2gnx Results

Evolutionary analysis

Figure 1
This image shows part of a complete alignment of the sequences used. Asterisks (*) indicate residues that are conserved across all sequences, and colons (:) indicate partial conservation across all sequences.

Figure 2
The phylogenetic tree shows how close the relationships between the sequences are. The longer the branches of the tree the more evolutionary divergent the sequences are. 2GNX A is the original protein being investigated and was a mouse protein. The branches with marked with * indicate that this branch arrangement occured more then 75% of the time.

Structural analysis

An analysis of the secondary structure of the protein from its amino acid sequence (Figure 3) shows the secondary structural arrangement of different regions of our protein

Figure 3
Secondary structure analysis of the 2GNX protein from Protein Data Bank.

Table 1 Dali analysis of the 2GNX protein

NR. STRID1 STRID2  Z   RMSD LALI LSEQ2 %IDE REVERS PERMUT NFRAG TOPO PROTEIN
 1: 3023-A 2gnx-A 42.9  0.0  280   280  100      0      0     1 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION 	hypothetical pro
 2: 3023-A 2cmr-A  5.7  3.5  114   192   11      0      0    11 S    IMMUNOGLOBULIN COMPLEX 	d5 (fab heavy chain) d5 (fab li
 3: 3023-A 1j3w-A  5.7  3.2   99   134   12      0      0     9 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION 	giding protein-m
 4: 3023-A 1jmr-A  5.5  3.0   94   246    9      0      0    12 S    
 5: 3023-A 1f5m-B  5.5  5.0  107   177    9      0      0    13 S    SIGNALING PROTEIN 	gaf 	(saccharomyces cerevisiae) yeas
 6: 3023-A 1vcs-A  5.0  4.7   82   102    9      0      0     8 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION 	vesicle transpor
 7: 3023-A 1kt0-A  4.9  2.8   81   357    6      0      0     7 S    ISOMERASE 	51 kda fk506-binding protein (fkbp51) Mutant
 8: 3023-A 1e2a-A  4.9  4.5   80   102    9      0      0     6 S    TRANSFERASE 	enzyme iia (enzyme iii, lactose-specific i
 9: 3023-A 2d2s-A  4.8  3.1   75   217   11      0      0     5 S    ENDOCYTOSIS/EXOCYTOSIS 	exocyst complex component exo84
10: 3023-A 2oew-A  4.7  2.8  119   358    8      0      0    12 S    PROTEIN TRANSPORT 	programmed cell death 6-interacting 
11: 3023-A 1h3q-A  4.7  4.2   92   140    4      0      0    11 S    TRANSPORT 	sedlin (sedl) 	(mus musculus) mouse 	S.B.Jan
12: 3023-A 2oev-A  4.5 36.5  151   697    7      0      0    14 S    PROTEIN TRANSPORT 	programmed cell death 6-interacting 
13: 3023-A 2cwy-A  4.5  2.4   82    92   20      0      0     7 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION 	hypothetical pro
14: 3023-A 2c5i-T  4.5  2.8   75    93   11      0      0     5 S    PROTEIN TRANSPORT/COMPLEX 	t-snare affecting a late gol
15: 3023-A 3nul    4.4  3.4   93   130    5      0      0    11 S    ACTIN-BINDING PROTEIN 	profilin i 	(arabidopsis thalian

A Dali analysis (Table 1) of the 2GNX protein was highly inconclusive and there were no significant structural matches to the hypothetical protein.

