So two days ago I celebrated my first paper ever to be published. For the entire 4 years of my PhD, my name has never been on anything and so this first paper means something very special to me. It marks the first time I have ever got a chance to contribute something, a small thing, to the knowledge body of the scientific community. But this blog isn't going to talk about what’s in the paper because you can read it here, and it’s good ;) https://www.cell.com/structure/fulltext/S0969-2126(20)30415-9
What I want to talk to you about today is the perk of being a scientist where you can make the most unexpected collaboration and a friendship that can never be replaced. It all started as a discussion between a few PhD students including me, my supervisor and a previous PhD student who was still in the lab then. We were talking about what to follow up from his project and it was all brainstorming and discussion at this point. We were discussing some hits that came up in a proteomic screen looking for the new binding interactors of CYRI-B and one of them was its cousin protein CYRI-A. It was a small but overlooked at the time. We didn’t fully understand what to make of this interaction. At the time, I was in my late second year and was just started to bite my teeth in working with this cousin protein CYRI-A. I said bite my teeth because CYRI-A is a protein that came from the deepest nightmare but more about it in another blog. So because CYRI-A came up in the screen, I thought to myself this could be an interesting angle to follow up and so I took on the challenge of determining why is there such dimerisation between CYRI proteins. It was a nerve-wracking decision because at the time, we didn’t have many tools to work with CYRI-A and we didn’t even know where this angle would lead to. But to me, it was interesting enough. And right at the beginning, I faced a fatal problem, the tag protein GST that we normally use for an assay called the pulldown assay to detect protein interaction didn’t work on CYRI-A. By “didn’t work” I mean it did not work at all. GST-tagged CYRI-A is completely insoluble in solution. We would purify tons of them from bacteria but the moment we centrifuged it down, the protein immediately precipitated and stuck in the pellet. For 3 months I was trying, and nothing worked, and I thought I was the reason, maybe I did something wrong, maybe the condition just not right, but again and again, I failed to even do the first step. That’s when I asked for help from a very senior person in the lab and we decided to test out all the different combinations of different conditions and different strains of bacteria. It was one of the biggest experiments I had to do and a part of me was hoping that at least one condition would work. But it was soon realised when we came to the final step that not a single one of the combinations produced any soluble protein, all of them produce the protein, but all the protein stuck in the cell pellet. It was a huge disappointment for me but at the same time, I finally knew that the tag we used would never work with CYRI-A. It saved me a lot of time tinkering with something that just wasn’t going to work. So at that point, I decided to change the tag into MBP and I was terrified because what if the second tag also not going to work? Working with such temperamental protein like this, there is no guarantee that this would work either. And so we did, cloning the first MBP-tag CYRI-A construct. I went back to the basic when it came to purify this tagged construct but there it was, I could see the protein in the solution! It was a surreal moment for me because up until this point, it was the first time I successfully cloned and purified CYRI-A and seeing it with my own eyes on the gel separated from the pellet was the best feeling ever. Of course, there was still a large portion of the protein in the pellet, but at least we could work with the rest of the protein in solution. But for the first time, I started to believe that this might just work, that all my effort might be paid off. On the day of doing the pulldown to first detect this dimerisation, it took me ages to make sure everything is in place for a smooth experiment. What I did was to stick the MBP-CYRI-A to beads, and then put the soluble GST-tagged CYRI-B in and hopefully, I could detect the signal. And yes, GST-CYRI-B is soluble, very soluble and it’s probably the easiest protein in the world to purify. It was an anxious 2-day experiment and the waiting for the result of the western blot. I was ready for the worst because the interaction might be too weak to even be detectable, but I was not disappointed, I saw it, the band that I have always wanted to see was right there on the screen. I repeated it multiple times and each and every time the band was still there. I was happy that I was right. Of course, the result was faced with a few criticisms, saying GST can also self-dimerise and might somehow influence the experiment. So, I went away and do again with untagged protein as well as another tag and it was then finally confirmed that the dimerisation does occur.
