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.
EtnR1: PsiPred – Secondary structure prediction
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.
EtnR1: SwissModel Homology Modelling
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
Related Information
Identical Proteins-Identical proteins to WP_005148475.1
Range 1: 1 to 580
EtnR1: BLAST alignments with three best matches and from well-studied reference species
CdaR family transcriptional regulator [Mycobacterium rhodesiae]
Sequence ID: WP_005148475.1 Length: 580 Number of Matches: 1
Related Information
Identical Proteins-Identical proteins to WP_005148475.1
Range 1: 1 to 580
| Score |
Expect |
Method |
Identities |
Positives |
Gaps |
| 1140 bits(2948) |
0.0() |
Compositional matrix adjust. |
560/580(97%) |
566/580(97%) |
0/580(0%) |
| Query |
Sequence |
Query 1 - 60
Sbjct 1 - 60 |
MTATSDVAHTETLVELREQLSNLQGLLMLAMLMTQSSDENKIVQLSTTSLPAFYRCPFVG
MTATSDVAHTETLVELREQLSNLQGLLMLAMLMTQS DENKI+QLSTTSLPAFYRCPFVG
MTATSDVAHTETLVELREQLSNLQGLLMLAMLMTQSGDENKIIQLSTTSLPAFYRCPFVG
|
Query 61-
120
Sbjct 61-
120
|
IYLNDGGWQKHLGARVDYSAAAEVDAQIATLGPSGGDLALGRYARCVALPLRGLDAHIGF
IYL DGGWQK LGARVDYSA AE+DA IATLGPSGG L LGRYA CVALPLRGLDAHIGF
IYLTDGGWQKALGARVDYSATAELDASIATLGPSGGVLTLGRYAWCVALPLRGLDAHIGF |
Query 121-
180
Sbjct 121-
180
|
FIVASDEEPSVGEQFLLRVLVQQTGVALANARLHRKEQASTEALRDSNNALAESISALEN
FIVASDEEPSVGEQFLLRVLVQQTG ALANARLHRKEQASTEALRDSN ALAES+SALEN
FIVASDEEPSVGEQFLLRVLVQQTGAALANARLHRKEQASTEALRDSNIALAESVSALEN |
Query 181-
240
Sbjct 181-
240
|
AARIHARLTEVAAKGNGEDGIATALHELTGLSVAIEDRFGNLRAWAGPDCPEPYPKDDAN
AARIHARLTEVAAKGNGE+GIA ALHELTGLSVAIEDRFGNLRAWAGPDCPEPYPKD+AN
AARIHARLTEVAAKGNGEEGIAIALHELTGLSVAIEDRFGNLRAWAGPDCPEPYPKDNAN |
Query 241-
300
Sbjct 241-
300
|
AREAMLQRCIRAGEPIRHAGRLSAVANPRVDIVGVVSLIDPDEGGGEQAKVALEHGTTIL
AREAMLQRCIRAGEPIRHAGRLSAVANPRVDIVGVVSLIDPDEGGGEQAKVALEHGTT+L
AREAMLQRCIRAGEPIRHAGRLSAVANPRVDIVGVVSLIDPDEGGGEQAKVALEHGTTVL |
Query 301-
360
Sbjct 301-
360
|
AMELARLRSLAEAELRLRRDLVEEVLLGTDDESALARAEALGHDLGTPHRVVIVESEGRC
AMELARLRSLAEAELRLRRDLVEEVLLGTDDESALARAEALGHDLGTPHRVVIVESEGRC
AMELARLRSLAEAELRLRRDLVEEVLLGTDDESALARAEALGHDLGTPHRVVIVESEGRC |
Query 361-
420
Sbjct 361-
420
|
ADMEKFFHGVRSAARHAHMGTLVVARSNTVVILSDADMNRDRFVSALVSQLGSDDCRVGV
ADMEKFFHGVR AARHAHMGTLVVARSNTVVILSDADMNRDRFVSALVS LGSDDCRVGV
