Design of protein-protein interfaces

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moody
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Message 75771 - Posted: 18 Jun 2013, 18:30:21 UTC

To our awesome Rosetta@Home contributors:

At the beginning of 2012 we told you that it looked like eight designed proteins stuck to EED as desired. Further experiments showed us that of our eight initial hits, only two were real, and those two proteins were too weak to be improved with laboratory techniques. We've gone back to the drawing board, using more aggressive modeling techniques to design better binding proteins to mimic Ezh2. It turns out that no known protein structure is similar enough to the Ezh2 binding helix to allow us to simply transplant key amino acids from Ezh2 to a new host protein, as we tried before. What were doing now is making new proteins from scratch that have the exact shape that we need to mimic Ezh2. The "EED" runs you'll now see on Rosetta@Home are structure prediction of designs to see if they have the desired curvature when Rosetta folds them up. Thanks again for all of your donated computer time!

For those new to this thread/message board, here is what I wrote previously about the EED project:

The second of these interactions involve a protein called EED and another called Ezh2. If you think about DNA as a ladder, then each of our cells has about six billion rungs worth of ladder. In order to keep all of that DNA from getting hopelessly tangled, our cells keep it on little spools, called histones, when not in use. Think of this like a massive film archive. It turns out that there is a lot of DNA that never gets used, so the cells put special flags on those spools (histones) that say something like "never use this section of DNA". EED is a large protein that attaches to those flags and brings along Ezh2, a "flagging machine" for the ride. EED makes sure that Ezh2 adds flags to histones that are supposed to get them, and not to histones carrying DNA the cell actually wants to use. Some cancer cells reduce the amounts of EED or Ezh2 to prevent flagging of DNA regions that they want to use to take over our bodies. Other cancer cells expand the amounts of EED or Ezh2 to flag DNA regions that are getting in the way. Scientists are working hard to better understand how EED, Ezh2, and friends work in normal cells and what goes wrong in cancer cells. Some are trying to develop drugs that prevent Ezh2 from attaching to EED. This means that the drugs have to stick to EED more tightly than Ezh2. Although Ezh2 attaches relatively loosely to a large patch on the surface of EED, their aren't any drugs yet available that do what we want.

I'm trying to make proteins that will do the same job. I used Rosetta@home to design a set of proteins that mimic Ezh2 in order to block it from attaching to EED. Just to give you a sense of how much computing I need for a project like this, I submitted just under two million work units to Rosetta@home, and donor's computers ran my protocol just under a billion times to give me about 54 designs to look at, from which fourteen were suitable for testing. This took about a month to run on Rosetta@home. From initial experiments, it looks like eight of the designs stick to EED, and one in particular sticks three times better than the Ezh2 found in our cells. Now I'm working to improve the best design at the lab bench and hope to send it to co-workers for testing in living cells. There's still a lot of work to be done to make sure that everything is working right with this design, but I want to give a big thank-you to all of you who donated your computer time to make this possible! For more information about EED, Ezh2, and related proteins, please visit:







Work units for this project carried the word "EED" in their name.
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Message 75781 - Posted: 20 Jun 2013, 10:31:35 UTC

Thanks for this totally inspiring update (I'm a recent returnee to R@H after a spell on another project).

While reading your update, my over-riding thought is how amazing it is that we (i.e. you, the scientists, or the human race in general) now know all this incredibly complex stuff, that wasn't known a few years ago. Whenever I read these updates about the mechanisms of proteins, bindings, targets, interactions, etc, I'm in awe of the scientific process - and every little new thing that is learned can never be taken away from us; it's a step on the road that will never be undone. I really believe that complete cures will be found one day, and it'll be because of work like this.

Told you I found it inspiring. Thanks again for the update.
Alver Valley Software Ltd - Contributing ALL our spare computing power to BOINC, 24x365.
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Message 75987 - Posted: 28 Aug 2013, 19:45:17 UTC - in response to Message 75635.  

