Laura Guerrero Ainhoa Plaza Nira Suárez Rosa Turbau

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

http://scop.mrc-lmb.cam.ac.uk/scop/

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


1 2 3

WW domain family


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

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


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...


1st: Phosphate binding pocket:

- side chains of Ser11and Arg12

- the backbone amide of Arg12

Phosphorilated residue

Hydrogen bonds

Ser11

Arg12


Located in loop I

2nd: Hibrophobic groove:

- Tyr18

- Trp29


constrain the proline at position +1 of the interacting phosphoserine


Trp29

Tyr18


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



Pro8

Pro9

pThr7

Ser11

Arg12

Arg12

Ser11

pThr7


Arg12

pThr7

Distance = 2.77 Å


Ser11

pThr7

Distance = 7.74 Å H bond via phosphate!

Trp29

Tyr18


Tyr18Trp29


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.


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.


Pro9-Trp29 interaction

Pro32-Trp6 interaction


Ser11

Arg12 Trp29

Tyr18

Ser11

Arg12

Trp29Tyr18


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

Arg – Arg

Distance = 5.06 Å

Ser - Ser

Distance = 3.98 Å


Pro – Pro

Distance = 0.91 Å

Trp – Trp

Distance = 1.40 Å



Tyr – Tyr

Distance = 1.78 Å

Trp – Trp

Distance = 1.63 Å


The non-phosphorylated variants of peptide ligands did not

induce modifications of the WW domain resonances, confirming

the phospho-dependence of the interaction.


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

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_Abstract&term=%22Zarrinpar+A%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_Abstract&term=%22Zarrinpar+A%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_Abstract&term=%22Lim+WA%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_Abstract&term=%22Lim+WA%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_Abstract&term=%22Huang+X%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_Abstract&term=%22Huang+X%22%5BAuthor%5D

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.

Laura Guerrero Ainhoa Plaza Nira Suárez Rosa Turbau

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