ADMEKFFHGVRRAARHAHMGTLVVARSNTVVILSDADMNRDRFVSALVSHLGSDDCRVGV |
Query 421-
480
Sbjct 421-
480
|
GGWCDRPRHLPRSYREAQLALKMQRRVGSRDPAPVTFYDELGVYRILAEVENQDSIERFV
GGWCDRPRHLPRSYREAQLALKMQRRVGSRDPA VTFYDELGVYRILAEVENQDSIERFV
GGWCDRPRHLPRSYREAQLALKMQRRVGSRDPAAVTFYDELGVYRILAEVENQDSIERFV |
Query 481-
540
Sbjct 481-
540
|
RQWLGPLLDYDAAKQSQLVATLSAYLECGGHHDATTAAIFVHRSTLKYRLSRIRTLLGLD
RQWLGPLLDYDAAKQSQLVATLSAYLECGGHHDATTAAIFVHRSTLKYRLSRIRTLLGLD
RQWLGPLLDYDAAKQSQLVATLSAYLECGGHHDATTAAIFVHRSTLKYRLSRIRTLLGLD |
Query 541 -580
Sbjct 541 - 580 |
VNDPDVRFNLQMATRAWKTLEHLGSGDGHMSHQVEIPPTL
VNDPDVRFNLQMATRAWKTLEHLGSGDGHMSHQVEIPPTL
VNDPDVRFNLQMATRAWKTLEHLGSGDGHMSHQVEIPPTL |
CdaR family transcriptional regulator [Mycobacterium sp. JS623]
Sequence ID: WP_015305844.1Length: 580Number of Matches: 1
Related Information
Identical Proteins-Identical proteins to WP_015305844.1
Range 1: 3 to 580
| Score |
Expect |
Method |
Identities |
Positives |
Gaps |
| 856 bits(2212) |
0.0() |
Compositional matrix adjust. |
424/579(73%) |
493/579(85%) |
1/579(0%) |
| Query |
Sequence |
Query 2 - 61
Sbjct 3 - 61 |
TATSDVAHTETLVELREQLSNLQGLLMLAMLMTQSSDENKIVQLSTTSLPAFYRCPFVGI
A DV+ TE+ +ELREQ+SNLQGLLML++LMTQS DENKI+QLS TS+P+FYRCPFVGI
VAQEDVS-TESYIELREQISNLQGLLMLSLLMTQSGDENKIIQLSITSVPSFYRCPFVGI
|
Query 62-
121
Sbjct 62-
121
|
YLNDGGWQKHLGARVDYSAAAEVDAQIATLGPSGGDLALGRYARCVALPLRGLDAHIGFF
+LND GWQ L E++ Q+ LGPSGG L RY+ CVALPLRGL+AHIG+F
HLNDRGWQNPLDPHDVSLERIELEPQLKALGPSGGVLNFTRYSWCVALPLRGLEAHIGYF |
Query 122-
181
Sbjct 122-
181
|
IVASDEEPSVGEQFLLRVLVQQTGVALANARLHRKEQASTEALRDSNNALAESISALENA
+VA++ EPS GEQFL+RVL QQTGVALANARLHRKEQASTEALR SN ALAES++ALE A
VVAAEAEPSTGEQFLIRVLAQQTGVALANARLHRKEQASTEALRRSNVALAESVTALEYA |
Query 182-
241
Sbjct 182-
241
|
ARIHARLTEVAAKGNGEDGIATALHELTGLSVAIEDRFGNLRAWAGPDCPEPYPKDDANA
A IHAR TE+A KG GE+GIATALHELTGL VAIEDRFGNLRAWAGPDCP+PYPKDD
ANIHARFTEIATKGQGEEGIATALHELTGLPVAIEDRFGNLRAWAGPDCPDPYPKDDPAT |
Query 242-
301
Sbjct 242-
301
|
REAMLQRCIRAGEPIRHAGRLSAVANPRVDIVGVVSLIDPDEGGGEQAKVALEHGTTILA
REAMLQRC++AGEPIR GRLSAVANPRVDI+GV+SLIDP G+QA+VALEHGTT+LA
REAMLQRCVQAGEPIRQGGRLSAVANPRVDIMGVLSLIDPQAVAGDQAQVALEHGTTVLA |
Query 302-
361
Sbjct 302-
361
|
MELARLRSLAEAELRLRRDLVEEVLLGTDDESALARAEALGHDLGTPHRVVIVESEGRCA
MELARLRSLAEAELRLRRDLVEE+LLGT DESALARAEALGHDLG HRV+IVE +GR A
MELARLRSLAEAELRLRRDLVEELLLGTGDESALARAEALGHDLGRCHRVLIVEPQGRTA |