To all of our wonderful Rosetta@Home contributors,

Here is an update about the p53-Mdm4 project: Using Rosetta@Home, we identified a set of 14 proteins that could be modified to stick to Mdm4 while ignoring Mdm4. We synthesized these proteins, tested them, and identified one that sticks to Mdm4 about 75 times better than it sticks to Mdm2. We sent this protein, called Mdm4 Binder 17 (MB17) to St. Jude's Childrens Research Hospital so the cancer experts could test it in cancer cells. So far, they have confirmed that MB17 strongly prefers to stick to Mdm4 over Mdm2 (about 170 times better, in their experiments). They also found that MB17 works correctly inside living cells, a major milestone for a designed protein. In the mean time, we've created new versions of MB17 that preference Mdm4 even more (about 370 times more than Mdm2), some that pinch-hit and prefer Mdm2 instead of Mdm4 (about 120 times more than Mdm4), and some that like both Mdm4 and Mdm2 equally well. The folks at St. Judes are starting to work with these improved variants as well. This is the first time that the cancer research community has ever had a tool to knock out just Mdm4 while leaving Mdm2 alone, so naturally, the folks at St. Jude's are pretty excited. They are gearing up to test MB17 in real cancer cells so we'll keep our fingers crossed. We'll keep you updated as new developments arise. I'm sorry for the long delays between posts. Things are pretty busy around here. For those of you seeing this thread for the first time, I've re-posted below what I posted previously about this topic:

Project 1:
The first of these interactions involves a protein called p53 and another called Mdm4. p53 is a communication hub in our cells, used to translate information about unwanted DNA mutations into an effective response by the cell. The activity of p53 is modulated by a pair of other proteins, Mdm4 and Mdm2, which act to shut off p53 when all is well. Cancer cells depend on DNA mutations to stay alive and so they find ways to shut off p53, either by reducing the amount available p53 or by expanding the amount of available Mdm4 or Mdm2. that way p53 doesn't rat out mutations that the cancer might need to survive. Scientists have spent a lot of time trying to understand how Mdm4 and Mdm2 work. They do this by shutting off Mdm2 and Mdm4 with drugs. There are even drugs that will shut off just Mdm2. There are no drugs, however, that will shut off just Mdm4. So I'm trying to make one. This is a tricky process since Mdm2 and Mdm4 are very similar, making it hard for proteins to tell them apart.

I aim to use Rosetta@home to design proteins that will attach to Mdm4 while ignoring Mdm2. I hope to provide a research tool to scientists that work on Mdm4, Mdm2 and p53, allowing them to better understand how this critical communication hub works. I hope that this leads to new cancer treatments. So far, I've used Rosetta@home to design 26 proteins that should attach to Mdm4 but not Mdm2. Many of the designed proteins do stick to Mdm4, but unfortunately, they also stick to Mdm2. I am working on a third round of design using a new approach that I hope will work. For more info about the proteins involved in this project, please visit the links below:


They are gearing up to test MB17 in real cancer cells so we'll keep our fingers crossed. We'll keep you updated as new developments arise.


Any news if the MB17 worked on the cancer cells?

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Message 75988 - Posted: 28 Aug 2013, 20:59:32 UTC - in response to Message 56754.  
Last modified: 28 Aug 2013, 21:11:55 UTC

Hello,

My name is Sarel Fleishman and I've been a postdoc in the Baker lab for the past two years. My project deals with structure prediction and design of protein-protein interfaces and you may have read a few messages from me on CAPRI and structure prediction. Now, I'm very excited to tell you that with minirosetta v1.40 we can do design work using the massive computational power of Rosetta @ Home.

A few words on protein-interface design. The primary challenge in this field is to be able to take a protein target and to design another protein that would bind to it in a specific way. Nature provides us with hundreds of thousands of examples of such protein-binding events. Such events are used for the amplification of signals within and between our cells in processes related to growth and development as well as for recognition, e.g., in the immune system. When such signals go awry, protein interactions become the center of events for uncontrolled cell growth, or cancer. Many pathogenic bacteria and viruses hijack molecular recognition processes to promote their growth and proliferation causing sickness and endangering lives.

These processes being so central to both health and disease it is hardly surprising that being able to manipulate them computationally is a major ongoing goal of molecular biology. Being able to target a protein and bind to it would open the way to novel therapies for a large number of diseases.

