SEA-PHAGES | Minor tail Protein on EA genomes

Link to this post \| posted 01 Aug, 2019 17:42
adiaz	I'm in the process of annotating YuuY and I noticed that one of the genes is called minor tail protein (Pham 11397, coordinates 12806-15232 in Yuuy)in almost every EA cluster genome even though it is larger than the tape measure protein (Pham 11400, coordinates 9625-12036) and much larger than the major tail protein (Pham 11479, coordinates 8110-8604). HHPRED shows 99% probability (36.7% coverage) that it matches to prophage endopeptidase tail but given the size of the gene I wanted to confirm that this is the right call. Thank you, Arturo

Link to this post | posted 01 Aug, 2019 17:42

I'm in the process of annotating YuuY and I noticed that one of the genes is called minor tail protein (Pham 11397, coordinates 12806-15232 in Yuuy)in almost every EA cluster genome even though it is larger than the tape measure protein (Pham 11400, coordinates 9625-12036) and much larger than the major tail protein (Pham 11479, coordinates 8110-8604).

HHPRED shows 99% probability (36.7% coverage) that it matches to prophage endopeptidase tail but given the size of the gene I wanted to confirm that this is the right call.

Thank you,

Arturo

Link to this post \| posted 01 Aug, 2019 18:00
adiaz	On a related note, gp22 (pham 17964) is also listed as a minor tail protein in most EA genomes but the HHPRED data matches Chitinase A (100% probability. I guess I'm having a hard time understanding how something that cleaves Chitin is related to a minor tail protein. Arturo

Link to this post \| posted 05 Aug, 2019 22:30
cdshaffer	I always think that an HHPRED of 100% probability is an indication that it is very confident that the protein you are searching with belongs to the same family of proteins it is matching. Chitin is a polymer of β-(1→4)-linkages of N-acetylglucosamine (NAG) while peptidoglycan is alternating residues of β-(1,4) linked N-acetylglucosamine and N-acetylmuramic acid. I always try to remind my students that functional annotation down to one specific substrate is very risky. In my mind it is easy to change just one or two amino acids at exactly the right place to radically change the binding properties of an enzyme to any one specific substrate. And one should be even more careful when there are known to be many different but related substrates [like polysaccharides]. So annotation of this type needs a "sanity check". Does it make sense that a phage would have an enzyme that cleaves chitin? No, not really, so I would not annotate this as a chitinase. One the other hand, it does make sense that some ancient protein that does something with N-acetylglucosamine could easily evolve into a chitinase along one branch and evolve into some other protein that somehow interacts with peptidoglycan in another branch. I would come away pretty confident that this phage protein either binds to NAG or cleaves β-(1→4)-linkages of NAG. Since there is no approved term for "protein either binds to NAG or cleaves β-(1→4)-linkages of NAG" I would stick with "minor tail protein" but I would also be pretty confident of that annotation given the structural similarity of chitin and peptidoglycan.

Link to this post | posted 05 Aug, 2019 22:30

cdshaffer

I always think that an HHPRED of 100% probability is an indication that it is very confident that the protein you are searching with belongs to the same family of proteins it is matching. Chitin is a polymer of β-(1→4)-linkages of N-acetylglucosamine (NAG) while peptidoglycan is alternating residues of β-(1,4) linked N-acetylglucosamine and N-acetylmuramic acid.

I always try to remind my students that functional annotation down to one specific substrate is very risky. In my mind it is easy to change just one or two amino acids at exactly the right place to radically change the binding properties of an enzyme to any one specific substrate. And one should be even more careful when there are known to be many different but related substrates [like polysaccharides].

So annotation of this type needs a "sanity check". Does it make sense that a phage would have an enzyme that cleaves chitin? No, not really, so I would not annotate this as a chitinase. One the other hand, it does make sense that some ancient protein that does something with N-acetylglucosamine could easily evolve into a chitinase along one branch and evolve into some other protein that somehow interacts with peptidoglycan in another branch. I would come away pretty confident that this phage protein either binds to NAG or cleaves β-(1→4)-linkages of NAG.

Since there is no approved term for "protein either binds to NAG or cleaves β-(1→4)-linkages of NAG" I would stick with "minor tail protein" but I would also be pretty confident of that annotation given the structural similarity of chitin and peptidoglycan.

Link to this post \| posted 06 Aug, 2019 19:29
adiaz	Hi Chris, Thank you for your thorough response, that was really helpful! I'm still familiarizing myself with phage biology, it's not unusual for the genes that encode for minor tail proteins to be larger than the major tail genes?

