I couldn’t decide whether to write this blog post. However, I sifted through the archives of BLOPIG and found in the original post this excerpt:

“And if your an athlete, like Anna (Dr. Lewis) who crossed the atlantic in a rowing boat or Eleanor who used to row for the blues – what can I say, this is how we roll, or row [feeble attempt at humour] – thats a non-scientific but unique and interesting experience too (Idea #8).  .”

Therefore I’ve decided that it might be an interesting post to look into what life is like when you are studying for a DPhil and also training for the blues. Rowing in particular is a controversial sport – I have heard of many stories advocating that rowing will be the absolute detriment to your DPhil. I’ve never felt pressured as part of OPIG to give up rowing – all of my supervisors have been very fair, in that if I get the work done then they accept this is part of my life. However, I realise all supervisors are not so understanding. I hope this blog post will give some insight into what it is like to trial for a Blues sport (in this case Women’s Lightweight Rowing), whilst studying for a DPhil at Oxford.

4:56 am – Alarm goes off. If its after September it’s dark, cold and likely raining. No breakfast as I will do the first training session fasted – just get dressed and go!

5:15 am – Leave the house with a bag full of kit, food for the day, laptop and papers to cycle to Iffley Sport’s Centre

5:45 am – Lightweight Women’s minibus leaves from Iffley to drive to Wallingford. Some girls try to study in the bus, but to be honest its too dark and we’re all a bit too sleepy.

6:15 am – Arrive at Wallingford. Get onto the water for a session in the boats. Although in the Boat Race we race in an 8 (8 rowers with one oar each, with a cox steering), we spend lots of time in different boats throughout the season. Perhaps unlike our openweight counterparts, we also do a lot of sculling (two oars per rower) as the only Olympic class boat for lightweight women is a sculling boat. We travel to Wallingford for a much longer, emptier stretch of river and normally get to see the sunrise.

8:10 am – We leave Wallingford to head back to Oxford. Start waiting in A LOT of traffic once you hit the ring road, and there’s a lot of panic in the bus about whether 9 am lectures will be made on time!

8:50 am – Arrive back at Iffley Sport’s Centre. Grab bike and cycle to the department.

9:00-9:15 am – Arrive at the Department. Quick shower to thaw frozen fingers and to not repulse my fellow OPIG members. I then get to eat warm porridge (highlight of the day) and go through my emails. I also check whether any of my jobs have finished on the group servers – one of the great perks of being in OPIG is the computational resources available to the group. Check the to-do list from yesterday and write a to-do list for today and get to work (coding, plotting results, reading papers or writing)!

11:00 am (Tuesdays & Thursdays) – Coffee morning! Although if it’s any time close to a race no bourbon biscuits or cake for me. This is a bit of an issue because at OPIG we eat a lot of cake. However, one member can usually be relied upon to eat my portion..

1:00 pm – Lunchtime! As a lightweight rower I am required to weigh-in at 59kg on the day of the Boat Race. If I am over that weight I don’t get to race. Therefore, I spend a portion of the year dieting to make sure I hit that target. The dieting lunch consists of soup and Greek yogurt. The post race non-dieting lunch consists of pasta from Taylors, chocolate and a Coke (yum!). OPIG members generally all have lunch at this time and enjoy solving the Times Cryptic Crossword. I’m not the best at crosswords so I normally chat to Laura and don’t concentrate.

2:00 pm – Back to work. Usually coding whilst listening to music. I normally start rushing to be able to submit some jobs to the group servers before I have to leave the office.

3:00 pm – Go to get a chocomilk with Clare. A chocomilk from the vending machine in our department costs 20p and is only 64 calories!

5:30 pm – Cycle to Iffley Sports Centre for the second training session of the day.

5:45 pm – If it’s light enough we hop in the minibus to go to Wallingford for another outing on the water. However, for most of the season its too dark and we head to the gym. This will either consist of weights to build strength, or we will use the indoor rowing machine (erg) to build fitness. The erg is my nemesis, so this is not a session I look forward to. Staring at a screen that constantly tells you how hard you are pushing, or if you are no longer pushing as hard I find to be psychologically quite tough. I’d much rather be gliding along the river.

8:35 pm – Leave Iffley after a long session to head home. Quickly down a Yazoo (strawberry milk) to boost recovery as I won’t be eating dinner until 45 minutes to an hour after the end of the session.