Table 2 Dali analysis of N-terminal domain

 NR. STRID1 STRID2  Z   RMSD LALI LSEQ2 %IDE REVERS PERMUT NFRAG TOPO PROTEIN
  1: 3256-A 2gnx-A 23.2  0.0  173   280  100      0      0     1 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION 	hypothetical pro
  2: 3256-A 1e2a-A  7.5  4.5   80   102    9      0      0     6 S    TRANSFERASE 	enzyme iia (enzyme iii, lactose-specific i
  3: 3256-A 1kt0-A  7.4  2.8   81   357    6      0      0     7 S    ISOMERASE 	51 kda fk506-binding protein (fkbp51) Mutant
  4: 3256-A 2d2s-A  7.3  3.1   75   217   11      0      0     5 S    ENDOCYTOSIS/EXOCYTOSIS 	exocyst complex component exo84
  5: 3256-A 1vcs-A  7.3  4.7   78   102    9      0      0     7 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION 	vesicle transpor
  6: 3256-A 2cmr-A  6.9  3.2  104   192   11      0      0     9 S    IMMUNOGLOBULIN COMPLEX 	d5 (fab heavy chain) d5 (fab li
  7: 3256-A 2c5i-T  6.9  2.8   75    93   11      0      0     5 S    PROTEIN TRANSPORT/COMPLEX 	t-snare affecting a late gol
  8: 3256-A 2h7o-A  6.8  3.0   81   270    5      0      0     7 S    SIGNALING PROTEIN 	protein kinase ypka fragment (protei
  9: 3256-A 2h7v-C  6.6  4.2   76   269   13      0      0     5 S    SIGNALING PROTEIN 	migration-inducing protein 5 (ras-re
 10: 3256-A 2dnx-A  6.5  4.9   80   130    6      0      0     6 S    TRANSPORT PROTEIN 	syntaxin-12 fragment 	(homo sapiens)
 11: 3256-A 1hg5-A  6.5  3.2   85   263    9      0      0     6 S     ENDOCYTOSIS 	clathrin assembly protein short form frag
 12: 3256-A 1a17    6.4  2.5   71   159    3      0      0     5 S    HYDROLASE 	serineTHREONINE PROTEIN PHOSPHATASE 5 fragme
 13: 3256-A 2if4-A  6.3  2.5   82   258    7      0      0     7 S    SIGNALING PROTEIN 	atfkbp42 fragment (twd1 (twisted dwa
 14: 3256-A 1owa-A  6.2  3.3   76   156   12      0      0     6 S    CYTOKINE 	spectrin alpha chain, erythrocyte fragment (e
 15: 3256-A 2oew-A  6.1  2.8  119   358    8      0      0    12 S    PROTEIN TRANSPORT 	programmed cell death 6-interacting 

A Dali analysis carried out separately with only the N-terminal domain (Table 2) of the protein also did not produce any significant structural matches.

Figure 4
2CMR-2GNX alignment (2CMR displayed in cyans and 2GNX displayed in green).

A CE alignment between IMMUNOGLOBULIN COMPLEX d5 (2CMR) and 2GNX was performed (Figure 4). The result revealed that the N-terminus of 2GNX matched 2CMR:A which was a TRANSMEMBRANE GLYCOPROTEIN, with Rmsd = 3.8Å and Z-Score = 3.7. The 3D figure showed that two proteins both had five-helix strucuture and they were well fitted. However, the function of this 5-helix stucture was not clear.

Table 3: Dali analysis of C-terminal domain

NR. STRID1 STRID2  Z   RMSD LALI LSEQ2 %IDE REVERS PERMUT NFRAG TOPO PROTEIN
  1: 3257-A 2gnx-A 24.3  0.0  118   280  100      0      0     1 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION 	hypothetical pro
  2: 3257-A 1jmr-A  7.6  3.0   94   246    9      0      0    12 S    
  3: 3257-A 1j3w-A  7.5  2.9   91   134   13      0      0     7 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION 	giding protein-m
  4: 3257-A 1f5m-B  6.8  2.9   95   177    9      0      0    10 S    SIGNALING PROTEIN 	gaf 	(saccharomyces cerevisiae) yeas
  5: 3257-A 1h3q-A  6.6  4.2   92   140    4      0      0    11 S    TRANSPORT 	sedlin (sedl) 	(mus musculus) mouse 	S.B.Jan
  6: 3257-A 3nul    6.3  3.4   93   130    5      0      0    11 S    ACTIN-BINDING PROTEIN 	profilin i 	(arabidopsis thalian
  7: 3257-A 1mc0-A  5.8  4.1   99   341    8      0      0    11 S    HYDROLASE 	3',5'-cyclic nucleotide phosphodiesterase 2a
  8: 3257-A 2h28-A  5.4  2.8   75   106    8      0      0    10 S    STRUCTURAL GENOMICS, UNKNOWN FUNCTION 	hypothetical pro
  9: 3257-A 2p7j-A  5.0  2.9   79   262   13      0      0    11 S    TRANSCRIPTION 	putative sensory boxGGDEF FAMILY PROTEIN
 10: 3257-A 2dmw-A  5.0  3.3   85   131    7      0      0    11 S    MEMBRANE PROTEIN 	synaptobrevin-like 1 variant fragment
 11: 3257-A 2avx-A  4.8  3.6   93   171    5      0      0    10 S    TRANSCRIPTION 	regulatory protein sdia Mutant 	(escheri
 12: 3257-A 2j3t-C  4.7  5.2   83   141    7      0      0     8 S    PROTEIN TRANSPORT 	trafficking protein particle complex
 13: 3257-A 2hj9-C  4.7  3.3   76   210    5      0      0     9 S    SIGNALING PROTEIN 	autoinducer 2-binding periplasmic pr
 14: 3257-A 2hje-A  4.6  3.0   75   210    5      0      0     9 S    SIGNALING PROTEIN 	autoinducer 2 sensor kinasePHOSPHATA
 15: 3257-A 2uv0-E  4.5  3.5   93   159    9      0      0    12 S    TRANSCRIPTION 	transcriptional activator protein lasr

However, a Dali analysis (Table 3) carried out with the C-terminal domain of the protein produced one significant structural match, this being the GAF signalling protein, i.e the 4th result in the Dali analysis.

