SEA-PHAGES | Usavi_draft_70: potentially ribbon-helix-helix?

Link to this post \| posted 21 Dec, 2022 05:41
tbouklas	Hello all, I am annotating phage Usavi (B1). The gene at stop 57734 (Usavi_draft_70) has some pretty good hits in HHpred to entries that mention ribbon helix helix, but I'm not sure if they are sufficient to call anything related to a RHH. None of the hits mention anything specifically about RHH, the currently approved function. But the hits were strong enough, and apppeared to cover enough of the sequence from Usavi, that I am curious. I've summarized the data I found in the attachment and also copied some key info below. I would appreciate any advice on this one? Usavi_draft_70: MLPIKCPPDVRQRLKIAAAEDGLTYAQLLVSLLDLRDDRHRRQRAAQASPLHRPRAETA Currently 7 members of pham call ribbon-helix-helix DNA binding domain protein. Phages DB: https://phagesdb.org/genes/Usavi_CDS_70/ HHpred results: https://toolkit.tuebingen.mpg.de/jobs/Usavi__69 Both 2BNW and 1IRQ link to manuscripts that discuss RHH. PDB: https://www.rcsb.org/structure/2BNW PDB: https://www.rcsb.org/structure/1IRQ *I don't see any HHpred hits that line up with the evidence entries for the example RHH genes, Chorkpop_70 and Pharaoh_67. Chorkpop_70 HHpred: https://toolkit.tuebingen.mpg.de/jobs/Chorkpop_70 Phareon_67 HHpred: https://toolkit.tuebingen.mpg.de/jobs/Chorkpop_71 Thank you, Tejas – Tejas Bouklas Lecturer, UCLA 636Kb

Link to this post | posted 21 Dec, 2022 05:41

Hello all,

I am annotating phage Usavi (B1). The gene at stop 57734 (Usavi_draft_70) has some pretty good hits in HHpred to entries that mention ribbon helix helix, but I'm not sure if they are sufficient to call anything related to a RHH. None of the hits mention anything specifically about RHH, the currently approved function. But the hits were strong enough, and apppeared to cover enough of the sequence from Usavi, that I am curious.

I've summarized the data I found in the attachment and also copied some key info below. I would appreciate any advice on this one?

Usavi_draft_70:
MLPIKCPPDVRQRLKIAAAEDGLTYAQLLVSLLDLRDDRHRRQRAAQASPLHRPRAETA

Currently 7 members of pham call ribbon-helix-helix DNA binding domain protein.

Phages DB: https://phagesdb.org/genes/Usavi_CDS_70/

HHpred results: https://toolkit.tuebingen.mpg.de/jobs/Usavi__69

Both 2BNW and 1IRQ link to manuscripts that discuss RHH.
PDB: https://www.rcsb.org/structure/2BNW
PDB: https://www.rcsb.org/structure/1IRQ

*I don't see any HHpred hits that line up with the evidence entries for the example RHH genes, Chorkpop_70 and Pharaoh_67.
Chorkpop_70 HHpred: https://toolkit.tuebingen.mpg.de/jobs/Chorkpop_70
Phareon_67 HHpred: https://toolkit.tuebingen.mpg.de/jobs/Chorkpop_71

Thank you,
Tejas

–
Tejas Bouklas
Lecturer, UCLA

Link to this post \| posted 21 Dec, 2022 21:01
Pollenz	Great work on this question. The ribbon-helix-helix is a DNA binding motif found in many proteins. It is typified by a B-sheet of 6-10 amino acids followed by a short spacer and two alpha helices. It can be found in many locations within a primary sequence of a protein, but must contain all THREE domains. In HHpred the alpha helices are noted by Hh and beta sheets by Ee in the ss_pred line of the alignments. Capital letters indicate high confidence in the secondary structure (ss) prediction. See PDB 6SBW_A for a good schematic of this type of ribbon HH domain (enclosed schematic). Note that Usavi70 has alignments to the subjects with Q1 to all hits. Amino acid 1 is the start of a helix and there is also a second helix in Usavi70, but the Usavi70 has NO beta sheet that precedes the hits compared to the queries. See that the deeper analysis of the PDB queries show that they are all aligning well past amino acid 1 and all have a 7-9 amino acid beta sheet that precedes the helix 1 that is aligned to Usavi70. Thus, the ribbon helix helix IS NOT supported for Usavi70 and should not be called. NCBI conserved domains also does not support either an HTH or DNA binding call as well. So best to leave a hypothetical NKF since all components of the domain are not present. Hope this is helpful RS Pollenz 485Kb

Link to this post | posted 21 Dec, 2022 21:01

Pollenz

Great work on this question.

The ribbon-helix-helix is a DNA binding motif found in many proteins. It is typified by a B-sheet of 6-10 amino acids followed by a short spacer and two alpha helices. It can be found in many locations within a primary sequence of a protein, but must contain all THREE domains. In HHpred the alpha helices are noted by Hh and beta sheets by Ee in the ss_pred line of the alignments. Capital letters indicate high confidence in the secondary structure (ss) prediction.

See PDB 6SBW_A for a good schematic of this type of ribbon HH domain (enclosed schematic).

Note that Usavi70 has alignments to the subjects with Q1 to all hits. Amino acid 1 is the start of a helix and there is also a second helix in Usavi70, but the Usavi70 has NO beta sheet that precedes the hits compared to the queries. See that the deeper analysis of the PDB queries show that they are all aligning well past amino acid 1 and all have a 7-9 amino acid beta sheet that precedes the helix 1 that is aligned to Usavi70.

