Redesigning IL-7R Binders
Hi Foldit players! We need your help redesigning protein binders!
I'm bcov, a graduate student in the Baker Lab. My PhD project is to make proteins that stick to other proteins. In my work, I’m given the model of a natural target protein and my task is to design a new protein that will bind to it. It turns out this problem is really hard because not only do my designed proteins need to bind to the target, but they have to properly fold first! Fortunately, I can use a high-throughput binding experiment that allows me to test 100,000 different proteins at once.
At the moment, I’m interested in studying the folding aspect of this problem. I have a clever experiment planned where I should be able to confirm the atomic accuracy of a designed protein even when it’s mixed with thousands of other proteins. For this experiment, I will need lots of binder designs that have different folds, but that share a common binding interface. I'm planning a series of Foldit puzzles in which players can redesign my binders while preserving the binding interface.
My designed binders target a protein called interleukin 7 receptor (IL-7R), which helps to regulate the human immune system, and is an important target for cancer therapy.
Here are the details of the experiment:
· I have 11 designed proteins that are confirmed to bind the target IL-7R
· I want to leave my designed binding interface the same, but redesign the rest of the protein
· In each puzzle, your task is to design the rest of the protein so that it folds the interface-side in precisely the right conformation
· Your designs will be tested for binding against IL-7R
· You will get a binding score based on how well your design binds in the wet lab
The binding score here is really cool actually. After we run the binding experiments at the end of the puzzle series, you will receive cold-hard data from the biochemistry lab about the binding strength of your design. Well-folded proteins that fold precisely into the puzzle structure will likely score the highest. Details about the binding score will be released later, but in general, there are three categories:
1. Your design did not bind to IL-7R
2. Your design bound to IL-7R but was worse than my design
3. Your design bound to IL-7R and was better than my design
If you end up in category 3, congrats! You beat me :P
Nearly all Foldit player designs will be tested experimentally. This is possible because we can test all the designs at the same time in our high-throughput binding experiment. Designs that look especially good will be tested multiple times with various mutations to increase data consistency.
Due to time constraints, puzzles in this series will be shorter than our normal week-long puzzles, and will only run for 4 days at a time. We'd like to generate as many variants as possible for the original 11 binders. So, don't worry if you miss a puzzle; there will be plenty more to follow up!
Check out the first puzzle of the series, Puzzle 1704: IL-7R Binder Redesign: Round 1, which is out now! Happy folding!
Update (8/16/2019): Read the followup post Protein Design Critique: IL-7R Binder Redesign
Protein Design Critique: Cubane FeS Binder
A few weeks ago, we challenged Foldit players to design a protein that could bind an iron-sulfur (FeS) cluster, in Puzzle 1688: Cubane FeS Binder Design. A cubane type [4Fe-4S] iron-sulfur cluster is a "cube" made out of alternating iron and sulfur atoms, and is bound by carefully-placed cysteine residues in a protein. Iron-sulfur metallo-proteins are responsible for electron transfer in light-harvesting, cellular respiration, many other processes. We'd like to design an iron-sulfur protein so that we can better understand electron transfer in proteins. By changing the environment around the iron-sulfur cluster, we could tune the electron transfer properties of the protein, which could open the door to metabolic engineering and new chemistry!
We asked Dr. Anindya Roy, the Baker Lab’s expert on redox proteins, to take a look at Foldit players’ designs from Puzzle 1688. Below are some comments from Anindya, which we hope players will take into account for the Round 2 puzzle, which is online now!
We were very excited about the structural diversity of designs by Foldit players, who developed a variety of different protein folds! Many natural redox proteins adopt a ferredoxin fold, with a secondary structure pattern of (β-α-β)2, and we were worried that Foldit players might also favor the same ferredoxin fold. We were happy to see lots of helical bundles and other α/β folds with different secondary structure patterns, because these folds might have properties that are not possible with the ferredoxins typically found in nature. We encourage players to keep exploring helical bundles and other folds!
Room for improvement
In these initial designs, the two main areas for improvement are excessive loops and incomplete burial of the FeS cluster.
