Part:BBa_K5246016

HB HfsD Part of export protein complex

Usage and Biology

Gene HfsD from Hirschia baltica encodes an integral outer membrane protein of 239 amino acids, that is essential for holdfast export and transfer to the anchoring proteins. Combination of HfsA, HfsB, HfaD and HfsE proteins together are resposible for holdfast association with the cell envelope.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 321
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Experimental characterization

Bioinformatic analysis

Conservative Domain Database analysis identified HfsD as part of the polysaccharide biosynthesis/export family, 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 is probably 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.

Considering HfsD's 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.

Due to AlphaFold 3's computational limitations, we were unable to fully assemble the entire export apparatus. However, the confidence in the predicted structures indicates that HfsA, HfsB, and HfsD 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.