Thus, the ribbon helix helix IS NOT supported for Usavi70 and should not be called. NCBI conserved domains also does not support either an HTH or DNA binding call as well. So best to leave a hypothetical NKF since all components of the domain are not present.

Hope this is helpful

RS Pollenz

Link to this post \| posted 21 Dec, 2022 21:19
tbouklas	Thank you for your quick response! – Tejas Bouklas Lecturer, UCLA

Link to this post \| posted 09 Jun, 2023 22:08
anders	As I tried to apply this to some proteins that we are annotating, I was confused because I couldn't replicate Rick's result with Q1 being in the first alpha helix. Pollenz Note that Usavi70 has alignments to the subjects with Q1 to all hits. Amino acid 1 is the start of a helix and there is also a second helix in Usavi70, but the Usavi70 has NO beta sheet that precedes the hits compared to the queries. See that the deeper analysis of the PDB queries show that they are all aligning well past amino acid 1 and all have a 7-9 amino acid beta sheet that precedes the helix 1 that is aligned to Usavi70. Thus, the ribbon helix helix IS NOT supported for Usavi70 and should not be called. NCBI conserved domains also does not support either an HTH or DNA binding call as well. So best to leave a hypothetical NKF since all components of the domain are not present. When I used the protein sequence that Tejas posted, I got HHPred alignments that did predict some beta sheet in front of the first alpha helix. Then I realized that Rick may have used the draft genome protein that began at the Glimmer auto-annotated start, which was downstream of the start that Tejas had used for the amino acid sequence she posted. If we use a start codon even further upstream, to make the protein longer at its N-terminus, we can get a pretty good HHPred alignment to ribbon-helix-helix structures that includes a full-length beta strand. I've attached an image of the HHPred alignment with the longer amino acid sequence. My interpretation is that HHPred alignments do support the ribbon-helix-helix domain, if translation begins at a start codon far enough upstream to contain the predicted beta strand. For Usavi, it will come down to which start codon the annotators choose as the most appropriate start. For the genes we're working on, we will pay close attention to the key features of ribbon-helix-helix that Rick nicely explained above! ~Kirk 87Kb

Link to this post | posted 09 Jun, 2023 22:08

anders

As I tried to apply this to some proteins that we are annotating, I was confused because I couldn't replicate Rick's result with Q1 being in the first alpha helix.

Pollenz
Note that Usavi70 has alignments to the subjects with Q1 to all hits. Amino acid 1 is the start of a helix and there is also a second helix in Usavi70, but the Usavi70 has NO beta sheet that precedes the hits compared to the queries. See that the deeper analysis of the PDB queries show that they are all aligning well past amino acid 1 and all have a 7-9 amino acid beta sheet that precedes the helix 1 that is aligned to Usavi70.

Thus, the ribbon helix helix IS NOT supported for Usavi70 and should not be called. NCBI conserved domains also does not support either an HTH or DNA binding call as well. So best to leave a hypothetical NKF since all components of the domain are not present.

When I used the protein sequence that Tejas posted, I got HHPred alignments that did predict some beta sheet in front of the first alpha helix.

Then I realized that Rick may have used the draft genome protein that began at the Glimmer auto-annotated start, which was downstream of the start that Tejas had used for the amino acid sequence she posted.

If we use a start codon even further upstream, to make the protein longer at its N-terminus, we can get a pretty good HHPred alignment to ribbon-helix-helix structures that includes a full-length beta strand.

I've attached an image of the HHPred alignment with the longer amino acid sequence.

My interpretation is that HHPred alignments do support the ribbon-helix-helix domain, if translation begins at a start codon far enough upstream to contain the predicted beta strand.

For Usavi, it will come down to which start codon the annotators choose as the most appropriate start. For the genes we're working on, we will pay close attention to the key features of ribbon-helix-helix that Rick nicely explained above! ~Kirk

Link to this post \| posted 15 Jun, 2023 02:10
debbie	Kirk, This pham is quite messy. I would call the longest ORF and call the r-h-h for this one even though other members of this pham cannot call it. debbie

Link to this post \| posted 20 Feb, 2024 18:24
EWisner	We have been using the information in this thread to try to determine if we shoudl call a ribbon helix-helix DNA binding domain We are annotated AW_Goat (AP4), gene stop 5933. It has good matches on phagesdb local blast and HHpred mentioning ribbon-helix-helix DNA binding domain protein / ribbon-helix-helix. We are uncertain if it has enough a.a. in a beta sheet sheet before the helix-helix in order to be called a ribbon helix-helix DNA binding domain. It has a B-Sheet indicated in the secondary structure, but it is only made up of 4 a.a. See attached HHPred. Edited 20 Feb, 2024 18:27 129Kb

Link to this post | posted 20 Feb, 2024 18:24

EWisner

We have been using the information in this thread to try to determine if we shoudl call a ribbon helix-helix DNA binding domain

We are annotated AW_Goat (AP4), gene stop 5933. It has good matches on phagesdb local blast and HHpred mentioning ribbon-helix-helix DNA binding domain protein / ribbon-helix-helix. We are uncertain if it has enough a.a. in a beta sheet sheet before the helix-helix in order to be called a ribbon helix-helix DNA binding domain. It has a B-Sheet indicated in the secondary structure, but it is only made up of 4 a.a. See attached HHPred.

Edited 20 Feb, 2024 18:27

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Usavi_draft_70: potentially ribbon-helix-helix?