Pyridoxal Phosphatase Results

Evolution

Multiple Sequence Alignment

Fig 1. Multiple sequence alignment (MSA) showing the conserved residues for Pyridoxal phosphatase.

Pyridoxal Phosphatase Multiple Sequence Alignment

Fig 2. Spatial alignment of conserved residues from the MSA.

The picture on the left shows the spatial alignment of the conserved residues from the multiple sequence alignment. The red highlighted residues are those that are conserved throughout the alignment while the yellow residues are those which have conservative substitutions in some of the organisms. The green residues denote those that had semi-conservative substitutions.

Phylogeny Tree

Fig 3. Rectangular cladogram generated from the BLAST and Multiple Sequence Alignment after boot-strapping.

Pyridoxal Phosphatase Phylogeny Diagram

Structure

General Structure of Pyridoxal Phosphatase & 2cfsA

Fig 4. Courtesy (and edited) of Almo et al., 2007. Structure of 2cftA - Human Pyridoxal 5'-Phosphate Phosphatase with its substrate. As mentioned in the "Introduction" section, the core domain consists of beta sheets sandwiched by alpha helices. 2cftA is similar to 2cfsA, with the differences being the presence of a PLP ligand and the type of metal ions (2cfsA has magnesium ions, 2cftA has calcium ions). Notice the similarities between the structure of 2cftA and the PyMOL-generated image of 2cfsA (Fig 5.).

Fig 5. The three-dimensional structure of 2cfsA, as generated by PyMOL

Red - Helix
Yellow - Sheet
Green - Loop
Green Dots - Water molecules

PDB

Based on the information provided in the website, Pyridoxal Phosphatase has the following characteristics:

• Pyridoxal Phosphatase is isolated from Homo Sapiens and is expressed in Escherichia Coli.
• Structure is similar to the Pyridoxal Phosphate Phospatase protein
• 1 (A) Chain
• Consists of Magnesium components
• Resolution of 2.40 angstroms. This means that the number of sidechains in the wrong rotamer is smaller as compared to proteins of a higher resolution (>2.5 angstroms). Other characteristics of proteins of similar resolutions are: (1) many small detectable errors, (2) correct folding, (3) fewer number of errors in the surface loops and (4) visible water molecules and small ligands.
  

http://www.rcsb.org/pdb/explore/explore.do?structureId=2cfs

DALI

Fig 6. Significant (and rejected) hits as generated from the DALI database

A total of 176 hits were generated, of which only the first 11 (as shown in Fig. 6) were predicted to be significant. The others were rejected on account that their respective lali scores (refer to the red, boxed section) were less than half of Pyridoxal Phosphatase's (nres: 296). The lali value is important as this refers to the number of structurally equivalent residues between the hit protein and the protein-of-interest. If two proteins are not structurally similar/related, chances are their functions will not be substantially similar.

http://ekhidna.biocenter.helsinki.fi/dali_server/results/20080506-034-babd470eae557046b605d8f91e8a16e5

It was noted that none of the hits actually matched 2cfsA. The closest was a Pyridoxal Phosphate Phosphatase (PDB ID 2oycA) which based on the results, was predicted to be highly similar to 2cfsA. Table 1 highlights the similarities between Pyridoxal Phosphatase (2cfsA) and the Pyridoxal Phosphate Phosphatase (2oycA)

Table 1 - 2oycA, as compared against 2cfsA

Using the PyMOL software, 2oycA was superimposed against 2cfsA, and both structures, as shown in Fig. 7, are structurally similar.

Fig 7. Superimposition of 2oycA against 2cfsA, as performed on PyMOL

The results obtained when 2cfsA was re-run against the DALI database

Towards the end of the study, 2cfsA was re-run against the DALI database. The rationale behind doing so was that the initial results yielded 2oyc as the query protein.

As opposed to the first run, 515 hits were generated. The top 25 protein hits were deemed significant, with the 26th protein hit onwards rejected for the same reason as that of the first batch - that their lali values were too low (less than half of 2cfs') for them to be considered as structurally significant to 2cfsA. More importantly was the observation that the DALI database identified 2cfsA as the query protein (unlike the first run). In addition, protein hit numbers 2 (2cftA) - 6 (2oycA) were ALL observed to belong to the Pyridoxal Phosphate Phosphatase family. Based on this, it was easier to narrow down the potential research candidates in terms of structural similarities. Due to time constraint however, not all the protein hits were researched on.

PDBsum

Fig 8. Three different views of 2cfsA, courtesy of PDBsum

Fig. 8 offers three different views of 2cfsA. The purple chains represent the amino acid chain of 293 amino acids while the green spheres represent the magnesium ions (x2).

By clicking the "Protein chain" link, the user was re-directed to a website containing information pertaining to the secondary structures of both 2cfsA and 2oycA.

Secondary Structures of 2cfsA (L, http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl) and 2oycA (R).