The cubane FeS cluster should be buried inside the protein core as much as possible. If the FeS cluster is to be used to catalyze a chemical reaction, then we want the active site to be protected from the water surrounding the protein. The top-scoring design by toshiue and Wilm, shown below, does a good job of burying the FeS cluster. The frozen FeS-binding loop is highlighted in blue and purple, with helices packing nicely against the cluster on three sides, shielding it from water.
If we zoom in on the FeS cluster, we can see some other nice features of this design. We like to see large, aromatic residues packed near the FeS cluster, like the TRP residue at the left of this protein. Players should try to design aromatic PHE, TRP, and TYR residues around the FeS cluster. Also, because the FeS cluster is negatively charged, it can be stabilized with complementary positively-charged residues, like the LYS residue shown beneath the cluster here. Players should also design positively charged LYS and ARG residues near the FeS cluster.
Unfortunately, we’re afraid this design has too many residues in loops, and not enough secondary structure. We can see in the first image that the frozen loop has been extended to make an even longer loop, which is unlikely to fold as intended. In order for these protein designs to fold up with high stability, we want to minimize the amount of loops in the structure. The more residues in helices and sheets, the better!
Below is a design by Galaxie and grogar7 that has a much smaller proportion of loop residues. The FeS-binding loop is flanked closely by long, stable helices on either side, and all of the other helices are connected by minimal loops. This design would have a much better chance of folding up into a stable structure.
However, in this design the FeS cluster is not completely buried, and will be exposed to the water surrounding the protein. This means we have less control over the electron transfer properties of the FeS cluster, which makes it harder to design an enzyme that can catalyze chemical reactions. One way to improve this design would be to extend the helices on either side of the FeS cluster in order to bury it away from the surrounding solvent.
This design also features lots of positively charged LYS and ARG residues at the binding site, which help to stabilize the negatively charged FeS cluster. Keep in mind that these charged residues have polar atoms that like to make hydrogen bonds. On the protein surface, they can make hydrogen bonds with the surrounding water; but if they’re buried away from solvent then they need to make hydrogen bonds within the protein!
Posted by bkoep 51 368 |
Fri, 07/19/2019 - 22:57 |
In summary, we encourage Foldit players to design more helical bundles and other folds, with a focus on minimizing loop residues and burying the FeS cluster in the protein core! Large aromatic residues (PHE, TRP, TYR) and positively charged residues (LYS, ARG) help to stabilize the FeS binding site! Play Puzzle 1701: Cubane FeS Binder Design: Round 2 now!
The Foldit protein design paper
Today, the scientific journal Nature published a paper titled De novo protein design by citizen scientists, all about the work of Foldit players!
The paper is written for an audience of professional scientists, and gets somewhat technical. This blog post is meant to summarize the main points of the paper, so that everyone can appreciate the significance of this achievement. If you have trouble accessing the paper on the Nature website, try this view-only online version or check the Baker Lab website.
What is 'de novo' protein design?
The Latin phrase de novo translates literally to “from the new”—we usually use it to mean “from scratch.” Veteran Foldit players will recognize this phrase from De-novo Freestyle Foldit puzzles, where players fold up a protein from a completely unfolded starting position (i.e. from scratch), rather than from a partially-folded starting position.
In the field of protein design, this phrase has a special meaning. De novo protein designs are created without referencing the sequences of natural proteins.
To illustrate, you could imagine designing a 3-helix bundle protein just by looking at the sequences of natural 3-helix bundles and choosing the most common amino acid at each position. Since we have lots of data about natural protein sequences, and powerful ways to extract patterns from data, this method is relatively easy. But it will only ever let us design proteins that are similar to natural proteins.
On the other hand, de novo protein design is much more difficult. Rather than relying on patterns in massive datasets, de novo design requires an understanding of the physical principles behind protein folding. The advantage is that we can use de novo methods to design brand new proteins that are unlike any proteins found in nature.
Why is protein design hard?
A designed protein must fold entirely on its own, without direction or instruction from any outside source.
The number of possible folds for a protein is huge, and a protein dissolved in solution is generally free to sample any of those possible folds. But if the protein sequence is chosen carefully, then the protein chain will have lower energy in one fold than in any other, and the protein will naturally prefer that lowest-energy fold.
