WW DOMAIN WEB PAGE
Summary

1. Sequence analysis

1.1 WW domains in Yeast
1.2 WW domain binding motifs
First type: PPXY motif
Second type: PPLP motif
Third group: PXXGMXPP motif
Fourth group: (Phospho-S/T)P motif
1.3 Phosphorylation
2. Structure



1. Sequence analysis

1.1 WW domains in Yeast

We did a sequence alignment of the 11 ww domains (smart architecture ) found so far in 8 proteins of the yeast proteome. These proteins are:

1.2 WW domain binding motifs

see here definition

We performed searches in yeast database looking for possible WW domain binding proteins on the basis of the presence in their sequences of one of the known WW domain binding motifs.

Methods used: 1. BLAST searching for exact matches against yeast database at the NCBI
2. Pattern Matching (PatMatch) against the yeast non redundant database (YNRDB) at the Saccharomyces Genome Database at the Stanford University


1. PatMatch using PPXY.
- results : 352 sequences retrieved.

2. BLAST using HTYLPPPYPG [EMBO J, 1999, 18, 2551].
- Results: the motif is not perfectly conserved in any of the yeast sequences.

3. PatMatch using PPPY [J. Virology, 1999, 37, 2921]
-results: Two proteins showing this motif could be of interest, DNA TOPOISOMERASE I (TOP1_YEAST ) and UBIQUITIN-CONJUGATING ENZYME (UBC6_YEAST ).

Location of the motifs in the structure
DNA TOPOISOMERASE I (156-159): the 3D structure of N-terminal fragment of this protein is known (1ois ). The motif is accessible to the solvent (fig.1 and table I). Note that the Tyr side chain is not very accessible however. Its side chain does not extablish Hbonds.

Fig. 1 Location of the PPPY motif (in green) in topoisomerase I from yeast. The protein contains two domains
represented in blue and red. The motif connects the two domains.

Table I. Acessibility of the residues in the motif (ABS=absolute, REL=relative in %)

REM RES _ NUM All-atoms Total-Side Main-Chain Non-polar All polar
REM ABS REL ABS REL ABS REL ABS REL ABS REL
RES PRO 156 26.03 19.1 18.25 15.2 7.79 48.0 18.25 15.1 7.79 51.3
RES PRO 157 104.04 76.4 100.89 84.1 3.15 19.4 100.89 83.4 3.15 20.8
RES PRO 158 104.23 76.6 95.88 80.0 8.36 51.5 95.88 79.3 8.36 55.0
RES TYR 159 67.44 31.7 48.84 27.5 18.60 52.6 46.55 34.1 20.89 27.4

Several other structures are known for topoisomerase I . In fig.2 we show the structural alignemnt of Human DNA topoisomerase in non-covalent complex with a 22 base pair DNA duplex (1a36 ) with the N-terminal fragment of the yeast enzyme. The two structures are rather similar, they also share high sequence identity in the alignment region. In yeast, binding to DNA is thus probably acompanied by minor conformational changes and the Tyr residue probably does not become more accessible upon binding as well. Binding to a ww domain could neverthless trigger larger conformational rearrangements. Those could block the function of enzyme (binding). Its is also interesting to note that the motif is quite conserved ( results )

Fig. 2 (top) Fitting of N-terminal fragment of DNA topoisomerase I from yeast (mangenta) with the human DNA topoisomerase I (red) complexed with a 22 base pair DNA (yellow). The motif is shown in cyan. (bottom) detailed view of the motif.


UBIQUITIN-CONJUGATING ENZYME (21-24): we built a model for this protein. The WHAT_CHECK report of the model presents problems in the packing of some residues. However, those residues are not located near the motif. The overall topology of the model should be correct nevertheless since ubiquitin-conjugating enzymes have a high sequence similarity. The motif is accessible (fig. 3 and table II).

Fig. 3 Location of the PPPY motif (in green) in UBIQUITIN-CONJUGATING ENZYME

Table II. Acessibility of the residues in the motif (ABS=absolute, REL=relative in %)

REM RES _ NUM All-atoms Total-Side Main-Chain Non-polar All polar
REM ABS REL ABS REL ABS REL ABS REL ABS REL
RES PRO 21 62.76 46.1 48.85 40.7 13.91 85.7 48.92 40.5 13.83 91.1
RES PRO 22 8.56 6.3 6.23 5.2 2.33 14.4 6.23 5.1 2.33 15.4
RES PRO 23 106.22 78.0 100.08 83.5 6.14 37.8 100.08 82.7 6.14 40.4
RES TYR 24 124.08 58.3 109.06 61.5 15.02 42.5 78.03 57.2 46.05 60.4


4. PatMatch analysis using different RSP5 binding peptides according to Kasanov's results [Chem Biol, 2001, 8, 231-241]. They defined the peptide ligand preferences for 14 WW domains knowing to bind the group 1 motif. Among them there are also RSP5 WW domains.
We used each peptide reported to bind RSP_1, RSP_2, RSP_3 as seed sequence in PatMatch retrieving no yeast sequence showing them.

