The ExPaSy ProtParam tool was used to derive basic information concerning EtnR1 based on its amino acid sequence.
It was calculated that:
• Number of amino acids: 580
• Molecular weight: 63306.7
• Theoretical pI: 5.64
It was also found that EtnR1 has an instability index of 38.13, classifying the protein as stable.
The NCBI BLAST tool was used to conduct a protein BLAST (Basic Local Alignment Search Tool) to identify proteins with close sequence similarity to the EtnR1 sequence. The SmartBLAST feature selects ‘the three best matches in the sequence database together with the two best matches from well-studied reference species, showing phylogenetic relationships based on multiple sequence alignment and conserved protein domains.’
In the case of EtnR1, the top hits were Mycobacterium CdaR family transcriptional regulators, though EtnR1 also showed minimal relationship to the E. Coli. CdaR which has been more thoroughly characterised. Figure 1 shows the phylogenetic tree constructed based on sequence similarities and conserved domains. The individual alignments can be found at the bottom of the page. >
Other candidates shown are regulatory protein [Streptomyces coelicolor A3(2)] (accession: NP_629806.1 GI:21224027), carbohydrate diacid regulon transcriptional regulator; autoregulator [Escherichia coli str. K-12 substr. MG1655] (accession: NP_414704.4, GI: 90111093), CdaR family transcriptional regulator [Mycobacterium tusciae] (accession: WP_006247394.1, GI: 493289684), CdaR family transcriptional regulator [Mycobacterium sp. JS623] (accession: WP_015305844.1, GI: 505118742) and CdaR family transcriptional regulator [Mycobacterium rhodesiae] (accession: WP_005148475.1, GI: 491290459).
The BLAST analysis also revealed a predicted PucR C-terminal helix-turn-helix domain (HTH-30) at the C-terminal end (Figure 2).
It is often found in PucR-like transcriptional regulators. This HTH-30 domain is likely to be where EtnR1 binds to DNA.
Figure 1. Phylogenetic tree produced by SmartBLAST with query: CdaR family transcriptional regulator [Mycobacterium chubuense] (accession: WP_014805817.1, GI:504618715).
Figure 2. Snapshot of BLAST results, showing putative HTH-30 domain at the C-terminal end of EtnR1.
The UCL Bioinformatics PsiPred tool was used to predict the secondary structure of EtnR1 based on its amino acid sequence (Figure 3).
Importantly, the four helices at the C-terminus show high confidence of prediction, and thus is very likely that the putative HTH-30 domain is present. Many of the other areas of secondary structure also show high levels of confidence.
Figure 3. Secondary structure prediction using PsiPred for EtnR1 based on amino acid sequence.
The SWISS-MODEL server [1-4] is an automated service that selects the best matching template to a submitted amino acid sequence, performs the alignment and builds a 3D model. The most important limiting factor of the quality of the model is generally the matching of the query sequence with an appropriate template.
SwissProt threaded amino acids 1 to 313 of EtnR1 onto a selected template protein: phosphodiesterase 5A GAF domain. It is a regulatory domain of the human PDE5. Notably, GAF domains are commonly found at the N-terminus in CdaR family transcriptional regulators, along with the HTH-30 domain at the C-terminus.
Based on the structure of the template, a homology model was constructed for this region encompassing residues 1-313 (Figure 4).
Figure 4. Homology model of residues 1-313 of EtnR1 based on phosphodiesterase 5A GAF domain.
Residues 313 to 580 were threaded onto a different template protein: regulator of polyketide synthase expression from Bifidobacterium adolescentis. This protein is a transcriptional regulator.
Based on the structure of the template, a homology model was constructed for this region encompassing residues 313-580 (Figure 5). The HTH-30 domain at the C-terminus can be visualised in this model.
Figure 5. Homology model of residues 313 to 580 of EtnR1 based on regulator of polyketide synthase expression BAD_0249 from Bifidobacterium adolescentis. HTH-30 at C-terminus is shown in cyan.
CdaR family transcriptional regulator [Mycobacterium rhodesiae]
Sequence ID: WP_005148475.1 Length: 580 Number of Matches: 1
Identical Proteins-Identical proteins to WP_005148475.1
Range 1: 1 to 580
CdaR family transcriptional regulator [Mycobacterium sp. JS623]
Sequence ID: WP_015305844.1Length: 580Number of Matches: 1
Identical Proteins-Identical proteins to WP_015305844.1
Range 1: 3 to 580
carbohydrate diacid regulon transcriptional regulator; autoregulator [Escherichia coli str. K-12 substr. MG1655]
Sequence ID: NP_414704.4
Gene-associated gene details
Identical Proteins-Identical proteins to WP_000929443.1
Range 1: 87 to 378
regulatory protein [Streptomyces coelicolor A3(2)]
Sequence ID: NP_629806.1
Gene-associated gene details
Identical Proteins-Identical proteins to WP_011030383.1
Range 1: 430 to 548
MTATSDVAHTETLVELREQLSNLQGLLMLAMLMTQSSDENKIVQLSTTSLPAFYRCPFVGIYLN DGGWQKHLGARVDYSAAAEVDAQIATLGPSGGDLALGRYARCVALPLRGLDAHIGFFIVASDEE PSVGEQFLLRVLVQQTGVALANARLHRKEQASTEALRDSNNALAESISALENAARIHARLTEVA AKGNGEDGIATALHELTGLSVAIEDRFGNLRAWAGPDCPEPYPKDDANAREAMLQRCIRAGEPI RHAGRLSAVANPRVDIVGVVSLIDPDEGGGEQAKVALEHGTTILAMELARLRSLAEAELRLRRD LVEEVLLGTDDESALARAEALGHDLGTPHRVVIVESEGRCADMEKFFHGVRSAARHAHMGTLVV ARSNTVVILSDADMNRDRFVSALVSQLGSDDCRVGVGGWCDRPRHLPRSYREAQLALKMQRRVG SRDPAPVTFYDELGVYRILAEVENQDSIERFVRQWLGPLLDYDAAKQSQLVATLSAYLECGGHH DATTAAIFVHRSTLKYRLSRIRTLLGLDVNDPDVRFNLQMATRAWKTLEHLGSGDGHMSHQVEIPPTL
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