Fascin Results

BLAST

Blast Results show that fascin 1 is very highly conserved throughout the Eukarya Domain providing 112 hits all with excellent E value and High scores

Figure 1:Fascin allignment showing identity to several other homologous proteins

Sample Fascin sequence (Text File)

Media:fascin1_fasta.txt

FASTA sequences from BLAST (Text File)

Media:FASTA_fascin1_all.txt

Multiple Alignment

From a possible 100 FASTA sequences, were cut down to 31. The process of elimination was conducted by the deletion of sequences with too many bp before and after the sample sequence, followed by the deletion of sequences with large segments of gaps in conserved and variable regions of the sequence alignment. Fascin domains were found via a protDOM search and compared to the remaining sequences.

Figure 2:Multiple allignment

Phylogeny Trees

Before any deletion of sequences occured, A tree was formed using 100-replications Dayhoff neighbor-joining method as a base line.

Figure 3:Non-specific phylogeny tree encompasing many species

Neighbor-method with Bootstrap values (1 = 100) Results were run at 500 replications

Figure 4:Neighbor-method phylogeny tree

Neighbor-method with Bootstrap values, compressed. Sequences with below 50 were deleted.

Figure 5: Specific phylogeny tree of classes insecta and anamalia

Fascin structure and domains

The Fascin protein is comprised of 8 repeats of the Fascin-like domain in total. It is made of two identical subgroups, each of which contains 4 Fascin-like domains linked together. The 2 subgroups are linked back to back slightly off-centre. The Fascin-like domain's tertiary structure adopts a beta-trefoil fold pattern with an internal threefold repeat. Secondary structure is predominantly Beta-sheet and random coil (loop) with small regions of alpha-helix. Structural similarity with several unrelated proteins is evident (eg. FGF).

Figure 6: Layout of primary and secondary sequences with domain annotations. (Finn et. al. 2006, Kabsch et. al. 1983, Murzin et. al. 2005)

Figure 7: 'Front' view showing secondary structure of Fascin.

Figure 8: 'Front' view of Fascin protein with each Fascin domain coloured. Red, Blue, Green and Yellow segments represent one subgroup while Aqua, Magenta, White and Orange represent the other. Grey colouration represents conserved residues found in sequence allignment.

Figure 9: 'Back' view of Fascin protein. Same colouration as above.

Structual allignments

A Dali database search returned several hundred similar structures to Fascin 1. The first page of results can be seen below. Unfortunately none of the structurally similar molecules were actin binding so could not help with actin binding regions. The molecule 1ijt (Fibroblast Growth Factor - FGF) was alligned with Fascin to show structural similarity (even though they are functionally very different and only contain 9% sequence identity). Structurally FGF has much more Beta sheet secondarty structure, but as said before they are still quite similar.

Figure 10: Dali output showing first 33 structurally similar protein results.

Figure 11: Pymol allignment of Fascin 1 (green) and FGF (aqua)

Similarity is between FGF and the Fascin domain (not the molecule).

Cleft/Pocket analysis

Pockets and clefts in the molecular surface of Fascin were analysed using the online service CASTp (Dundas J et. al. 2006). Three major pockets corresponding to the beleived actin binding sites for Fascin are shown with both a front and back view. Conserved regions from 'across species' multiple sequence allignment are outlined in the sequence window. Note how many of the residues involved in the pocket are from these conserved regions.

Figure 12: Putative actin binding pocket regions on Fascin 'front view'.

Figure 13: Putative actin binding pocket regions on Fascin 'back view'.

Electrostatic surface model

Using the online PDB2PQR resource in conjunction with the APBS feature in pymol an electrostatic surface model for Fascin was generated. Regions corresponding to our putative actin binding sites were circled. Note the positively charged regions encapsulating a negatively charged centre.

Figure 14: Front view of electrostatic surface model. Putative actin binding site 1 (front) circled in green.

Figure 15: Back view of electrostatic surface model. Putative actin binding site 2 (back) circled in green.

Hydrophobicity Plot

A hydrophobicity plot was generated and showed that overall the Fascin-1 protein is hydrophobic.

Figure 16: Plot showing the hydrophobicity of fascin-1 protein and second plot showing hydrophilicty plot which acts as a peptide property calculator. From these results we can overall state that this protein overall is hydrophobic.

Important molecular regions

The focus so far has been on binding of Fascin to its ligand actin. Fascin actually interacts with many other protein molecules to undergo its function. One such molecule is the phosphorylating molecule phosphokinase-C (PKC). Phosphorylation of serine residues 39 on a Fascin subgroup alters the actin binding properties of the molecule. From our research we have also found that although it is known Fascin binds actin, the site of binding is still unknown. Through multiple sequence allignment across species, electrostatic modeling and pocket analysis we have tried to determine the actin binding site on the Fascin molecule. The image below shows Fascin's overall composition and these 2 important regions.

Figure 17: 360 degree view of Fascin. Putatitive front (1) and back (2) actin binding sites are shown in grey and Serine 39 residues on both Fascin subgroups are shown in red.

Putative actin binding sites were localised to residues 136-143 and 386-395.

Protein Interaction and Cellular Location

Interaction of other proteins with Fascin-1 and its concentrations levels in different regions of the body were analysed and represented below.

Figure 18: Image showing various predicted functional partners that interact with Fascin-1.

Figure 19: Image showing expression in various human cell types

Abstract | Introduction | Methods | Results | Discussion | Conclusion | References
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