Figure 5
Dotlet analysis for 2GNX.

The Dotlet analysis (Figure 5) showed that there was no internally homologous repeats in the C-terminus of 2GNX.

 USR1:A  185/392   QVAKNLFTH---LDDVSVLLQEIITEARNLSNAEICSVFLLDQ-----------------
 USR2:A  181/283   TASEXKALTAKANPDLFGKISSFIRKY------DAANVSLIFDNRGSESFQGHGYHHPHS
 USR1:A  225/432   ----------NELVAKVFDGGVVDDESYEIRIPADQGIAGHVATTG----------QILN
 USR2:A  235/#44   YREAPKGVDQYPAVVSLP----------SDRPVXHWPNVIXIXTDRASDLNSLEKVVHFY
 USR1:A  265/472   IPDAYAHPLFYRGVDDSTGFRTRNILCFPIKNENQEVIGVAELVNKINGPWFSKFDEDLA
 USR2:A  285/387   DDKV-------------------QSTYFLTRPEP-HFTIVVIFESK---------KSERD
 USR1:A  325/532   TAFSIYCGISIAHSLL
 USR2:A  316/418   SHFISFLNELSLALKN

Figure 6: CE predicted structural alignment. USR1 = 1MC0(PDB code), Regulatory Segment of Mouse 3',5'-Cyclic Nucleotide Phosphodiesterase 2A, Containing the GAF A and GAF B Domains. USR2= 2GNX

The conserved residues of the ligand binding site in 1MC0 were not consistent with the aligned residues in 2GNX.

Zoraghi R. et al. (2003) indicated a fingerprint of the ligand binding site in 1MC0, which was the following patterns:

SX(13-18)FDX(18-22)IAX(21)[Y/N]X(2)VDX(2)TX(3)TX(19)[E/Q]

Figure 7
Fingerprint of the ligand binding site in 1MC0 (Zoraghi et al).The identical residues were coloured in red and the underline residues were the ones that missing in the PDB file.

The alignment above (Figure 7) indicated that the published patterns roughly fit into the protein sequence of 2GNX. The 3D structure analysis (figure ) revealed that some residues (in yellow) were likely not within the ligand binding pocket, however other residues (in red) were still potential ligand binding site.

Figure 7.1
Ligand Binding Site Predicted by Q-siteFinder

The result from Q-siteFinder confirmed that there were probably protein binding pocket in the predicted region. However, the volume of the two pockets were small compare to a normal cGMP binding site (Zoraghi R, 2003).

Figure 8
Potential ligand binding sites in 2GNX.

The figure above (Figure 8) shows the residues that are identical to the published patterns. The residues in red are the potential ligand binding residues and the residues in yellow were the residues that matched the published data but are not likely to be in the ligand binding pocket in 2GNX.

Functional Analysis

STRING and CDART returned no results for the submitted protein data.

BlastP Results

BlastP returned results however the results were limited to hypothetical proteins that gave no added information.