Link to this post \| posted 07 Aug, 2019 19:10
cdshaffer	I believe the "major" and "minor" terms relate back to very early papers where they purified phage proteins and ran them out on protein gels. So a "major" proteins would be the very intense band on the gel and would presumably be high copy number. There would also be weak or even very faint banks on the gel and these would be the "minor" proteins. The terms then had less to do with size but rather abundance.

Link to this post | posted 07 Aug, 2019 19:10

cdshaffer

I believe the "major" and "minor" terms relate back to very early papers where they purified phage proteins and ran them out on protein gels. So a "major" proteins would be the very intense band on the gel and would presumably be high copy number. There would also be weak or even very faint banks on the gel and these would be the "minor" proteins. The terms then had less to do with size but rather abundance.

Link to this post \| posted 28 May, 2020 22:13
vcw0	Members of the SMART 2020 team recently had a discussion about functional annotation of minor tail proteins in subcluster EA1 Microbacterium phages. Of particular interest is the functional assignment of the gene that is directly upstream of the endolysin gene (called lysin A in some older annotations, but should be called endolysin if only one lysin is annotated in a genome - see official functional assignment list for this update). This gene (Pham 17919 in Phamerator Actino_Draft 358; e.g., gp23 in TeddyBear and Pherferi or gp24 in Martin) should be called as a minor tail protein, along with the all the other proteins encoded by genes located between the tape measure and endolysin genes. BLAST data support the "minor tail" call. HHpred data, however, show a strong hit with several hydrolases, including glycoside hydrolases. "Glycoside hydrolase" is an official function on the list. (Please note that the reference example on the official function list for a phage with an annotated glycoside hydrolase will soon be updated by Debbie). But why is that particular function not called for that gene in the final annotation that should be submitted for a subcluster EA1 phage? Note that at least four "large" minor tail proteins are essential to construct a flexible phage tail. Based on BLAST and HHPred, some of the genes may be assigned as "hypothetical proteins"; yet, these genes can be assigned as minor tail proteins based on synteny even when a BLAST or HHpred hit may not be indicated. Difficulty with crystallization of some of the smaller proteins in this group is known to account for the lack of functional calls as minor tail components in HHpred. Many minor tail proteins are known to include enzymatic functions, and the presence of glycoside hydrolase activity is certainly consistent with an enzymatic activity that would assist in breaking down glycosidic bonds in complex sugars within bacterial cell walls. BUT, in the end, the minor tail function usurps the glycoside hydrolase function. So for now, call that gene (just upstream of the endolysin gene) a minor tail protein. At some point, a revision may come to the official function list that includes more functional specificity (e.g., minor tail protein/glycoside hydrolase domain).

Link to this post | posted 28 May, 2020 22:13

vcw0

Members of the SMART 2020 team recently had a discussion about functional annotation of minor tail proteins in subcluster EA1 Microbacterium phages. Of particular interest is the functional assignment of the gene that is directly upstream of the endolysin gene (called lysin A in some older annotations, but should be called endolysin if only one lysin is annotated in a genome - see official functional assignment list for this update). This gene (Pham 17919 in Phamerator Actino_Draft 358; e.g., gp23 in TeddyBear and Pherferi or gp24 in Martin) should be called as a minor tail protein, along with the all the other proteins encoded by genes located between the tape measure and endolysin genes. BLAST data support the "minor tail" call. HHpred data, however, show a strong hit with several hydrolases, including glycoside hydrolases. "Glycoside hydrolase" is an official function on the list. (Please note that the reference example on the official function list for a phage with an annotated glycoside hydrolase will soon be updated by Debbie). But why is that particular function not called for that gene in the final annotation that should be submitted for a subcluster EA1 phage?

Note that at least four "large" minor tail proteins are essential to construct a flexible phage tail. Based on BLAST and HHPred, some of the genes may be assigned as "hypothetical proteins"; yet, these genes can be assigned as minor tail proteins based on synteny even when a BLAST or HHpred hit may not be indicated. Difficulty with crystallization of some of the smaller proteins in this group is known to account for the lack of functional calls as minor tail components in HHpred.

Many minor tail proteins are known to include enzymatic functions, and the presence of glycoside hydrolase activity is certainly consistent with an enzymatic activity that would assist in breaking down glycosidic bonds in complex sugars within bacterial cell walls. BUT, in the end, the minor tail function usurps the glycoside hydrolase function. So for now, call that gene (just upstream of the endolysin gene) a minor tail protein. At some point, a revision may come to the official function list that includes more functional specificity (e.g., minor tail protein/glycoside hydrolase domain).

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Minor tail Protein on EA genomes