It is difficult to choose the sequence because there are also many possible protein sequences (more than there are atoms in the universe!). And, once we choose a sequence for our target fold, we cannot check all the possible folds to ensure that our target fold has the lowest energy.
For a deeper discussion about the difficulties of protein design, see this previous blog post.
How can computer gamers design proteins?
Figure 1 below shows the Foldit game interface. Foldit players have a number of tools that allow them to change both the fold and the sequence of a virtual protein. The player's score is calculated from the energy of the virtual protein, with a state-of-the-art energy function developed by academic protein scientists. By competing with one another to reach the highest score, Foldit players arrive at virtual proteins with extremely low energies (a high Foldit score corresponds to a low protein energy).
Since energy alone is not enough for protein design, the Foldit team has had to make some adjustments to the Foldit score function. Every step of the way, we’ve relied on the work of Foldit players to expose problems with our score function. Foldit players are excellent at exploring new kinds of protein folds that are unlike anything seen in nature. For this reason, Foldit players are incredibly helpful for identifying unanticipated weaknesses in our energy function, and ultimately can improve our understanding of protein folding.
How do Foldit players actually design proteins?
Figure 2 shows that Foldit players design proteins much differently than automatic protein design algorithms. From start to finish, players will routinely accept huge penalties (high-energy spikes; colored traces in panel 2a), that ultimately pay off with low-energy designs.
Automatic algorithms, on the other hand, can only accept very small penalties, and they do so less frequently (gray traces in panel 2a).
How do virtual Foldit designs behave in real life?
Figure 3 shows data from the lab tests that we perform on protein designs from Foldit players.
The first thing to note, in panel 3a, is that these proteins are extremely diverse and span many different protein folds. Due to the amount of planning and creativity required to conceive a protein fold, a protein engineer will usually focus on a small number of protein folds for a given task. This paper reports a greater number of protein folds than any other protein design paper to date—including a brand new fold that is not observed in any natural proteins!
Panels 3c-f show that these proteins are very well-behaved both on the computer and in the lab. The plots in panel 3c show that Rosetta@home computer simulations predict the designs will fold accurately (details here).
Panels 3d-e show that the proteins don’t aggregate together, and are rigidly structured in solution. And panels 3f-g show that the proteins do not unfold except in extremely harsh conditions (read more here. Most natural proteins unfold with only 3-5 kcal/mol of energy; many of the designed proteins are hyper-stable and require >10 kcal/mol!
How do we know that the proteins fold up as designed?
Since proteins are smaller than the wavelength of visible light, we can’t see them directly under a microscope. However, in some cases we can use very intensive techniques to determine the structure of a protein indirectly (read more here and here). We used these techniques to solve high-resolution structures of 4 proteins designed by Foldit players.
Figure 4 shows the exact placement of atoms in the real-life protein structures, which is nearly identical to the virtual protein design in every case.
So, what does this all mean?
This is a huge accomplishment for Foldit players! De novo protein design is a very new field, and already citizen scientists are making significant contributions—not just by designing new proteins, but also by helping us improve our understanding of protein design. We hope that scientists in other fields will be able to find similar ways to engage public creativity and enthusiasm, to increase our understanding of the world.
Now that Foldit players can accurately design high-quality proteins from scratch, we can start to challenge Foldit players with more applied protein design problems. We’d like Foldit players to help us design new proteins that can assemble into multi-component structures and materials, or that can bind to biological targets as potent medicines, or that can degrade toxic chemicals!
Because Foldit depends on the cooperation and competition of its player community, our scientific ability grows rapidly with the number of Foldit players. We look forward to expanding the Foldit community and recruiting more creative and curious Foldit players!
Help us design a protein for cancer treatment right now, by playing Puzzle 1683: Integrin Antagonist Design!( Posted by bkoep 51 368 | Wed, 06/05/2019 - 18:11 | 6 comments )
New Custom Contests feature
We are excited to announce a new feature in Foldit: Custom Contests! As you may know, contests have been a feature that allows anyone to host their own private Foldit puzzle, chosen from a limited, pre-selected list. Now, you can make your own custom Foldit puzzle of whatever you choose and host it as a contest. We designed the Custom Contest feature especially for educators, who can now tailor their Foldit puzzles to their exact curriculum. There are plenty of other uses as well, including private contests that research groups can use to brainstorm new ideas, or even Foldit parties!
