Difference between revisions of "Part:BBa K5246004"
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Using the DeepTMHMM tool to analyze its transmembrane structure, it was predicted that HfsD most probably is globular and does not cross the inner membrane. Instead, it is positioned outside of it in the periplasm. This is consistent with the CDD findings of similarities between HfsD and periplasmic Wza proteins. | Using the DeepTMHMM tool to analyze its transmembrane structure, it was predicted that HfsD most probably is globular and does not cross the inner membrane. Instead, it is positioned outside of it in the periplasm. This is consistent with the CDD findings of similarities between HfsD and periplasmic Wza proteins. | ||
| − | + | AlphaFold 3 resulted in a high-quality structure of HfsD with some less well-characterized regions (orange and yellow). A pTM score above 0.5 suggests that the predicted overall structure may closely resemble the true protein fold, while ipTM indicates the accuracy of the subunit positioning within the complex. Values higher than 0.8 represent confident high-quality predictions (Fig.1). | |
<center> https://static.igem.wiki/teams/5246/registry/hfsd-1.png </center> | <center> https://static.igem.wiki/teams/5246/registry/hfsd-1.png </center> | ||
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<center> <b> Fig. 2. </b> Alphafold 3 structure showing </center> | <center> <b> Fig. 2. </b> Alphafold 3 structure showing </center> | ||
| − | Due to the computational limitations of | + | Due to the computational limitations of AlphaFold 3, we were unable to fully assemble the entire export apparatus. However, the confidence in the predicted structures indicates that HfsA, HfsB, and HfsD together form a tunnel-like complex in the membrane for polysaccharide export. |
| − | Conservative Domain Database and protein BLAST analyses identified HfsD as part of the polysaccharide export family, similar to E. coli Wza proteins. DeepTMHMM predicted that HfsD is globular and located in the periplasm. The predicted | + | Conservative Domain Database and protein BLAST analyses identified HfsD as part of the polysaccharide export family, similar to E. coli Wza proteins. DeepTMHMM predicted that HfsD is globular and located in the periplasm. The predicted Alphafold 3 structures suggest that HfsA, HfsB, and HfsD form a tunnel-like complex for polysaccharide export. Our findings correspond with earlier research. [1][2][3] |
===References=== | ===References=== | ||
Revision as of 13:29, 27 September 2024
CB2/CB2A HfsD Part of export protein complex
Introduction
Usage and Biology
Gene HfsD from Caulobacter Crescentus encodes an integral outer membrane protein of 247 amino acids, that is essential for holdfast export and transfering to the anchoring proteins. HfsD is a polysaccharide secretin Wza
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 337
Illegal NgoMIV site found at 442 - 1000COMPATIBLE WITH RFC[1000]
Experimental characterization
Bioinformatic analysis
Conservative Domain Database analysis identified HfsD as part of the polysaccharide biosynthesis/export family, which is linked to PEP-CTERM system proteins involved in polysaccharide export. Additionally, HfsD shows significant similarity to E. coli Wza periplasmic proteins, which play a role in cell membrane and wall formation.
Protein BLAST indicated similarities with several E.Coli export channel proteins.
Using the DeepTMHMM tool to analyze its transmembrane structure, it was predicted that HfsD most probably is globular and does not cross the inner membrane. Instead, it is positioned outside of it in the periplasm. This is consistent with the CDD findings of similarities between HfsD and periplasmic Wza proteins.
AlphaFold 3 resulted in a high-quality structure of HfsD with some less well-characterized regions (orange and yellow). A pTM score above 0.5 suggests that the predicted overall structure may closely resemble the true protein fold, while ipTM indicates the accuracy of the subunit positioning within the complex. Values higher than 0.8 represent confident high-quality predictions (Fig.1).
Considering HfsD similarity with PEP-CTERM-associated proteins and the fact that it should be a part of the export complex, we tried to fold it together with HfsA and HfsB (Fig.2).
Due to the computational limitations of AlphaFold 3, we were unable to fully assemble the entire export apparatus. However, the confidence in the predicted structures indicates that HfsA, HfsB, and HfsD together form a tunnel-like complex in the membrane for polysaccharide export.
Conservative Domain Database and protein BLAST analyses identified HfsD as part of the polysaccharide export family, similar to E. coli Wza proteins. DeepTMHMM predicted that HfsD is globular and located in the periplasm. The predicted Alphafold 3 structures suggest that HfsA, HfsB, and HfsD form a tunnel-like complex for polysaccharide export. Our findings correspond with earlier research. [1][2][3]
References
1. Toh, E., Kurtz, Harry D. and Brun, Y.V. (2008) ‘Characterization of the Caulobacter crescentus holdfast polysaccharide biosynthesis pathway reveals significant redundancy in the initiating glycosyltransferase and polymerase steps’, Journal of Bacteriology, 190(21), pp. 7219–7231. doi:10.1128/jb.01003-08.
2. Javens, J. et al. (2013) ‘Bypassing the need for subcellular localization of a polysaccharide export‐anchor complex by overexpressing its protein subunits’, Molecular Microbiology, 89(2), pp. 350–371. doi:10.1111/mmi.12281.
3. Smith, C.S. et al. (2003) ‘Identification of genes required for synthesis of the adhesive holdfast in Caulobacter crescentus’, Journal of Bacteriology, 185(4), pp. 1432–1442. doi:10.1128/jb.185.4.1432-1442.2003.