Query 362-
421
Sbjct 362-
421
|
DMEKFFHGVRSAARHAHMGTLVVARSNTVVILSDADMNRDRFVSALVSQLGSDDCRVGVG
DM+KFFH VR AAR+A +G+L+VAR++TVVILSDA+++R++F+SA+ + +G D+ RVGVG
DMDKFFHAVRRAARNAQLGSLIVARASTVVILSDAEVDREKFLSAISTSVGDDNFRVGVG |
Query 422-
481
Sbjct 422-
481
|
GWCDRPRHLPRSYREAQLALKMQRRVGSRDPAPVTFYDELGVYRILAEVENQDSIERFVR
GWCDRP PRSY EAQLALKMQRR G+ A V FYDELGVYRILAEVENQ SIE FVR
GWCDRPEDFPRSYHEAQLALKMQRRSGTATAAGVIFYDELGVYRILAEVENQQSIESFVR |
Query 482-
541
Sbjct 482-
541
|
QWLGPLLDYDAAKQSQLVATLSAYLECGGHHDATTAAIFVHRSTLKYRLSRIRTLLGLDV
QWLGPLLDYDAAK SQ+VATL+ YL+CGGH+D TTAA+++HRSTLKYRLSRIR LLG+D+
QWLGPLLDYDAAKGSQMVATLAGYLQCGGHYDTTTAALYIHRSTLKYRLSRIRDLLGIDI |
Query 542 -580
Sbjct 542 - 580 |
NDPDVRFNLQMATRAWKTLEHLGSGDGHMSHQVEIPPTL
NDP+ RFNL++A RAW TLE L +G GHMS E+ P++
NDPEARFNLELAARAWGTLEELAAGQGHMSPPAEVDPSV |
carbohydrate diacid regulon transcriptional regulator; autoregulator [Escherichia coli str. K-12 substr. MG1655]
Sequence ID: NP_414704.4
Related Information
Gene-associated gene details
Identical Proteins-Identical proteins to WP_000929443.1
Range 1: 87 to 378
| Score |
Expect |
Method |
Identities |
Positives |
Gaps |
| 75.9 bits(185) |
0.0() |
Compositional matrix adjust. |
85/305(28%) |
143/305(46%) |
35/305(11%) |
| Query |
Sequence |
Query 271 - 330
Sbjct 87 - 144 |
DIVGVVSLIDPDEGGGEQAKVALEHGTTILAMELARLRSLAEAELRLRRDLVEEVLLGTD
+IVGV+ L E + ++ T + +E +RL L + RLR +LV ++ +
EIVGVIGLTGEPENLRKYGELVCM--TAEMMLEQSRLMHLLAQDSRLREELVMNLIQAEE
|
Query 331-382
Sbjct 145-
202
|
DESALAR-AEALGHDLGTPHRVVIVESEG-------RCADMEKFFHGVRSAARHAHMGTL
+ AL A+ LG DL P V IVE + A++++ + + + R+ +
NTPALTEWAQRLGIDLNQPRVVAIVEVDSGQLGVDSAMAELQQLQNALTTPERNNLVA-- |
Query 383-
431
Sbjct 203-261
|
VVARSNTVVILS--------DADMNRDRFVSALVSQL---GSDDCRVGVGGWCDRPRHLP
+V+ + VV+ DA+ +R R V L++++ G RV +G + P +
IVSLTEMVVLKPALNSFGRWDAEDHRKR-VEQLITRMKEYGQLRFRVSLGNYFTGPGSIA |
Query 432 - 489
Sbjct 262 - 313
|
RSYREAQ--LALKMQRRVGSRDPAPVTFYDELGVYRILAEVENQDSIERFVRQWLGPLLD
RSYR A+ + + QR SR FY +L + +L + R PL
RSYRTAKTTMVVGKQRMPESR----CYFYQDLMLPVLLDSLRGDWQANELAR----PLAR |
Query 490 - 548
Sbjct 314-373
|
YDAAKQSQLVA-TLSAYLECGGHHDATTAAIFVHRSTLKYRLSRIRTLLGLDVNDPDVRF
+ L+ TL+A+ AT+ A+F+HR+TL+YRL+RI L GLD+ + D R
LKTMDNNGLLRRTLAAWFRHNVQPLATSKALFIHRNTLEYRLNRISELTGLDLGNFDDRL |
Query 549-553
Sbjct 374-378
|
