Part:BBa_K554007
Hemolysin B - HlyB
HlyB is part of the hemolysin secretion system ([http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Project#Device_3:_Secretion_system Device 3, Protein Secretion System]), very important to export the proteins produced inside bacteria and that must act in targets outside (such as IL-12 and IL-10). HlyB acts as a ATP-binding cassette, and recognizes the substrate via its secretion signal (like HlyA) and is responsible for the specificity of the secretion system process. This system is composed of 4 essential parts: the C-terminal signal sequence of alpha-hemolysin (HlyA, which will be linked to the export target protein), the two specific translocator proteins HlyB and HlyD and the outer membrane protein [BBa_K554009 TolC].
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1301
Illegal BglII site found at 1964 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 231
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
You can see a representation of this device acting in the schema below: HlyB gene and product are shown as a symbolic cilinder in orange.
Representation of device 3, the protein secretion system, in a Jedi bacteria that contains Device 1 (Adrenaline sensor/IL-12 producer). To export a protein, the bacteria must have the HlyD, HlyB and TolC proteins and the target protein must have a signal sequence (HlyA tail). In this case, the target protein to be secreted is IL-12.
A more realistic schema of ABC transport system is shown below:
Three-dimensional structure representation
You can find below a tridimensional structure of ATP-binding domain of hemolysin B from Escherichia coli (retrieved from PDB [http://www.pdb.org/pdb/explore/explore.do?structureId=1mt0 1MT0] (Schmitt et al. 2003)) solved by crystallography and X-ray diffraction at 2.5 A resolution. This is a jmol applet, in which you can interactively see the protein structure of HlyB:
MIT_MAHE 2020
Usage and Biology HlyB is part of the Type I hemolysin secretion system and is very important to export the proteins produced inside bacteria and that must act in targets outside. HlyB acts as an ATP-binding cassette (ABC) and recognizes the substrate via its secretion signal (like HlyA) and is responsible for the specificity of the secretion system process. HlyB has various molecular functions like ATPase activity, ATPase-coupled transmembrane transporter activity, ATP binding and peptidase activity. Together with the inner membrane protein HlyD and the outer membrane protein TolC, HlyB is responsible for transport of the 107 kDa toxin HlyA from the cytoplasm, across both membranes of the cell envelope of Escherichia coli, directly to the medium (F. Zhang et al., 1993).
Type I secretion is sec-independent and bypasses the periplasm. This widespread pathway allows the secretion of proteins of diverse sizes and functions via a C-terminal uncleaved secretion signal. This C-terminal secretion signal specifically recognizes the ABC protein, triggering the assembly of the functional trans-envelope complex. (P. Delepelaire, 2004).
The X-ray crystal structure of the nucleotide-binding domain (NBD) of HlyB shares the common overall architecture of ABC-transporter NBDs. However, the last three residues of the Walker A motif adopt a 310 helical conformation, stabilized by a bound anion. In consequence, this results in an unusual interaction between the Walker A lysine residue and the Walker B glutamate residue (Lutz Schmitt et al., 2003).
A study to test the ability of HlyB to complement a defect in the alpha-factor transporter in Ste6, expressed in Saccharomyces cerevisiae, showed that HlyB was not able to restore mating ability to a Ste6 deletion strain. The HlyB protein did not co‐fractionate with Ste6 on sucrose gradients, indicating that improper localization of the HlyB protein could contribute to the lack of complementation. Immunofluorescence experiments suggest that HlyB is localized to structures derived from the endoplasmic reticulum (ER). The HlyB‐expressing cells revealed a perinuclear staining typical of ER‐localized proteins and intensely staining ring‐like structures (HlyB‐bodies). Double‐label immunofluorescence experiments show that the HlyB structures also contain the ER binding protein (BiP), the product of the kAR2 gene (Ralf Kölling et al., 1996).
Another recent study established an optimized purification protocol for HlyB and the negative influence of free detergent on the basal ATPase activity of HlyB. In this experiment for the purification of HlyB, membranes of 0.5 L cell culture were diluted with buffer P to a protein concentration of 10 mg/mL and solubilized with 0.5% (w/v) fos-choline 14 for 1h at 8 °C. Solubilized membranes were filtered (0.45 µm), diluted two-fold using buffer P supplemented with 2 mM imidazole and loaded on Zn2+-charged immobilized metal-ion affinity chromatography (IMAC) column (5 mL HiTrap Chelating HP, GE Healthcare).
The column was washed with 8 mL of buffer P including 0.015% (w/v) DDM and 2 mM imidazole. Non-specifically bound proteins were removed by washing with 18 mL buffer P supplemented with 0.015% (w/v) DDM and 40 mM imidazole. HlyB was eluted with buffer P containing 0.015% (w/v) DDM and 25 mM EDTA. It was found that detergent exchange is more efficient while the protein is bound to IMAC resin than during SEC, as the washing can be largely extended. Furthermore, empty detergent micelles are of considerable size and often migrate through the SEC column close to or even with the protein. Both DDM and LMNG yielded approximately 6 mg of pure and homogeneous HlyB per litre of bacterial cell culture. The homogeneity after purification was assessed by SEC and the elution profile was comparable for DDM and LMNG-purified protein (Kerstin Kanonenberg et al., 2019).
References
Barbara D. Tzschaschel, Carlos A. Guzmán,, Kenneth N. Timmis and Victor de Lorenzo. An Escherichia coli hemolysin transport system-based vector for the export of polypeptides: Export of shiga-like toxin IIeB subunit by Salmonella typhimurium aroA. Nature Biotechnology 14, 765 - 769 (1996) [http://www.nature.com/nbt/journal/v14/n6/abs/nbt0696-765.html Article link]
P. Delepelaire. Type I secretion in Gram-negative bacteria. Biochimia et Biophysica Actca 1694, 149-161 (2004) [http://www.ncbi.nlm.nih.gov/pubmed/15546664 Link to PubMed]
Ivaylo Gentschev, Guido Dietrich and Werner Goebel. The E. coli α-hemolysin secretion system and its use in vaccine development. Trends in Microbiology 10, 39-45 (2002) [http://www.ncbi.nlm.nih.gov/pubmed/11755084 Link to PubMed]
Schmitt, L., Benabdelhak, H., Blight, M.A., Holland, I.B., Stubbs, M.T.Crystal structure of the nucleotide-binding domain of the ABC-transporter haemolysin B: identification of a variable region within ABC helical domains.Journal: (2003) J.Mol.Biol. 330: 333-342 [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=12823972 Link to PubMed]
Zhang, F., Sheps, J. A., & Ling, V. (1993). Complementation of transport-deficient mutants of Escherichia coli alpha-hemolysin by second-site mutations in the transporter hemolysin B. The Journal of biological chemistry, 268(26), 19889–19895.
Kölling, R., & Hollenberg, C. P. (1996). The hemolysin B protein, expressed in Saccharomyces cerevisiae, accumulates in binding-protein (BiP)-containing structures. European journal of biochemistry, 239(2), 356–361. https://doi.org/10.1111/j.1432-1033.1996.0356u.x
Kanonenberg, K., Smits, S., & Schmitt, L. (2019). Functional Reconstitution of HlyB, a Type I Secretion ABC Transporter, in Saposin-A Nanoparticles. Scientific reports, 9(1), 8436. https://doi.org/10.1038/s41598-019-44812-0| None |