We have selected a small number of protein targets for which we want to design protein binders. As an example, one such target that I have been working on is cholera toxin. This protein is a crucial component of the process by which the cholera bacterium causes cells in the gut to excrete large amounts of water, which causes death from dehydration (see the following wiki page for more details: http://en.wikipedia.org/wiki/Cholera). We have developed a computational strategy that allows us to design proteins to bind to the cholera toxin and disable it.

We are now in the process of testing this and similar design methodologies on a number of other targets. But expanding the number of targets we quickly realized that we need a lot more computational resources to adequately address this problem. This is why we have turned to Rosetta @ Home and to you for your help in this exciting project.

The simulations that you will see in protein interface design will be quite different from one another. In each case we tailor the design strategy to the particular protein target, stressing, for instance, the formation of interactions with a specific key region on the protein surface. In general, though, the simulations will involve docking steps, where the protein binder moves with respect to the target and design, where amino acid residues on the surface of the protein binder change in order to better attach to the target. Promising protein designs are synthesized in our lab and tested for binding to the target protein.

I am working on this project with my colleagues Eva and Jacob, and for each target that we test using Rosetta @ Home we will provide background material on the target and why we selected it on this thread.

These design simulations tend to use more memory than many prediction runs (typically at most 800Mb). We will test different ways of reducing this memory load so that our simulations could run on all participating computers in the Rosetta @ Home project, but initially we will only run these simulations on computers that can handle this memory restriction. Please report any problems that you might have with this simulation.

Thank you very much for participating in this project! I'm looking forward to getting feedback and results from you.
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Message 76458 - Posted: 19 Feb 2014, 23:32:24 UTC

Hi all - as the IPD/Baker lab technical writer, I will be regularly updating R@home message boards with research updates. I've created a new thread for this (R@h Research Updates) but will also be trying to update project-specific threads such as this one!

Computational Design of an Enzyme-Based Protein Inhibitor

Computational design of protein-protein interactions to generate new binding proteins for any specified site or surface of interest on a target protein can lead to a number of novel therapeutic and biochemical tools. As an example, in recent work by Sarel and collaborators, novel proteins have been designed to bind to a conserved epitope on influenza hemagglutinin.

Computational design of a protein that binds polar surfaces, however, has not been previously accomplished. In a September 2013 paper published in the Journal of Molecular Biology, Procko et al describe the computational design of a protein-based enzyme inhibitor that binds the polar active site of hen egg lysosome (HEL). A hot spot design approach first identified key, conserved interaction residues that contribute to much of the binding energy to HEL within a large interface. Rosetta software then identified a protein scaffold that supported the hot spots while also optimizing contact with surrounding surfaces to obtain a high affinity protein binder.

Follow this this link to read more about this exciting work: http://depts.washington.edu/bakerpg/drupal/Computational-design-of-a-protein-based-enzyme-inhibitor-pub
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Message 76472 - Posted: 20 Feb 2014, 22:46:26 UTC - in response to Message 76458.  

Awesome news :)

Does IPD = Institute for Protein Design?
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Message 76481 - Posted: 21 Feb 2014, 21:08:02 UTC - in response to Message 76472.  

Awesome news :)

Does IPD = Institute for Protein Design?


Yes! IPD is the Institute for Protein Design. You can find more research updates in the news section of the IPD's website here:

http://depts.washington.edu/ipd/
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Message 76584 - Posted: 3 Apr 2014, 17:48:58 UTC - in response to Message 75987.  

Tom,

I apologize for the long delay in getting back to you. The top MB17 variants bind Mdm4 (Mdmx) or Mdm2 tightly enough to yank them out of human cells and trigger the self-destruction pathway (p53 apoptosis pathway) in colon cancer cells. We're now looking at other types of cancer cells. We're now working on making the MB17 variants bind more tightly to Mdm4 or Mdm2. We're also studying how the p53 pathway works using the MB17 variants. The work is on-going so we don't have the full story yet. MB17 and its variants were intended to aid as cancer research tools and we're cautiously optimistic that they'll work to that end. We're hoping that the things we learn will fuel the development of new kinds of cancer drugs and treatment strategies. You likely have recently seen Rosetta@Home jobs with either Mdm4, Mdm2, or MB17 in the name. These represent ongoing efforts to design improved Mdm4 and Mdm2 binding proteins using new tools in the Rosetta software suite.

