S.tar, by iGEM Technion 2016

Computational Design of Ligand Binding Sites

The bacterial world offers a relatively small selection of chemoreceptors in comparison to the vast number of possible ligands. These receptors evolved specifically to recognize substances which benefit or harm the organism in some way. On top of that the fact that the majority of known receptors today are not well characterized meant that we had very few options of creating chimeric receptors like we initially planned.

In light of the above we had to turn to a new path – redesigning the Tar chemoreceptor to bind a different ligand using computational biology - The Rosetta software.

Rosetta

Rosetta is a bioinformatics software suite for macromolecular modeling and design built by the RosettaCommons organization - a collaboration between several universities and research groups from around the world.

Rosetta development began in the laboratory of Dr. David Baker at the University of Washington as a structure prediction tool but since then has been expanded to solve many different computational macromolecular problems.
As of 2016, Rosetta algorithms have been used to predict, design and analyze almost every set of biomolecular systems: proteins, RNA, DNA, peptides, small molecules and non-canonical amino acids.

Local installation of Rosetta

We quickly discovered that for heavy duty tasks such as redesigning a protein, Rosetta requires more computational power than a regular PC has to offer. While searching for possible computing resources, we came across the local Technion grid of WLCG.

The Worldwide LHC Computing Grid (WLCG) is a global collaboration of more than 170 computing centers in 42 countries, linking up national and international grid infrastructures. It was launched in 2002 to provide a resource to store, distribute and analyze the 15 petabytes (15 million gigabytes) of data generated every year by the world’s largest and most powerful particle accelerator - The Large Hadron Collider (LHC).
In Israel, there are three computing centers connected to the grid, located in the Technion Institute, Tel Aviv University and Weizmann Institute.

WLCG supports not only the particle accelerator, but also allows casual users to benefit from this amazing project. We contacted the local Technion grid administrator and received a temporary user on the Atlas server (one of four particle accelerator components). This granted us access to vast computational power, much more than was necessary. With the help of David Cohen, grid computing specialist from Physics department, we successfully installed Rosetta and all required programs.

Fig. 1: WLCG computing grid.

Fig. 2: worldwide computing grid distribution. Yellow dot represent different computing center.

Designing a binding site

To redesign the Tar chemoreceptor we followed the protocol presented in "Rosetta and the Design of Ligand Binding Sites", (1). The purpose of the protocol is to design a binding site around a selected small molecule ligand. The general steps of the protocol can be seen in the flowchart to the right.

Using this protocol we managed to create a library of mutated Tar receptors that theoretically bind a substance in a novel way and activate the chemotaxis pathway in response to it. For each design we ran 3-5 iterations of the protocol to assure optimal results.

Fig. 3: Flowchart of the ligand binding domain design protocol.

Filtering Process

The output of the protocol is a library of variants, ranging from dozens to even thousands of protein PDB files, depending on the parameters of the design run. This fact means that filtering the results is an extremely crucial part of the process.
Rosetta is able to predict which protein designs are likely to have improved protein activity, this is done by measuring every aspect of the protein complex such as binding energies, interactions between amino acids, backbone angles, hydrogen bonds and more. After the calculation process the user can decide which parameters are relevant and drop the results which scored the lowest on these. The specific filters we used in our designs can be seen in this attachment.

First run - Benchmark Test

Before running ahead into complex designs, we had to make sure that Rosetta can "handle" the Tar protein, meaning it does not create unnecessary or drastic changes. To do this we ran the protocol with aspartic acid – which is the receptor's native ligand, to see if Rosetta outputs the native Tar LBD or a result as similar to it as possible.

From this design process we received four output structures (after filtering) with 3-5 mutations each, all of which in the binding pocket. These results proved that Rosetta can recognize and work with the Tar LBD.

Fig. 4: Native Tar results.

Protocol automation

For the purpose of our work, we automated the different steps of the protocol, including the filtering process, turning it into a single main script file complete with well-documented instructions. This script also enables easy modification of the filtering parameters to suit the specific ligand being used in the design. For more information see our software tool.

Redesigning for new ligands

Histamine

As a proof of concept we redesigned the Tar LBD to bind Histamine. This ligand is a derivative of Histidine, which is also an amino acid as the native Tar ligand, aspartic acid. This increases the chances of a successful result. Beside the molecular considerations, Histamine is known to be found in food poison, especially in rotten fish.
The next figure and video demonstrate the library we got after running few cycles and filtration:

Fig. 5: Alignment of the ligand binding domain (LBD). The alignment presents the 11 variants in the library with the native Tar (wild type).

