Results - NANP

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Query Sequence

The amino acid query sequence of 2gfh protein (Figure 3) from Mus musculus is obtained from Genbank.

<1 mgsdkihhhh hhmglsrvra vffdldntli dtagasrrgm levikllqsk yhykeeaeii

61 cdkvqvklsk ecfhpystci tdvrtshwee aiqetkggad nrklaeecyf lwkstrlqhm

121 iladdvkaml telrkevrll lltngdrqtq rekieacacq syfdaivigg eqkeekpaps

181 ifyhccdllg vqpgdcvmvg dtletdiqgg lnaglkatvw inksgrvplt sspmphymvs

241 svlelpallq sidckvsmsv>

Figure 3. The 260 amino acid sequence of 2gfh protein.

Sequence Homology

From the BlastP similarity was used for comparison as these had shown higher homology to the query sequence sequence search, a total of 500

proteins were yielded.Only a total of 38 proteins, in contrast with the remainder of the search results.These proteins were chosen according to

their bit scores and E-values. Two more outlier partial sequences contributing to poor overall alignment (huge deletion gaps) were subsequently

removed. The remaining 36 sequences were used for the generation of the phylogenetic tree (and bootstrapped tree as well).

Multiple Sequence Alignment

The following multiple sequence alignment (MSA) was obtained (Figure 4). From the alignments, gi|10888xy and

gi|10888yz are representative of gi|108881764 and gi|108881765 respectively. Both these

hypothetical proteins belong to the mosquito Aedes aegypti.

The identifier numbers for these two proteins were initially changed to an alpha-numeric one, due to the inability of Phylip to generate a tree

from the original identifiers. This was due to the fact that the programme only took the first five numeric digits (10888), thereby resulting

in a programme error prompt which listed both proteins as duplicates (from the identifier numbers). Both these identifiers were subsequently

renamed for the final phylogenetic tree.

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Figure 4. MSA of query (top-most sequence – No.1) and related sequences.

From the MSA, it can be observed that there are generally slight domain conservations throughout the protein sequences. Small insertion and

deletion gaps were noticeable along the alignment as well. A particularly large insertion gap was observed between amino acids 91 to 114.

The organisms with the large insertion gaps were as identified below:

Bacillus licheniformis

Bacillus subtilis

Bacillus halodurans

Bacillus clausii

Symbiobacterium thermophilum

A highly conserved (with invariant) section of amino acids (LV)–(LVA)–(LIV)–(LIV)-T-N-G was observed in all the sequences from amino acid 211

to 217 in the alignment. Downstream of this conserved portion of genes are 5 more invariant positions (1 or 2 amino acids in length).From these

short conservation regions, the functions or even structure of the encoded proteins could have significance in its evolutionary pattern.

Phylogenetic Tree

The tree was plotted to obtain the phylogenetic lineage (Figure 5).

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Figure 5. (A) Phylogenetic tree showing organisms with related protein sequence homology in Radial Tree view. (B) Rectangular

Cladogram view with related protein sequence homology.

From the Rectangular Cladogram view, it could be observed that there are four distinct separate groups involving fishes, mammals (where the

query protein is also mapped), bacteria and insects.


Bootstrapping values obtained were analysed. Branch values occurring below 75% (<75%) would be indicated by an asterisk (*),

as shown in Figure 6.

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Figure 6. Branch bootstrap values in Rectangular Cladogram view. Branches with strap values <75% were indicated with

asterisks (*)

DALI Searching

SUMMARY: PDB/chain identifiers and structural alignment statistics NR. STRID1 STRID2 Z RMSD LALI LSEQ2 %IDE REVERS PERMUT NFRAG TOPO PROTEIN

