTES D’ASSAIG
1. Segons els resultats dels aliniaments estructurals:
- STAMP --- rmsd : 1.62
- STAMP avançat --- rmsd : 1.72
- XAM --- rmsd : 2.02
Per quin motiu ens podriem quedar (i ens quedem) amb l’aliniament que ens fa el XAM (tot i els resultats del RMSD)?
Tot i que l’aliniment que ens fa el XAM té el pitjor dels RSMDs obtinguts amb altres programes de superposició (2.02), hem de tenir en compte de que es tracta de un valor acceptable i que en les altres superposicions obtingudes per l’STAMP no aliniem la lamina beta-3, fet que ens pot distorsionar els resultats. Per aixó fem una superposició manual on ens asegurem de que tots els residus rellevants de l’estructura i que en mantenen conservats estiguin superposats.
2. Explica breument el plegament dels dominis WW, les seves carácterístiques i a quin tipus de seqüències s’uneix.
És una fulla beta amb tres cadenes antiparal·leles. La seva seqüència és altament conservada en tota la familia i es caracteritza per dos triptofans (W) separats per uns 20-22 aa. Les seqüències reconegudes pel domini són riques en prolines (P), tot i així, cada proteïna de la familia segons el grup del que formi part reconeixerà diferents motius.
3. Explica com s’uneix un lligand fosforilat a la proteïna Pin1 i les diferències respecte a un no fosforilat.
El lligand fosforilat (en el residu Tre o Ser) s’unieix mitjançant els residus Ser11 i Arg12 del phosphate binding pocket i pel Trp29 del solc hidrofòbic. El residu no fosforilat no s’unirà al ww domain de Pin1.
4. Explica quins dos residus es veuen més afectats pel canvi de conformació en comparar la Pin1 complexada amb la no complexada i expleca per què.
El dos residus més afectats són la Ser11 i la Arg12, que formen el fosfat binding
pocket, situat al loop I, donat que és un lloc d’unió al lligand fosforilat.