Fig. 6: 3D imaging of the 11 variants in the library with the native Tar (wild type), each color represent different variant. As expected, the mutations can be seen near the binding pocket.

Analyzing the results illustrating two main regions of mutations, one around amino acid number 34 in the LBD sequence and the second around the 115^th amino acid. Those results led us to design and perform a two-step cloning assay (link to Histamine cloning assay), in each step we insert the mutations with single PCR reaction.

Lactose and Glucose

These days many people are allergic to milk products because of their sensitivity or intolerance to Lactose. We want to offer them a detection solution based on our system and a chip, therefore we redesign the Tar LBD to bind Lactose.
The next figures demonstrate the library we got for Lactose after running few cycles and filtration:

Fig. 7: Alignment of the ligand binding domain (LBD). The alignment presents the 11 variants in the library with the native Tar (wild type).

Fig. 8: 3D imaging of the 7 variants in the library with the native Tar (wild type), each color represent different variant.

As this is a novel design of ligand binding domain to bind sugar molecule, we decide to have proof of concept with smaller and easier sugar, one of the Lactose component- Glucose. Glucose is well known monosaccharide and it is the main compound used in the production of energy in living organisms. For this reason we can find existing chemoreceptors for Glucose (2), but redesigning Tar LBD to bind Glucose was perform as one step before redesigning Tar LBD to bind Lactose.
The next figure and video demonstrate the library we got for Glucose after running few cycles and filtration:

Fig. 9: Alignment of the ligand binding domain (LBD). The alignment presents the 8 variants in the library with the native Tar (wild type).

Fig. 10: 3D imaging of the 8 variants in the library with the native Tar (wild type), each color represent different variant.

Rohypnol

Rohypnol, also known as Flunitrazepam, used in some countries to treat insomnia. But Rohypnol known better as 'date rape drug', meaning it used for drugging other person, incapacitate him and make him more vulnerable to sexual assault such as rape.
As one application of our project, we like to offer a simple rape drug test based on our system to help men and women defend themselves when going out. Redesign the Tar LBD to bind Rohypnol take as one step closer to achieve this goal.
The next figures demonstrate the library we got for Rohypnol after running few cycles and filtration:

Fig. 12: Alignment of the ligand binding domain (LBD). The alignment presents the 4 variants in the library with the native Tar (wild type).

Fig. 13: 3D imaging of the 4 variants in the library with the native Tar (wild type), each color represent different variant

Ampicillin

Target bacteria toward antibiotics may seem redundant because than the bacteria will die, but it can be also used as an effective kill switch- small amount of antibiotics can kill more bacteria if those attract to it. To expand our novel redesigning method we redesigned the Tar LBD to bind Ampicillin antibiotics.
The next figures demonstrate the library we got for Ampicillin after running few cycles and filtration:

Fig. 14: Alignment of the ligand binding domain (LBD). The alignment presents the 13 variants in the library with the native Tar (wild type).

Fig. 15: 3D imaging of the 13 variants in the library with the native Tar (wild type), each color represent different variant.

Histamine chemotaxis results

The design run produced 870 results, after filtering we remained with 11 possible variants which we decided to clone and test with microscope (link to chemotaxis microscope assay). Only one variant was discovered as attractant for histamine.

Fig. 16: microscope results of chemotaxis activity for variant His_9 with 10mM of Histamine. On the left shown the activity after 0 minutes (when the Histamine added). On the right shown the activity after 20 minutes.

Rosetta Guide for the iGEM beginner:

During our work with Rosetta we stumbled into quite a few challenges that required us to browse through the official documentation and the Rosetta support forums and also consult with experts in computational design. These problems made us realize how difficult Rosetta can be to completely new users, especially undergraduates lacking the necessary knowledge.
To make Rosetta more accessible to the iGEM community we decided to team up with iGEM TU Eindhoven and compile a quick start guide complete with important links, protocols and information we gathered from our experience with Rosetta.
We hope that this guide will help future iGEM teams and novice Rosetta users in general.

Click here to see the full guide.

Referances

1. Moretti, R., Bender, B.J., Allison, B. and Meiler, J., 2016. Rosetta and the Design of Ligand Binding Sites. Computational Design of Ligand Binding Proteins, pp.47-62.

2. Adler, Julius, Gerald L. Hazelbauer, and M. M. Dahl. "Chemotaxis toward sugars in Escherichia coli." Journal of bacteriology 115.3 (1973): 824-847.