During the COVID-19 pandemic, due to inaccessibility of labs and experimentation, we couldn’t perform mathematical modelling for our experiments. Hence, we chose bioinformatics modelling for validating our results. It serves as a strong proof-of-concept of our fusion protein design and we believe that it’ll closely resemble the results expected through experiments.

What is protein modelling?

Bioinformatics tools can be used to model a protein structure if the sequence of the protein is known. Computational protein structure prediction relies on principles of protein structures obtained through X-ray crystallography, NMR spectroscopy and other physical energy functions to predict the three-dimensional structures of proteins. It uses various Machine Learning algorithms to develop protein structures. There are three methods for modelling proteins:

Methods

Our Approach:

Protein Sequence

R2 tail fiber protein sequence comprises 691 amino acids. The AP22 bacteriophage tail fiber protein that targets A. baumannii is 271 amino acids in length. However, through literature review, it was found that the R2 pyocin- NTF (N-truncated fragment) (G-443 to R-691) is sufficient to bind to the bacterial surface. Therefore, only the N-truncated fragment was modelled.
Through preliminary bioinformatics analysis during fusion protein design, we identified the restriction sites needed to create an R2 pyocin-AP22 fusion protein. It was inferred that removing the last 134 amino acids of the R2 pyocin tail fiber protein sequence and ligating the last 137 amino acids of the AP22 bacteriophage sequence to this truncated pyocin sequence would create a functional fusion product.

>R2-NTF tail fiber Sequence
GSFTKEADGELPGGVNLDSMVTSGWWSQSFTAQAASGANYPIVRAGLLHVYAASSNFIYQTYQAYDGESFYFRCRHSNTWFPWRRMWHGGDFNPSDYLLKSGFYWNALPGKPATF PPSAHNHDVGQLTSGILPLARGGVGSNTAAGARSTIGAGVPATASLGASGWWRDNDTGLIRQWGQVTCPADADASITFPIPFPTLCLGGYANQTSAFHPGTDASTGFRGATTTTAVIRNGYFAQAVLSWEAFGR

>AP22 tail fiber protein
MANPIFIPMAFAANGIKNLIQKVRQVGQDPEDFTWDEGAPLITMTKIEDGGKAPKGQDFNGVLNALSEHVIYGQNGNRYTWSQDVVDEFGGYALGAIVQSNDTTKEFRSLIANNTVNPNNGLGGAWEVYSGQGS IPTATSTTAGITKVLNVLNSNDVGSALSAAQGKVLNDKFNFQNSKNQSGYVRLGDSGLIIQWGVFTSTKTQSNLIFPLAFPNALLSITGNLNSNTPDVIGIDFDLSTATKTSIKTGAAQVGASWLSGKKISWIAIGY

The last 134 amino acids from the R2-NTF pyocin were removed and the last 137 amino acids of the AP22 phage were inserted in the remaining sequence to generate the R2-NTF-AP22 fusion tail fiber protein sequence.

>R2-NTF-AP22 Fusion tail fiber
GSFTKEADGELPGGVNLDSMVTSGWWSQSFTAQAASGANYPIVRAGLLHVYAASSNFIYQTYQAYDGESFYFRCRHSNTWFPWRRMWHGGDFNPSDYLLKSGFYWNALPGKPATF IPTATSTTAGITKVLNVLNSNDVGSALSAAQGKVLNDKFNFQNSKNQSGYVRLGDSGLIIQWGVFTSTKTQSNLIFPLAFPNALLSITGNLNSNTPDVIGIDFDLSTATKTSIKTGAAQVGASWLSGKKISWIAIGY

We model this sequence of the fusion tail fiber using all the three modelling methods discussed above.
In homology modelling, we use the sequence input for modelling. After entering the sequence of the fusion protein in the fasta format we searched for templates on the platform. 6cl6 was chosen as a template structure as it had a GMQE score of 0.71 and identity of 63.64 and the target model was predicted to be a homo-trimer. The GMQE (Global Model Quality Estimation) score is a quality estimation which combines properties from the target–template alignment and the template search method. The resulting GMQE score is expressed as a number between 0 and 1, reflecting the expected accuracy of a model built with that alignment and template and the coverage of the target. Higher numbers indicate higher reliability. It also takes into account the QMEAN score to increase the reliability of the quality estimation.

For threading and ab-initio modelling, we submitted our target sequence on the I-Tasser & Robetta Baker Lab’s modelling server respectively and results were obtained via email.

Protein Structure

The overall structure of the trimeric R2-NTF is a barbell-like protein, with a three-domain organization consisting of a “head”, medial “shaft”, and “foot”. The head (G443-M525) and foot domains (P598-R691) are globular and connected by an intertwined, helical, and fibrous-looking shaft (W529-V597).

We modelled our protein through all the three methods and compared the results of our models. The models obtained through threading were monomers and since our native structure is a trimer, we didn’t consider the monomeric models. The models obtained through homology modelling & ab-initio were trimers and were considered & further compared to choose the best model.

Obtained Models:

Homology Modelling Threading/Fold-Recognition Ab-initio Modelling