Part:BBa_K4990009
Bacteria-Targeting Peptide
TO KNOW ABOUT IT!
In the wake of advancements in molecular biology, numerous proteins and peptides with active functions have been discovered or invented for pharmaceutical applications. Recombinant technology, characterized by its high expression levels and ease of operation, has been extensively applied in biomedicine and related fields. To obtain multi-target, multifunctional active proteins, it is requisite to link and fuse two or more proteins with known functions. This method of obtaining bifunctional or multifunctional fusion proteins has become one of the new approaches for developing new drugs and researching bioproducts, especially widely used in the preparation of bispecific single-chain variable fragments (scFv) or antibody-drug conjugates[1-5].
Fusion proteins are principally composed of two parts: the functional protein and the linker peptide. The functional protein is the original protein intended for fusion, typically with known structure and function, posing no issues in selection. However, due to the significance of the linker peptide in the overall structure of the fusion protein, its selection and design require thoughtful research to ensure the overall activity of the fusion protein remains unchanged[2]. Consequently, research on linker peptides has gradually come into focus.
Usage in short
You can use it to target and kill Fn.
What is it
This is the structure of (Bacteria-Targeting Peptide)BTP, which is consited of B-domain, Linker A, FK-13.
B-domain targets the pilus of Fn, Linker A is a rigid and cleavage linker, FK-13 has antimicrobial activity.
How does it work?
The following are detailed microscale contacts involved in the self-assembly of B-domain and mFadA.
Notice
1.The residue numbering is based on the complete mFadA sequence, rather than renumbering after truncation of domain A or B.
2.The protein sequence of mFadA can be referred to in this article: Han YW, Ikegami A, Rajanna C, et al. Identification and characterization of a novel adhesin unique to oral fusobacteria. J Bacteriol. 2005;187(15):5330-5340. doi:10.1128/JB.187.15.5330-5340.2005
3.The information regarding contacts related to self-assembly mentioned above comes from this article: Nithianantham S, Xu M, Yamada M, Ikegami A, Shoham M, Han YW. Crystal structure of FadA adhesin from Fusobacterium nucleatum reveals a novel oligomerization motif, the leucine chain. J Biol Chem. 2009;284(6):3865-3872. doi:10.1074/jbc.M805503200
Two mFadA monomers were sourced from reference [1]. The mFadA protein sequence from Fn ATCC10953 was selected, and structural prediction was conducted using Colabfold. Employing the Rosetta local_docking method, a total of 100,000 rounds of Monte Carlo-based repeated docking were performed, leading to the successful identification of the optimal self-assembly outcome. The obtained assembly closely resembles the binding mode described in reference [2], as illustrated in the diagram below：
We further identified the microscale contacts between the two mFadA monomers. These contacts are categorized into primary and secondary hydrophobic interactions, as well as salt bridge interactions, as depicted in the diagram below
It is noteworthy that while the hydrophobic structure formed by leucine chains makes a significant contribution to the self-assembly, the salt bridge shell formed by four pairs of acidic and basic residues envelops these hydrophobic centers, providing stability to the binding.
Wu et al. [6] found that through their research on chitosan-fused rigid linker peptides, that rigid linker peptides exhibit self-cleaving properties under specific conditions. At pH=6-7, cleavage occurs. It's hypothesized that the EAAAK amino acid arrangement can form a stable hydrophilic α-helical structure. The forces at play can be attributed to the Glu-...Lys+ salt bridges between Glu and Lys. Hydrogen bonds are pivotal in forming these salt bridges, so pH greatly influences the stability of the salt bridges. At neutral pH, the strength of salt bridges is at its weakest, making cleavage more likely. Conversely, in high salt environments, the ionic strength can stabilize these bridges, preventing cleavage [7].
FK-13 exhibits antibacterial effects against both Gram-positive and Gram-negative bacteria. Through electrostatic interactions, the positively charged FK-13 connects with negatively charged bacteria, inserting into bacterial cell membranes and leading to the leakage of their contents, resulting in cell death.
Currently, two hypotheses support the mechanism by which FK-13 disrupts cell membranes: the carpet model, based on FK-13's cationic amino acids interacting with the phospholipid head groups in the cell membrane, which can form a carpet-like structure on the membrane surface, ultimately compromising membrane integrity. Changes in helical structure, charge, and hydrophobicity influence its antibacterial activity. On the other hand, the toroidal pore model suggests that due to membrane surface tension and curvature, FK-13 induces the formation of toroidal pores in bacterial membranes, causing leakage of bacterial contents and leading to bacterial death.
For mammalian cells, whose membranes are mostly neutral, FK-13's interaction with membranes is comparatively weak, and cells are less susceptible to damage. Furthermore, FK-13 can enhance the rigidity of epithelial cell membranes and reduce their permeability, thus minimizing bacterial attacks. Additionally, FK-13 possesses an amphipathic helical structure, which under hydrophobic conditions promotes oligomerization. The potency of the immune response is directly proportional to the α-helical structure of the oligomers. When there are more α-helices present, the antibacterial activity is stronger[8].
Reference
[1] Tao, L., Gao, M., & Zhou, H. (2015). Research progress on novel antibody-chemotherapy drug conjugates. Journal of Pharmaceutical Biotechnology, 22(3), 253-258.
[2] Gustavsson M，Lehtio J，Denman S，et al． Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in pichia pastoris［J］． Protein Eng，2001，14（9）:711-715.
[3] Wang SH，Zheng CJ，Liu Y，et al． Construction of multiform scFv antibodies using linker peptide［J］． J Genetics and Genomics，2008，35:313-316.
[4] Zhang JH，Yun J，Shang ZG，et al． Design and optimization of a linker for fusion protein construction［J］． Progr Natur Sci，2009，19: 1197-1200.
[5] Shan D，Press OW，Tsu TT，et al． Characterization of scFv-Ig constructs generated from the Anti-CD20 mAb 1F5 using linker peptides of varying lengths［J］.J Immunology，2014，162:6589-6595.
[6]Guo N，Zheng J，Wu L，et al. Engineered bifunctional enzymes of endo-1，4-β-xylanase/endo-1，4-β-mannanase were constructed for synergistically hydrolyzing hemicellulose[J]. Journal of Molecular Catalysis B：Enzymatic，2013，97：311-318.
[7]Arai R，Wriggers W，Nishikawa Y，et al. Conformations of variably linked chimeric proteins evaluated by synchrotron X-ray small-angle scattering[J]. Proteins：Structure，Function，and Bioinformatics，2004，57（4）：829-838.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 31
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 31
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 31
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 31
- 1000COMPATIBLE WITH RFC[1000]
| None |