4.1 PatMatch using RSP5_1 consensus [WXX(W/Y)LXPPXY].

4.2 PatMatch using RSP5_2 consensus (BXXPPPY). 4.3 PatMatch using RSP5_3 consensus (PPPYXXB). 1. BLAST search using PPPLP motif.
- results : 12 proteins showing the exact match, 39 containing the PPPL motif and 67 containing the PPLP motif.
Analysis of the 12 exact matches
1.2 Domain omposition and function
Sequence ID
Definition
Residues
Function
Domains
PSI-BLAST results (NRDB)
Q06604 Ypr171w 186-190 unknown none Conv. 2nd iteration. No homologous.
P53933 Ynl094w 507-511 unknown none Conv. 3rd iteration. Only hypothetical proteins, none showing the motif.
P40021 YER033C 120-124 unknown none Conv. 1st iteration. No homologous.
P28003 YAL034C or FUN19 245-249 unknown none Conv. 3rd iteration. Under threshold, only hypothetical proteins, none showing the motif.Below threshold, protein kinase PKNbeta (JC7083, E=0.041, PPPKP)
Q00453 RGM1 155-159 Probable transcription repressor. The Pro-rich region (95-211) is able to repress the DNA expression. ZnF_C2H2 (19-44; 50-73 Zinc Finger domain)
S64993 YLR144C 24-28 Necessary for actin polymerization in permeabilized cells (Science, 1999 285, 901-6) none
P40450 BNI1 related protein 1 777-781
803-807
824-828		 Involved in microtubules organization. Potential target for RHO proteins. FH2 (Formin Homology 2 Domain, 868-1332)
P40453 Ubiquitin carboxyl-terminal hydrolase 7 530-537 Hydrolysis of ubiquitin C-terminal thioester RHOD (Rhodanese Homology Domain 318-447)
UCH-1(Ubiquitin carboxyl-terminal hydrolases 1 609-640)
UCH-2 (Ubiquitin carboxyl-terminal hydrolases 2 994-1068)
P32521 PAN1 protein 1402-1406 Cytoskeletal adaptor (GO) EH (Eps15 homology domain)
Efh (EF-hand, calcium binding motif)
P37370 Verprolin 353-357 Involved in cytoskeletal organization and cellular growth. It can bind SH3 domain and is very rich in Pro. WH2 (Wiskott Aldrich syndrome homology region 2/actin binding 30-47, 87-106)
P41832 BNI1 protein 1305-1309 Involved in microtubules organization. Potential target for RHO proteins DNA_topoIV (DNA gyrase/topoisomerase IV, subunit A 756-789)
FH2 (Formin Homology 2 Domain 1348-1824)
P32491 MAP kinase (MKK2) 88-93 Ser/Thr protein kinase involved in a signal trasduction pathway important in yeast cell morphogenesis and growth. S_TKc (Ser/Thr protein kinases, catalytic domain 214-481)

1.2 Fasta search against the PDB database using the 12 proteins showing the perfect match.
results : in most of the cases the motif is outside the region of alignment, in other cases it is not 100% conserved. The results' file also contains links to SacchDB and SWISS_PROT entries for each of the 12 proteins.
2. PatMatch using PPPLP motif.
- Results: Same results than using BLAST.

3. Pattern Search against PBD for PPLP motif containing proteins.
- Results: 31 structures retrieved. Most of them are peptides or don't show homologous when used as seed sequences against YNRDB. No yeast homologous sequences have been retrieved mantaining a conserved PPLP motif.


1. PatMatch using PXXGMXPP motif
-Results:
SequenceName
MatchPattern
Function
MatchStartCoord
MatchStopCoord
Q05455 PLIGMAPP unknown 24 31
P45976 PMPGMMPP Pre-mRNA polyadenylation factor that directly interacts with poly(A)polymerase. 306 313

1.3 Phosphorylation

PROSITE finds a number of phosphorylation patterns in the yeast ww domain sequences (see results). A casein kinase II phosphorylation pattern
is found in the late ww domains (near the HECT domain) of all E3 ubiquitin ligases analysed (deposited in SMART). Phosphorylation at a
conserved Thr was also confirmed using an alternative approach NetPhos [JMB (1999), 294,1351-1362]. The analysis was mostly carried out for
ESS1 and RSP5 since the function and homologues of these proteins are well characterized. It is also interesting to note that the phosphorylation site occurs at the binding interface in the structure of the 3rd ww domain of nedd4 from Rattus norvegicus. (pdb code 1i5h )-see fig. 4.

Fig. 4 Location of the putative phosphorylation site (mangenta) in the 3rd ww from nedd4 of Rattus norvegicus. The PPPY motif is shown in red.
The structure of PIN WW domain from Homo sapiens has been determined both in the free state (pdb code 1pin) as well as bound to a double phosphorylated peptide (pdb id code 1f8a). Two Arg interact with the phosphorylated groups in the 1f8a structure. The first Arg is located in strand1 and the second in loop1. The first Arg is found both in ESS1 as well as in the three ww of domains of RSP5, but not in PR40. The second Arg is not found in any domain. The corresponding position in ESS1 sequence is however also a positivelly charged residue, Lys. (see here the alignment ).

RSP5, PR40 and ESS1 WW domains have been suggested to bind phosphorylated proteins, although experimental data on the interaction of the
domains with phosphorylated proteins is not very clear. For example, it appears that RSP5 has the capacity to interact with both the phosphorylated and unphosphorylated carboxy-terminal domain (CTD) of RNA plymerase II.
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