9:00 pm – Arrive home. I “cook” dinner which when I’m dieting consists of chucking sweet potato and healthy sausages from M&S in the oven while I pack my kit bag for the next day.

9:30 pm – Wolf down dinner and drink about a pint of milk, whilst finally catching up with my boyfriend about both our days.

10:00 pm – Bedtime at the latest.

Repeat!

In my last presentation, I talked about the article by King et al. describing a method for computationally removing T-cell receptor epitopes from proteins. The work could have significant impact on the field of designing protein therapeutics, where immunogenicity is a serious obstacle.

One of the major challenges when developing a protein therapeutic is the activation of the immune system by the drug and subsequent production of antibodies against it, rendering the therapeutic ineffective. This process is known as immunogenicity. Immunogenicity is triggered by T-cells recognition of peptide epitopes displayed on the MHC (major histocompatibility complex). This recognition can be impeded by designing the protein therapeutic to remove the potential T-cell epitopes from its surface. There has been some success in experimental T-cell epitope removal, but the process remains resource and time consuming.

In this work, King et al. created a function which assigns to each residue a score that measures its propensity to be a part of a T-cell epitope. The score consists of three parts. The first part is based on a SVM (Support Vector Machine) score calculated over each 15-residue long window, that attempts to predict how likely is the corresponding peptide sequence to bind the MHC. The SVM has been trained on the available immunological data from the Immune Epitope Database (IEDB). The second part of the score is calculated on each 9-residue window and compares the frequency of the 9-mer in the host genomic data and in the known epitope data (a sequence occurring with a high frequency in a human genome would be rewarded while the opposite is true for sequences occurring in the known epitope data). The third part penalizes any deviations from the original charge of the protein. These three parts are combined with a standard Rosetta score that measures the stability of the protein. The weights assigned to each segment were calibrated on existing protein structures. The combined score would be used to score the mutations in the sequence of the protein of interest, according to their propensity of reducing immunogenicity. The top scoring mutations would then be combined in a greedy fashion.

The authors tested their method on fluorescent reporter protein superfolder GFP (sfGFP) and the toxin domain of the cancer therapeutic HA22. In the case of sfGFP the authors targeted the four top-scoring T-cell epitopes. They created eight different proteins designs, out of which all preserved the function of the original protein (fluorescence). The authors selected the top scoring design for experimental immunogenicity testing. The experiments have shown that the selected design had a significantly reduced immunogenicity in comparison to the original protein. In the case study of HA22 the authors created five designs, out of which three displayed cytotoxicities at the same level or higher than the original protein. The two most cytotoxic designs were further characterized experimentally for their propensity to induce immune response. The authors have found that the two mutants elicited a significantly reduced T-cell response.

Figure 1: Reduction of immunogenicity without loss of function. A) Three of the five designs show cytotoxicity at the same level or higher than the original protein. B) Two of the three cytotoxicity-preserving designs show reduced immunogenicity

Overall, this very interesting study showed that computational methods can be successfully used for reducing immunogenicity of protein therapeutics, opening new avenues for computational protein design.

This blogpost is be about the “Computational design of an epitope-specific Keap1 binding antibody using hotspot residues grafting and CDR loop swapping” by Liu et. al. that I presented at group meeting in May.

Antibody design is a subject that I am closely interested in, especially methods that have an important computational step. So far the go-to methods for designing an antibody used by industry are animal model immunisation and/or phage display, with little or no use of computational methods. In the past few years, however, a few computational methods for rational design of antibodies have been making a showing. Firstly, there are the ones where a structure of the docked antibody-antigen already exists, and the antibody is further refined computationally to increase binding affinity. Then there are the ones where the paratope of the antibody is proposed by the designer against a specific target. The paper I am summarizing here by Liu et. al follows the latter idea in a neat way.

Liu et. al. show that if a specific motif is important for binding a certain target, i.e. there is a crystal structure which shows that the motif is buried in the target and/or you predict that its residues are important for binding, it is worthwhile trying to graft that that motif in the CDR area of antibody (the one which is responsible for antibody specificity and affinity). Grafting of entire CDR loops has been long used for antibody humanisation, with many examples where CDR loops maintaining conformation and binding specificity when being transferred from a non-human scaffold to a human scaffold. This is somewhat  aided by the fact that the starting and end points of the area being grafted is stable (i.e. the anchors are  conformationally the same in all the antibody structures that we observe), which is not the case in Liu et al where they graft a four residue motif. The cool thing they do which makes it more probable for the motif to maintain conformation is identify an antibody which has in one of its CDR loops a fragment with the same backbone conformation with the motif they are trying to graft.  They then just replace the residue types to the ones that are known to bind the target. For the Nrf2 motifs (that binds Keap1) they managed to create 5 potential designs. These were further expanded, using rational point mutations on the rest of the antibody in order to increase possibility of binding, to 10. Out of the 10 two showed binding.