We just published a paper that can be found here that describes the Custom Contests in depth. If you’re interested in making Custom Contests, please email mail.fold.it |at| gmail.com for access.( Posted by beta_helix 51 868 | Mon, 03/04/2019 - 16:53 | 8 comments )
New Foldit tool: Pick Sidechains
We're introducing a new Foldit tool for folding protein side chains, called Pick Sidechains. Pick Sidechains is currently available to devprev users for testing, and soon will be released for all users!
To use Pick Sidechains, select a segment of your protein and click on the Pick Sidechains button. This starts the tool, which runs continuously until you stop it with the Stop button in the upper left corner of the screen (just like Shake, or Remix).
Pick Sidechains displays a cloud of all possible side chain positions for the selected segment. Each possibility is called a rotamer. Use the mouse to hover over the cloud and highlight individual rotamers. Left-click on a rotamer to apply it and see how it affects the score of your solution.
When you start Pick Sidechains, a new panel will appear that shows the segment you selected, along with a list of rotamer options for that segment.
Every rotamer in the list is labeled with a shorthand name, using the letters ‘m’, ‘p’, and ’t’ to describe the rotation of each bond in the side chain (for “minus”: -60º, “plus": 60º, or “trans": 180º).
The panel also includes a gauge for each rotamer’s prevalence. Certain rotamers are more common than others, and these are typically preferred for protein folding. A full gauge indicates a common rotamer, while a rare rotamer will have an empty gauge. More common rotamers will usually score better, but sometimes a rare rotamer can make an excellent hydrogen bond or fill a void to gain more points!
After you have picked a rotamer, stop Pick Sidechains with the Stop button in the upper left corner of the screen.
A tool for H-bond Networks
In most cases, the Shake tool will still be the fastest and most effective way to fold the side chains of your protein. Pick Sidechains is meant for special situations where Shake performs poorly. One example is designing H-bond Networks.
Shake is not very good for designing H-bond Networks, because it doesn’t know about puzzle Objectives—Shake can only optimize the base Foldit score (without Objective bonuses). This means Shake will sometimes ignore a potential H-bond Network because other rotamers improve the base score, even if the network would yield a huge Objective bonus for more points overall.
In these situations, it’s up to Foldit players to design H-bond Networks by hand. Pick Sidechains should give players improved manual control over side chains, and hopefully will help players design better H-bond Networks!
An example of a well-satisfied H-bond Network designed by fiendish_ghoul in Puzzle 1561. This network spans the interface between the two symmetric units, and is located in the core of the protein, with nine polar atoms that need to form hydrogen bonds (numbered 1-9). Since these atoms are buried in the protein core and cannot make H-bonds with the water around the protein, they need to form H-bonds with each other. Note that there are un-bonded hydrogens on atoms 3 and 5, so this network is not completely satisfied—still, this network is over 80% satisfied, which is very impressive! The Shake tool is unlikely to find well-satisfied H-bond Networks on its own, and may require guidance from Foldit players. Pick Sidechains can help Foldit players build H-bond Networks.
Tips for using the Pick Sidechains
You can select more than one segment for Pick Sidechains, to view multiple rotamer clouds at once.
Some side chains (ASN, GLN, HIS, THR) have different atoms with different bonding abilities, but it can be hard to tell these atoms apart in the default Foldit view. If you have enabled "Show advanced GUI" in General Options, then you can change your View Options to one of the “CPK" Colors (like "Score/Hydro+CPK”). This will color oxygen atoms red and nitrogen atoms blue.
Some side chains (CYS, SER, THR, TYR) have a hydrogen that can be rotated to form hydrogen bonds in different directions. However, this hydrogen is hidden by default. In the View Options menu, change the View Hydrogens setting to “Show bondable hydrogens” to see these hydrogens in your protein. If hydrogens are visible, then Pick Sidechains will display extra rotamers so you can control the position of the hydrogen.bkoep 51 368 | Mon, 12/03/2018 - 19:49 | 5 comments )