Part:BBa_K4990004
mFadA
Usage in short
You can use it to validate the self-assembly of mFadA!
What you need to know!
Many bacteria have long chain fiber-protein complexes on their surfaces, which are called pili or fimbriae. These pili are composed of individual pilus monomers that link together end-to-end in the extracellular environment, self-assembling into long chain fibers with high physical strength.
For Fusobacterium nucleatum, its pili are referred to as FadA (Fusobacterium adhesin A). The monomers that make up these pili come in two forms: ①pre-FadA, which serves as an anchoring structure, attaching the entire pilus to the bacterial inner membrane. ②mFadA (mature FadA), which can link head-to-tail and self-assemble into a long filament.
In our project, the aim is to achieve bacteria-bacteria targeting. To accomplish this, we intend to leverage the self-assembly property of mFadA. We have fused a bacterial pilus monomer onto a membrane protein of the engineered bacterium, which we call the "fishing rod protein" . The membrane protein acts as the fishing rod, the linker serves as the fishing line, and the bacterial pilus monomer functions as the bait. By utilizing surface display techniques to display the fishing rod protein, our engineered bacteria can essentially "fish" for target bacteria, enabling precise bacteria-bacteria targeting.
This is our initial bacteria-bacteria targeting strategy. However, displaying the entire bacterial pilus monomer directly on the surface would lead to a range of issues, including steric hindrance, nonspecificity, and metabolic waste. Therefore, we truncated the mFadA to address these concerns.
What is it?
Below is the structure of the mFadA, which is the monomer of the pili of Fn:
What can it do?
It can self-assemble into Fn pili. Engineered mFadA can also participate in the self-assembly of pili.
How does it work?
The following are detailed microscale contacts involved in the self-assembly of 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.
Molecular dynamics analysis results of mFadA.| a. RMSD of mFadA protein complexes; b. Combined Rg and RMSD analysis of mFadA protein complexes. c. RMSF of the mFadA protein complex. d. Analysis of protein slewing in the space of mFadA protein complexes. e. Covariance matrix of mFadA protein complexes. f. Ramachandran diagram of the mFadA protein complex (The dramatic fluctuations in the figure are caused by the cyclic boundary of the water box ).
FadA was obtained by washing F.n with hot PBS.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 31
Illegal PstI site found at 178 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 31
Illegal PstI site found at 178 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 31
Illegal PstI site found at 178 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 31
Illegal PstI site found at 178 - 1000COMPATIBLE WITH RFC[1000]
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