NLQMA
L +A
LLYVA |
regulatory protein [Streptomyces coelicolor A3(2)]
Sequence ID: NP_629806.1
Related Information
Gene-associated gene details
Identical Proteins-Identical proteins to WP_011030383.1
Range 1: 430 to 548
| Score |
Expect |
Method |
Identities |
Positives |
Gaps |
| 71.2 bits(173) |
1e-11() |
Compositional matrix adjust. |
43/124(35%) |
69/124(55%) |
5/124(4%) |
| Query |
Sequence |
Query 433 - 492
Sbjct 430 - 484 |
SYREAQLALKMQRRVGSRDPAPVTFYDELGVYRILAEVENQDSIERFVRQWLGPLLDYDA
+Y++AQ AL + RR G ++ + +L + + D++ F L L D+DA
AYKQAQQALSVARRRGRV----CVEHEHVAAGSVLPLLAD-DAVRAFADGLLRALRDHDA
|
Query 393-552
Sbjct 485-
544
|
AKQSQLVATLSAYLECGGHHDATTAAIFVHRSTLKYRLSRIRTLLGLDVNDPDVRFNLQM
+ LVA++ A+L G DA A + VHR TL+YR+ R+ +LG ++DPDVR L +
TGRGDLVASVRAWLSRHGQWDAAAADLGVHRHTLRYRMRRVEEILGRSLDDPDVRMELWL |
Query 553-
556
Sbjct 545-548
|
ATRA
A +A
ALKA |
For reference: EtnR1 Amino Acid Sequence
MTATSDVAHTETLVELREQLSNLQGLLMLAMLMTQSSDENKIVQLSTTSLPAFYRCPFVGIYLN
DGGWQKHLGARVDYSAAAEVDAQIATLGPSGGDLALGRYARCVALPLRGLDAHIGFFIVASDEE
PSVGEQFLLRVLVQQTGVALANARLHRKEQASTEALRDSNNALAESISALENAARIHARLTEVA
AKGNGEDGIATALHELTGLSVAIEDRFGNLRAWAGPDCPEPYPKDDANAREAMLQRCIRAGEPI
RHAGRLSAVANPRVDIVGVVSLIDPDEGGGEQAKVALEHGTTILAMELARLRSLAEAELRLRRD
LVEEVLLGTDDESALARAEALGHDLGTPHRVVIVESEGRCADMEKFFHGVRSAARHAHMGTLVV
ARSNTVVILSDADMNRDRFVSALVSQLGSDDCRVGVGGWCDRPRHLPRSYREAQLALKMQRRVG
SRDPAPVTFYDELGVYRILAEVENQDSIERFVRQWLGPLLDYDAAKQSQLVATLSAYLECGGHH
DATTAAIFVHRSTLKYRLSRIRTLLGLDVNDPDVRFNLQMATRAWKTLEHLGSGDGHMSHQVEI
PPTL
References
1. Marco Biasini, Stefan Bienert, Andrew Waterhouse, Konstantin Arnold, Gabriel Studer, Tobias Schmidt, Florian Kiefer, Tiziano Gallo Cassarino, Martino Bertoni, Lorenza Bordoli, Torsten Schwede. (2014). SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Research; (1 July 2014) 42 (W1): W252-W258; doi: 10.1093/nar/gku340.
2. Arnold K., Bordoli L., Kopp J., and Schwede T. (2006). The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling. Bioinformatics, 22,195-201.
3. Kiefer F, Arnold K, Künzli M, Bordoli L, Schwede T (2009). The SWISS-MODEL Repository and associated resources. Nucleic Acids Research. 37, D387-D392.
4. Guex, N., Peitsch, M.C., Schwede, T. (2009). Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: A historical perspective.Electrophoresis, 30(S1), S162-S173.