 1: 3033-A 2gfh-A 41.1  0.0  246   246  100      0      0     1 S    HYDROLASE        haloacid dehalogenase-like hydrolase domain
 2: 3033-A 1fez-A 18.1  3.5  178   256   22      0      0    13 S    HYDROLASE        phosphonoacetaldehyde hydrolase         (bacillus c 
 3: 3033-A 2hsz-A 17.9  3.3  168   222   23      0      0    13 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION    novel predicted
 4: 3033-A 1qq5-A 17.3  3.1  198   245   19      0      0    12 S    HYDROLASE        l-2-haloacid dehalogenase       (xanthobacter aut 
 5: 3033-A 1o03-A 17.0  5.0  188   221   20      0      0    11 S    ISOMERASE        beta-phosphoglucomutase         (lactococcus lactis
 6: 3033-A 2b0c-A 16.4  2.6  184   199   20      0      0    13 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION    putative phospha 
 7: 3033-A 2fdr-A 15.8  4.4  190   214   19      0      0    15 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION    conserved hypoth
 8: 3033-A 2p11-A 15.7  2.9  194   211   16      0      0    20 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION    hypothetical pro 
 9: 3033-A 1te2-A 15.7  3.6  170   211   19      0      0    15 S    HYDROLASE        putative phosphatase    (escherichia coli o157
10: 3033-A 1yns-A 15.3  4.0  169   254   11      0      0    13 S    HYDROLASE        e-1 enzyme (enolase-phosphatase e1)     (homo s 
11: 3033-A 1qyi-A 15.0  3.5  198   375   19      0      0    17 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION    hypothetical pro
12: 3033-A 2i6x-A 14.9  3.1  176   199   19      0      0    18 S    HYDROLASE        hydrolase, haloacid dehalogenase-like family 
13: 3033-A 1u7p-A 14.3  2.9  144   164   18      0      0    14 S    HYDROLASE        magnesium-dependent phosphatase-1 (mdp-1)       (
14: 3033-A 1ymq-A 14.1  2.3  130   260   16      0      0    14 S    TRANSFERASE      sugar-phosphate phosphatase bt4131      (bacte 
15: 3033-A 1j8d-A 13.1  2.5  141   180   11      0      0    12 S     HYDROLASE       deoxy-d-mannose-octulosonate 8-phosphate ph
16: 3033-A 2ho4-A 12.9  2.4  131   246   19      0      0    14 S    HYDROLASE        haloacid dehalogenase-like hydrolase domain 
17: 3033-A 1pw5-A 12.7  2.3  136   246   21      0      0    12 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION    nagd protein, pu
18: 3033-A 1nf2-A 12.7  2.6  127   267   13      0      0    11 S    STRUCTURAL GENOMICS/UNKNOWN FUNCTION     phosphatase     (the 
19: 3033-A 1rlm-A 12.4  2.8  131   269   13      0      0    14 S    HYDROLASE        phosphatase Mutant      (escherichia coli) bacte
20: 3033-A 1f5s-A 12.1  3.5  159   210   14      0      0    15 S     HYDROLASE       phosphoserine phosphatase (psp)         (methanoco 
21: 3033-A 1cr6-B 12.0  3.8  177   541   18      0      0    18 S    HYDROLASE        epoxide hydrolase       (mus musculus) mouse expr
22: 3033-A 1rku-A 11.9  3.6  172   206   11      0      0    18 S    TRANSFERASE      homoserine kinase       (pseudomonas aeruginosa 
23: 3033-A 2b30-A 11.8  2.7  134   284   16      0      0    12 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION    pvivax hypotheti
24: 3033-A 1kyt-A 10.5  2.5  122   216   13      0      0    15 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION    hypothetical pro 
25: 3033-A 2o2x-A 10.3  3.6  139   204   17      0      0    14 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION    hypothetical pro
26: 3033-A 1u02-A 10.1  2.7  128   222   16      0      0    12 S    STRUCTURAL GENOMICS      trehalose-6-phosphate phosphatase 
27: 3033-A 2fea-A 10.0  3.5  167   219    7      0      0    21 S    HYDROLASE        2-hydroxy-3-keto-5-methylthiopentenyl-1- pho
28: 3033-A 2hx1-A  9.6  3.2  130   275   24      0      0    19 S    HYDROLASE        predicted sugar phosphatases of the had supe 
29: 3033-A 1mh9-A  9.2  3.2  146   194   15      0      0    15 S    HYDROLASE        deoxyribonucleotidase (mitochondrial 5'(3')-

Figure 7. The DALI search results that were returned through e-mailed. The highlighted (yellow) shows the query protein. With a z value

of 41.1 and a root mean standard deviation of 0.0 and %IDE of 100, shows that it is a HAD family protein. The highlighted (green) shows

significant similarities of query protein as a hydrolase phosphatase as Z values are more then 1, RMSD still of low values and %IDE of more

then 20.Z

From the DALI search (Figure 7), Neu5Ac phosphatase is a haloacid dehalogenase-like hydrolase. This family is structurally different from the

alpha/ beta hydrolase family. It has L-2-haloacid dehalogenase, epoxide hydrolases and phosphatases. This family consists of two domains of

structure. One is an inserted four helix bundle, which is the least well conserved region of the alignment, between residues 16 and 96 of (S)-2-

haloacid dehalogenase I. The remaining of the fold is composed of the core alpha/beta domain. It is classified as a hydrolase found in mouse.

