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.
Self-assembly of FadA (Self-assembly of FadA is time-dependent)
Though mFadA demonstrated outstanding adhesive stability in model predictions, we still aspired for it to possess superior self-assembly and adhesive capabilities. Consequently, the CPU-China team decided to modify the wild-type mFadA monomer, hoping that the modified mFadA monomer's B-chain (the longer orange chain in Figure 3.) would achieve a lower energy when binding with the A-chain (the turn of the purple chain in Figure 3.) of the wild-type mFadA monomer. In essence, the modified mFadA protein exhibits enhanced stability when adhering to the wild-type mFadA, bolstering the engineered bacterium's ability to target colorectal cancer cells.
Initially, we utilized Alphafold2 in Google Colab to fold two adhering mFadA proteins to their optimal conformations, choosing "template_mode" as pdb and selecting the best solution which ranks the first from five relax results as the lowest energy docking conformation.The lowest energy docking conformation was uploaded to the Rosetta Online Server that Includes Everyone (ROSIE). We leveraged its stabilize-pm feature to asymmetrically saturate mutate the mFadA segment displayed by the engineered bacterium, selecting mutations that reduce the overall energy.
Saturate mutation of mFadA homodimer. a. position from 1-20 b. position from 21-40 c. position from 41-60 d. position from 61-80 e.position from 81-99 f. 100-111
3. The mFadA protein displayed by the engineered bacterium was subjected to mutations generated in step 2. Using Google Colab, the modified protein and the wild-type protein were folded again via Alphafold2 to obtain the optimal docking conformation, resulting in a design of 35 mutants in total.
4. The 35 mutants were uploaded to ROSIE, and the score feature was used to evaluate the docking results. Referring to the mutation docking results and the wild-type docking results “Total score”. Total score is a scoring function to measure the interface energy between two interacting molecules, a lower total score value indicates a more favorable interaction. Based on the output total scores, we ranked the 36 dimers, selecting the 5 dimers with the lowest scores.
5. The 5 mutated dimers obtained in step 4 underwent dynamic simulation on Gromacs, where their binding energies were calculated.
The mutants obtained are as follows: Mutant 1: 9G→R (Total score=-494.085) Mutant 2: 10E→L (Total score=-497.952) Mutant 3: 9G→K (Total score=-494.072) Mutant 4: 6S→Q, 9G→R (Total score=-495.48) Mutant 5: 9G→R, 10E→L, 19A→R (Total score=-498.652) Benchmark: Wild-type (Total score=-479.283)
In addition to the thermodynamic analysis of mFadA adhesion, we also performed molecular dynamics analysis of the interaction between our modified protein and wild type mFadA. Through Gromacs simulations, we obtained the binding energies for the mutants in conjunction with the wild-type mFadA.
As a simple, efficient, and stable-connecting protein, mFadA is undoubtedly poised to shine in future applications such as tumor targeting, bacterial adhesion, and bacterial localization. Therefore, our ambitions for mFadA modification extend far beyond our current achievements. Presently, our modifications to the mFadA protein are confined to the first 20 amino acids, capable of producing strong interactive forces, and we have only conducted saturation mutagenesis for these 20 positions. In future research, we aim to delve deeper into mFadA, modifying other amino acid residues, anticipating the derivation of even more stable complexes between mutant mFadA and wild-type mFadA from alternative amino acid sites. We also plan to elongate the leucine zipper of the mFadA protein, aiming to achieve more stable aggregates through enhanced amino acid-hydro interactions. We look forward to the forthcoming experimental successes of the CPU-China team and anticipate that the modified mFadA will offer even more exciting surprises and applications for humanity.
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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