The next challenge was then to determine how this dimeric complex formed? What is the binding site of this complex? The difficult thing at the time was that we had no structural information about how the protein really looks like. All we had was a predicted structure and we had to make use of it as much as we could. I remember sitting down looking at the structure, trying to fit all the little details that we knew so far about the protein and how it worked together. I came up with a few ideas, but they were all quite complicated and perhaps overly thinking ideas. So the senior scientific officer came to me and suggested to think a bit simpler, you would never know you might be surprised. I took his advice and started to focus on what we knew, then by using a prediction software online to come up with a model of how the dimer would look and surprisingly it also predicts an interesting prediction, that this dimerisation site is similar with the site for Rac1 and CYRI would interact. We knew this site, it has been shown before composing of two conserved arginine residues so to test this would be very simple. We only needed to mutate these arginines into something else and redid the pulldown. If these residues are involved in the binding, we should see at least a decrease in the dimer signal. And that was exactly what we saw. For the first time, I actually got to taste the feeling of predicting something and then went on to prove it right. It was electrifying and I felt validated and more confident in my scientific ability than ever.
I wanted to take it a step further by measuring the affinity between the monomers but to do this, I would need someone with expertise in surface plasmon resonance. This is when the unexpected collaboration happens. Shehab, a group leader in the Beatson introduced me to his postdoc, Tamas, for help. My first impression when I saw him was a quiet person sitting at the end of the last cubicle. But then I sort of realising who he was. I saw him before, but we never talked. He was on the same train with me to work a few times, I remember him holding a giant black umbrella with his quiet and often relaxing pace of walking. His beard and his hair screamed out the typical scientist figure that you would see in a stock photo with a quick search on Google. He sat there with his headphone on focusing on some protein structure on the screen. I walked towards him just to say hi and wanted to tell him about the idea that I would want his help with. In my mind, I would imagine his voice to be quiet and calm like the typical voice of a professor, but no, it was clearer than I thought it was, like what you would hear from a confident MC on the stage, but with a touch of hesitation here and there. He said he would be happy to help me and that was our first of the many days of our collaboration. I remember guessing exactly where he was originally from based on his accent and I was so proud of it. We discussed a lot about the project, but also lab business and the future. It was these moments like this that helped us understand each other better and how each of us work. I guess I’m the bubbly and loud one while he’s more like a gentle giant, but for some reason, it worked. He taught me how to use the giant scary metallic machine to grind up cells, I’m still terrified of it til now. Through our conversations, we realise that no one has actually solved the structure of CYRI-B before. I mean for such an easy protein where you could purify “a potato of protein” as we usually said, it was a surprise that nobody has attempted to solve for its structure. So one thing leads to another, we all agreed that he should go for it. It was a nerve-wracking time because we realised that we’re not the only one in this race and that other labs were also working on the same thing. But we soon realised that it wasn’t the structure of CYRI-B that was the aim, but it was the structure of the complex between CYRI-B and Rac1 that was more important. Tamas was a competent crystallographer and by using his knowledge and a few tricks up his sleeves, he was able to optimise and solve the complex structure in just 3 months. It was absolutely incredible and the first time seeing the structure of the complex was quite a moment to remember for all of us in that room. It was also from his structure, that we saw the dimer for the first time, not as a predicted structure, but a real one.
Combining the data I had and Tamas’s, we found we could really fit them together. Rac1 binds to CYRI, CYRI dimerise with another CYRI so what if this dimerisation is what inhibited Rac1 binding? We then decided to do a competition assay, where we let CYRI molecules to dimerise, and then slowly introduced Rac1 in with an increasing concentration. If the two compete for the same binding site, we should see the decrease in the dimer signal. And that was exactly what we saw. It was a concrete experiment that answered us a question that we have always wondered. And the rest was history.
I've always been so proud of this graphical abstract :) There you can see the dimer and the CYRI-Rac1 complex at the top.
I never thought I was going to be exposed to the world of crystallography and structural biology. I was trained as a biochemist but felt in love with cell biology. But it was a fresh breeze of air to learn something completely unexpected and completely new to me. I was only taught this at uni, but to be able to actually do it, design the experiments to test it, examining the structure and forming hypotheses, it was everything. We started this project with little to nothing, it was a curiosity-driven project and we never expected to be able to show the world what we have achieved. For many, starting a completely new project is terrifying, and it really is. But just by keep moving forwards, even if it’s step by step, you will eventually get there. Maybe your eureka moment will be near, maybe it is not until you almost give up, but if you don’t keep moving forward, you will never discover it. This project has also taught me the importance of collaboration in science. It can start small, a seed, an idea, but with the combined effort of everyone involved, we could grow it into a tree. And more importantly, you get to meet new people, friends that never thought you would, you get to learn from them the things you never thought you would ever have a chance to. So to all of those PhD students out there who are struggling to find your feet, never forget that by always moving forward and by asking for help, you are already making progress!
Until next time :)