The chemical components would be phosphate ion, sodium ion, 1,2-ethanediol, chloride ion. PO4 and EDO are ligands while

Na and Cl are metals.

Protein Structure

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Figure 8. Secondary structure of 2gfh protein with residue interaction and the catalytic residues marked out in red boxes. (

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Figure 9. (A) Main, bottom and right view of 2gfh protein, the spheres represent the element/chemical components. (B) 2gfh protein viewed using KiNG. (C) Topology diagram of 2gfh showing the beta and alpha strand. (

The structure of 2gfh protein was determined to a be polypeptide(L) with 260 residues. Secondary structure (Figure 8) comprises of 56% helical

(13 helicals; 146 residues) and 11% beta sheet (8 strands; 31 residues)

Protein Folding

Table 1. Matching folds detected by SSM and Dali, with scores values between the Neu5Ac-9-P phosphatase and other proteins.(

Hit Z-score No. 
1 16.6 16 0.00 100.0% 2gfhA Crystal structure of protein c20orf147 homolog (17391249) from Mus musculus at 1.90 a resolution
2 9.4 16 2.34 23.0% 1x42A Crystal structure of a haloacid dehalogenase family protein (ph0459) from Pyrococcus horikoshii ot3
3 9.2 10 1.63 26.0% 1swwA Crystal structure of the phosphonoacetaldehyde hydrolase d12a mutant complexed with magnesium and substrate phosphonoacetaldehyde
4 9.3 10 1.66 26.0% 1swvA Crystal structure of the d12a mutant of phosphonoacetaldehyde hydrolase complexed with magnesium
5 7.1 11 1.75 24.4% 1fezA The crystal structure of Bacillus cereus phosphonoacetaldehyde hydrolase complexed with tungstate, a product analog
6 6.3 12 2.34 20.5% 2p11A Crystal structure of hypothetical protein (yp_553970.1) from Burkholderia xenovorans lb400 at 2.20 a resolution
8 7.5 11 1.96 26.8% 1rqlA Crystal structure of phosponoacetaldehyde hydrolase complexed with magnesium and the inhibitor vinyl sulfonate
9 7.3 11 1.96 26.8% 1rqnA Phosphonoacetaldehyde hydrolase complexed with magnesium
10 6.7 13 2.44 22.1% 2b0cA The crystal structure of the putative phosphatase from Escherichia coli

The high score values between Neu5Ac phosphatase and the other proteins (Table 1), proving that the folding of the different proteins match.

The Z-score measures the statistical significance of a match in terms of standard Gaussian statistics. It is based on the quality of the match

between the query and target structures and assumes a Gaussian distribution of quality scores would be obtained from a large enough databases

of protein structures. The higher the Z-score, the higher is the statistical significance of the match is the number of matched secondary

structure elements, examples; helices and strands between the two structures.

Sequence Similarity

Hydrolase: domain 1 of 1, from 18 to 224: score 96.2, E = 1e-25

                     ++av+FD+D+TL+d+ + + ++ + e+ ++l  + + +++ ++  + 
      query    18    VRAVFFDLDNTLIDT-AGASRRGMLEVIKLLQSkyhykeeaeIICDK 63   
                  v   l +++ ++    ++   t ++ +   +++++ ++++  ++    ++
      query    64 VQVKLSKECfHpYSTCITDVRTSHWEeaiqetkggadnrklaeecYfLWK 113 
                   ++ ++  l +++++ l +L++  +++ +lT+gdr++++++ ea+++ ++
                  fd+i++++e +        KP+p if + ++ lgv+p ++    +mVGD+
                  + +++ +  +AG+++++++n +   
      query   203 LETDIQGGLnAGLkATVWINKS    224  

Figure 10. The alignments of the top-scoring domains of 2gfh protein (query) using Pfam 21.0 (Janelia Farm). (

A search of using Pfam (Figure 10) matched the query sequence in this case Neu5Ac-9-P phosphatase with hydrolase. The E value of 1e-25 gives

significant results proving that it is not by chance nor random that the match made was a hydrolase.