Topology diagrams of 2cfsA (Fig 9.1. http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2cfs&template=protein.html&r=wiring&l=1&chain=A) and 2oycA (Fig 9.2). The topology diagram is a simplified version of the secondary structure information as provided above, and is an indication of the location of the alpha helices (represented by the red cylinders) and beta strands (represented by the pink arrows).

Fig 9.1. Topology Diagram of 2cfsA

Fig 9.2. Topology diagram of 2oycA

Cleft Analyses of 2cfsA (Fig 10.1. http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2cfs&template=clefts.html&pdbcode=2cfs&r=speedfill) and 2oycA (Fig 10.2).

Fig 10.1. Cleft Analysis of 2cfsA

Fig 10.2. Cleft Analysis of 2oycA

Cleft Analysis via PyMol

Fig 11.1. Front View of 2cfsA & its potential binding sites, as generated using PyMOL

Fig 11.2. Front View of 2oycA & its potential binding sites, as generated using PyMOL

Fig 11.3. Back View of 2cfsA & its potential binding sites, as generated using PyMOL

Fig 11.4. Back View of 2oycA & its potential binding sites, as generated using PyMOL

PROFUNC (2cfsA)

Related Protein Sequences in the PDB (SAS)

Matches to existing PDB Structures

Secondary Structure Matching (SSM)

Nest Analysis

Summary of Predicted Function

Based on the results obtained via the local alignment method (Smith-Waterman method), it was noted that 2fcsA and 2oycA were indeed similar in terms of sequence and structure. However, contrary to the implications of the DALI-generated results, 2oycA was no longer the most closely-related protein to 2cfsA. That role now belonged to 2cftA. Based on the results generated by Profunc, 2cftA's sequence and structure, as provided by PDB, were identical to that of 2cfsA. The secondary structure matching, however, showed that 2cfsA and 2cftA had differing (albeit minimally) Q scores (1.000 and 0.981 respectively). 2cfsA was described as a "Crystal structure of human pyridoxal 5'-phosphate phosphatase", while 2cftA was described as a "Crystal structure of human pyridoxal 5'-phosphate phosphatase with its substrate". In addition, 2oycA was rated 4 tiers below 2cftA (second in the scoring table).

PROFUNC (2oycA)

A search on 2oycA was also carried out via PROFUNC, and consistency in the results confirmed that 2cfsA and 2oycA were indeed structurally similar. This lends weight to the original hypothesis that they could be functionally related. In fact, based on the Nest Analysis method, 2oycA was observed to be sharing a large number of active sites. The results of the Nest Analysis (2oycA) are as shown below. Notice the similarities between the active sites of both 2oycA and 2cfsA.

In addition, the predicted function of 2oycA was similar to 2cfsA.

Fig 12. The LIGPLOT of interactions involving the PLP ligand in 2cftA. This was hypothesized to be the location of 2cftA's active site. The Mg 1296(A) ion of 2cfsA is located in the same position as the calcium ion of 2cftA. Therefore, there could be a possibility that the active site of 2cfsA could be at Mg 1296(A).

Fig 13. The LIGPLOT of interactions involving the Mg 1296(A) ion in 2cfsA. Notice the similarity of its location with relation to the calcium ion in 2cftA

Fig 14. The LIGPLOT of interactions involving the Mg 1297(A) ion in 2cfsA. While it does not seem to share any similarities with the PLP ligand in 2cftA, the information provided by the LIGPLOT will be useful when visualizing the catalytic site of 2cfsA via PyMOL.

Fig 15. The catalytic site of 2cftA, with its PLP ligand and inhibitory calcium ion. As illustrated, the calcium ion is hepta-coordinated and participates in a bidentate interaction with the active site nucleophile Asp-25, resulting in a loss of activity. Diagram and caption courtesy of Almo et al., 2007.

Fig 16. The three-dimensional visualization of the catalytic site of 2cfsA. This was generated based on the location of the residues constituting the catalytic site, as provided in Figs 13 and 14.

Based on the above-mentioned results, it was concluded that 2cfsA, 2cftA and 2oycA were structurally similar, and that further functional studies could indeed uncover the function of the protein-of-interest: 2cfsA

Function

In order to predict the function of Pyrixol Phosphotase, sequence homology, structural similarities, neighbouring genes, expression levels in tissue and protein-protein interactions were analysed.

General Annotation

Sequence homology

To check for function among similar homologs, 2cfs_A sequence was run against FASTA, Pfam and PSI Blast non-redundant database. A search using Pfam and BLAST revealed that 2cfs_A belonged to the superfamily, Haloacid dehalogenase-like hydrolase. The top 15 search results from BLAST

http://blast.ncbi.nlm.nih.gov/Blast.cgi

Structural Similarities

Protein-Protein Interactions

http://string.embl.de/newstring_cgi/show_network_section.pl?taskId=ph89gQxcLucL&allnodes=1