Once again I want to give a big thank-you to you and all of our Rosetta@Home contributors!
_______

[quote]To all of our wonderful Rosetta@Home contributors,

Here is an update about the p53-Mdm4 project: Using Rosetta@Home, we identified a set of 14 proteins that could be modified to stick to Mdm4 while ignoring Mdm4. We synthesized these proteins, tested them, and identified one that sticks to Mdm4 about 75 times better than it sticks to Mdm2. We sent this protein, called Mdm4 Binder 17 (MB17) to St. Jude's Childrens Research Hospital so the cancer experts could test it in cancer cells. So far, they have confirmed that MB17 strongly prefers to stick to Mdm4 over Mdm2 (about 170 times better, in their experiments). They also found that MB17 works correctly inside living cells, a major milestone for a designed protein. In the mean time, we've created new versions of MB17 that preference Mdm4 even more (about 370 times more than Mdm2), some that pinch-hit and prefer Mdm2 instead of Mdm4 (about 120 times more than Mdm4), and some that like both Mdm4 and Mdm2 equally well. The folks at St. Judes are starting to work with these improved variants as well. This is the first time that the cancer research community has ever had a tool to knock out just Mdm4 while leaving Mdm2 alone, so naturally, the folks at St. Jude's are pretty excited. They are gearing up to test MB17 in real cancer cells so we'll keep our fingers crossed. We'll keep you updated as new developments arise. I'm sorry for the long delays between posts. Things are pretty busy around here. For those of you seeing this thread for the first time, I've re-posted below what I posted previously about this topic:

Project 1:
The first of these interactions involves a protein called p53 and another called Mdm4. p53 is a communication hub in our cells, used to translate information about unwanted DNA mutations into an effective response by the cell. The activity of p53 is modulated by a pair of other proteins, Mdm4 and Mdm2, which act to shut off p53 when all is well. Cancer cells depend on DNA mutations to stay alive and so they find ways to shut off p53, either by reducing the amount available p53 or by expanding the amount of available Mdm4 or Mdm2. that way p53 doesn't rat out mutations that the cancer might need to survive. Scientists have spent a lot of time trying to understand how Mdm4 and Mdm2 work. They do this by shutting off Mdm2 and Mdm4 with drugs. There are even drugs that will shut off just Mdm2. There are no drugs, however, that will shut off just Mdm4. So I'm trying to make one. This is a tricky process since Mdm2 and Mdm4 are very similar, making it hard for proteins to tell them apart.

I aim to use Rosetta@home to design proteins that will attach to Mdm4 while ignoring Mdm2. I hope to provide a research tool to scientists that work on Mdm4, Mdm2 and p53, allowing them to better understand how this critical communication hub works. I hope that this leads to new cancer treatments. So far, I've used Rosetta@home to design 26 proteins that should attach to Mdm4 but not Mdm2. Many of the designed proteins do stick to Mdm4, but unfortunately, they also stick to Mdm2. I am working on a third round of design using a new approach that I hope will work. For more info about the proteins involved in this project, please visit the links below:


They are gearing up to test MB17 in real cancer cells so we'll keep our fingers crossed. We'll keep you updated as new developments arise.


Any news if the MB17 worked on the cancer cells?
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Message 76599 - Posted: 8 Apr 2014, 13:34:26 UTC

Thanks for the update: always good to see our crunching has practical application.
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Message 77641 - Posted: 11 Nov 2014, 12:05:09 UTC - in response to Message 76584.  

The top MB17 variants bind Mdm4 (Mdmx) or Mdm2 tightly enough to yank them out of human cells

Hi moody, that sounds very exciting - can you expand a bit on how exactly this occurs?
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Message 80390 - Posted: 18 Jul 2016, 10:27:03 UTC

Hi all!

Sorry if this is not a right thread to post it, but...

Will this article be useful in rosetta's calculations methods?

http://www.pnas.org/content/early/2016/07/12/1603929113.abstract


From Siberia with love!
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Message 80413 - Posted: 22 Jul 2016, 12:18:28 UTC - in response to Message 80390.  

Will this article be useful in rosetta's calculations methods?

It looks like something you might be able to do on a GPU.

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Message boards : Rosetta@home Science : Design of protein-protein interfaces



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