Surface Properties

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Figure 11. Molecular surface of 2gfh colored by electrostatic potential shown using Pymol.

Using the PDB file name 2gfh, a model was constructed using Pymol showing the electrostatic potential of the molecular surface. As shown in

Figure 11, the red color portions are negatively charged while the blue would be positively charged region. The charge ranges from -63.539 to


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Figure 12. (A) Molecular structure of 2gfh showing the possible binding sites with the different colors represent classes of amino

acids. (B) Results from Profunc show that 2gfh comprises of 2 ligands: phostphate ion (PO4) and ethylene glycol (EDO).

Profunc helps to identify the likely biochemical function of a protein from its 3 dimensional (3D) structure. It uses fold matching, residue

conservation, surface cleft analysis, and functional 3D templates, to identify both the protein’s likely active site and

possible homologues in the PDB. The search provided information on the possible binding sites and important identification of potential ligands

like PO4 and EDO. Based on comprehension and research, EDO (Figure 14) could most likely be a chemical compound widely used to

crystallize protein from its native form and used as automotive antifreeze. Finding of the PO4 ligand (Figure 13) was important as

it would most likely be an active site. As Neu5Ac-9-P phosphatase is a hydrolase, the PO4 could well be involved in the mechanism

and function of the protein.

Document7 07.png Figure 13. (A) Molecular structure of 2gfh with the ligand PO4. (B) Molecular and chemical structure of PO4. (C) Ligand interaction involving PO4.

Document7 11.png Figure 14. (A) Molecular structure of 2gfh with the ligand EDO. (B) Molecular and chemical structure of EDO. (C) Ligand interaction involving EDO.

Table 2. Number 3 shows siginificant scores implying possible convserved residues in N-acetylneuraminic acid phosphatase.

No Score Number
of residues
Cleft Average accessibility Average conservation Residues
1 3.770 3 3 2 0.437 Ser212(A), Gly213(A), Arg214(A)
2 3.579 3 3 - 0.913 Ala201(A), Gly202(A), Leu203(A)
3 3.483 3 3 - 0.816 Leu177(A), Gly178(A), Val179(A)
4 3.000 3 3 2 1.000 Asn15(A), Thr16(A), Leu17(A)
5 0.646 4 4 - 0.646 Cys145(A), Ala146(A), Cys147(A), Gln148(A)

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Figure 15. Molecular structure of Neu5Ac-9-P was determined using RasMol, showing the conserved region of asparagine, threonine and

leucine with EDO molecule in grey and PO4 in yellow.

Profunc also provided information of the conserved residues in Neu5Ac-9-P phosphatase. By using nest analysis whereby, nests are structural

motifs that are often found in functionally important regions of protein structures and given a score value. When a score is above 2.0, it

implies that the nest is a functionally significant one. The results were tabulated showing the nest’s start and end residues

residues making up the nest. Residue conservation was given to each nest residue. The score ranges from 0.0 to 1.0 which signifies that the

residue is not at all conserved or perfectly conserved respectively. It is determined from a multiple sequence alignment of the

protein’s sequence against BLAST hits from UniProt sequence database. Results (Figure 15) show 2 highly conserved

region asparagine, threonine and leucine as the residue conservation score was 1.0.

Functional analysis

The MSA (Figure 16) for the query sequence and the other 35 sequences shows several conserved motifs. The 1st conserved motif

consists of almost invariant region of aspartic acid (D), only the 33rd protein (gi: |45552117|)

showing gap. The 2nd motif shows conserved and invariant of leucine (L), threonine (T), asparagine (N) and glycine (G). The

3rd motif shows 2 invariant amino acid residues of lysine (K), proline (P), valine (V), glycine (G), aspartic acid (D) and

isoleucine (I). This correlates with the study done by Maliekal et al and strongly suggested that the query protein is a phosphatase.

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Figure 16. MSA of the query protein Neu5Ac phosphatase with 35 others proteins. Only the 60th – 70th and the

210th -300th amino acid sequence were shown to illustrate the conserved and invariant regions. The 3 boxed-up sequences

were either conserved or invariant regions.