One of the potential issues in a real scenario however is the fact that not an entire binding site is copied on antibody, the motif being a subset of the whole, which means the possibility of a low affinity and/or low chances of competing with the original protein (i.e. Nrf2) from which the motif was copied. This actually turned out to be the case, with the initial designs showing low mM affinity. Liu et. al. further worked on improving the initial designs, and they did so by computationally swapping the H3 CDR of the initial designs to a set of other H3 structures that have been seen in other solved antibodies using the Rosetta design protocol. They retained the ones that had a predicted buried SASA of > 2000 A^2, a change in energy of more than 20 REU and a shape complementarity greater than 0.6. These were then tested experimentally with a few of them showing nM affinities, a result which at this time should make you very happy if your entire design phase was done computationally.

Some concepts in science are counter-intuitive, like the Monty Hall problem or the Mpemba effect. Occasionally, this is also true for terminology, despite the best efforts of scientists to ensure that their work can be explained unambiguously to newcomers. Specifically, in our field of protein structure prediction, the word “decoy” has been used to mean one of many conformations generated by a de novo modelling protocol such as Rosetta, or alternative conformations of loops produced by an ab initio program e.g. Sphinx. Though slightly baffled by this usage when I started working in the field, I have now become so familiar with its strange new meaning that I have to remind myself to explain it in talks to a more general audience, or simply aim to avoid the term altogether. Nonetheless, following a heated discussion over the term in a recent group meeting, I thought it would be interesting to trace the roots of the new meaning.

Let’s begin with a definition from Google:

decoy

So we start with the idea of something distracting, resembling the true thing but with the intent to deceive. So how has this sense of the word evolved into what we use now? I attempted to dig out the earliest mention of decoy for a computationally generated protein conformation with a Google scholar search for “decoy protein”, which led to the work of Thomas and Dill published in 1996. Here the authors describe a method of distinguishing the native fold of a protein from the sequence threaded, without gaps, onto alternative structures from the PDB. This problem of discrimination between native and non-native had been carried out previously, but Thomas and Dill chose to describe the alternatives as “decoy conformations” or just “decoys”.

A similar problem was commonly attempted over the following years, of separating native structures from sets of computationally generated conformations. Due to the demands of conformer generation at this time, some sets were published themselves in online databases to be used as a resource for training scoring functions.

When it comes to the problem of de novo protein structure prediction, unfortunately it isn’t as simple as picking out the correct answer from a population of incorrect answers. Even among hundreds of thousands of conformations generated by the best methods, the exact native crystal structure will not be found (though a complication here that the protein is dynamic and will occupy an ensemble of native conformations). Therefore, the aim of any scoring function in structure prediction is instead to select which incorrect conformation is closest to the native structure, hoping to obtain at least the correct fold.

It is for this reason that we move towards the idea of choosing a model from a pool of decoys. Zhu et al. (2003) use “decoy” in precisely this way:

“One strategy for ab initio protein structure prediction is to generate a large number of possible structures (decoys) and select the most fitting ones based on a scoring or free energy function”

This seems to be where the idea of a decoy as incorrect and distracting is lost, and takes on its new meaning as one of a large and diverse set of protein-like conformations, which has continued until now.

So is it ever helpful to refer to “decoys” as opposed to “models”? What is communicated by “decoy” that is not achieved by using the word “model”? I think this may come down to the impression which is given by talking about a pool of decoys. People would not generally assume that each decoy on its own has any effective use for prediction of function. There is a sense that this is not the final result of the structure prediction pipeline, there is work yet to be done in refining, clustering, and making human judgments on the suitability of the output. Only after these stages would I feel more comfortable using the word “model”, to express the greater confidence we have in the structure (small though that may be in the de novo structure prediction world). However, the inadequacy of “model” does not alone justify this tenuous usage of “decoy”. Perhaps we could speak more often about populations of “conformations”. In any case, “decoy” is widespread in the community, and easily understood by those who are most likely to be reading, reviewing and editing the literature so I think we will be stuck with it for a while yet.