Table 4: BlastP Results

			Score (Bits)	E Value
ref	XP_001163972.1	PREDICTED: similar to FLJ32549 protein [Pan	850	0.0
ref	XP_001116860.1	PREDICTED: hypothetical protein isoform 1 [M	848	0.0
ref	NP_689653.3	hypothetical protein LOC144577 [Homo sapiens...	847	0.0
gb	AAH36246.1	FLJ32549 protein [Homo sapiens]	846	0.0
ref	XP_001116875.1	PREDICTED: hypothetical protein isoform 3 [M	843	0.0
ref	XP_531657.2	PREDICTED: hypothetical protein XP_531657 [Cani	827	0.0
ref	XP_615557.3	PREDICTED: hypothetical protein [Bos taurus]	823	0.0
gb	EDL24424.1	cDNA sequence BC048403, isoform CRA_a [Mus muscul	803	0.0
ref	NP_766610.2	hypothetical protein LOC270802 [Mus musculus...	803	0.0
ref	XP_576234.2	PREDICTED: hypothetical protein [Rattus norv...	802	0.0
ref	XP_001364942.1	PREDICTED: hypothetical protein [Monodelphis	797	0.0
ref	XP_416063.1	PREDICTED: hypothetical protein [Gallus gallus]	796	0.0
dbj	BAC39804.1	unnamed protein product [Mus musculus]	760	0.0
ref	XP_001116868.1	PREDICTED: hypothetical protein isoform 2 [M	743	0.0
ref	NP_001085035.1	hypothetical protein LOC432102 [Xenopus l...	697	0.0
ref	NP_001025261.1	hypothetical protein LOC555715 [Danio rer...	665	0.0
ref	NP_001076454.1	hypothetical protein LOC100005809 [Danio ...	661	0.0
ref	XP_001331282.1	PREDICTED: hypothetical protein [Danio rerio	598	2e-169
emb	CAG12393.1	unnamed protein product [Tetraodon nigroviridis]	593	8e-168
pdb	2GNX	A Chain A, X-Ray Structure Of A Hypothetical Protein...	554	3e-156
dbj	BAE41440.1	unnamed protein product [Mus musculus]	508	2e-142
ref	NP_001038719.1	hypothetical protein LOC692281 [Danio rer...	357	1e-96
ref	XP_624797.1	PREDICTED: hypothetical protein [Apis mellifera	235	3e-60
ref	XP_974676.1	PREDICTED: hypothetical protein [Tribolium cast	232	5e-59
ref	XP_001193974.1	PREDICTED: hypothetical protein [Strongyloce	208	5e-52
ref	XP_797380.2	PREDICTED: hypothetical protein, partial [St...	207	2e-51
dbj	BAE37112.1	unnamed protein product [Mus musculus] >dbj B...	134	2e-29
gb	EDL24425.1	cDNA sequence BC048403, isoform CRA_b [Mus muscul	132	6e-29
ref	XP_642387.1	hypothetical protein DDBDRAFT_0205477 [Dicty...	87.8	1e-15
emb	CAJ08583.1	hypothetical protein, conserved [Leishmania majo	36.6	3.5

Method Predicted Subcellular Location Evaluation

Locate analysis predicted that the protein is a soluble non-secreted protein. Localisation data was diverse as follows:

Table 5: Method Predicted Subcellular Location Evaluation

Method	Location	Score
CELLO	Mitochondrion	1.34
CELLO	Extracellular region	1.08
pTarget	Endoplasmic reticulum	93.90
Proteome Analyst	No prediction	0.00
WoLFPSORT	Cytoplasm	13.00
WoLFPSORT	Nucleus	12.00
WoLFPSORT	Golgi apparatus	3.00
MultiLoc	Peroxisome	0.49
MultiLoc	Mitochondrion	0.23
MultiLoc	Extracellular region	0.09

BC048403 Symatlas Expression Profile

Pfam, Profunc, Proknow, and Interpro all returned no results for the protein 2gnxA. However, Symatlas did provide an interesting lead. The expression data is presented in the following diagram. However, the significant results were the number of olfactory receptors with correlated expression profiles.

Figure 9
Symatlas Expression Profile.

Co-occurring Motifs Corresponding to BC048403

Olfactory receptors were also encountered when the protein was submitted to cis-RED to retrieve the corresponding cis-regulatory motif patterns. All fourteen motif patterns or modules, corresponding to the BC048403 protein are also motif patterns that are found in many different olfactory receptors. Motifs are predicted by cisRED with p-values < 0.005.

In total, the fourteen motifs corresponded to 120 different olfactory receptors. The following table lists the olfactory receptors with 3 or more co-occurring motifs. The header row lists the fourteen modules. Highlighted in orange (nine co-occurring modules) and green (7 co-occurring modules), are the olfactory receptors having the most modules in common with the BC048403 protein.

Table 6: Co-occurring Motifs Corresponding to Olfactory Receptors

Number of Motifs Corresponding to each Olfactory Receptor

The following graph represents the number of co-occurring motifs across the entire range of 120 corresponding olfactory receptors.

Figure 10
Graph of the number of motifs corresponding to each olfactory receptor.

These motifs were searched for in the other species databases of cis-RED however they were not found as there is no inter-species search tool. Unfortunately, micro-array expression data for the olfactory receptors with the most co-occurring motifs, were unavailable.

Micro-array Expression Profiles Similar to FLJ32549

The following micro-array data was found by browsing through the profile neighbours of the human ortholog using GEO Profiles.

Figure 11
Neighbouring expression profiles to FLJ32549.

Other interesting motifs found to appear in the Bc048403 protein were motifs that corresponded to the cadherin family.