Magnetic moments of dancing proteins in major Danish research project

There is nothing like overturning a scientific paradigm to get researchers to open their eyes.

For Birthe B. Kragelund from the Department of Biology at the University of Copenhagen, the great paradigm shift took place in about 2000, when the mapping of the human genome clearly showed that 30% of our proteins do not behave as described in the classical textbooks on molecular biology.

Instead of being folded into well-defined structures, which until then had been indisputable in any discussion on the form and function of proteins, they are disordered and dynamic like boiling strands of dancing spaghetti. A century of textbook understanding of proteins instantly vaporized.

The discovery also opened up a whole new field of research on these intrinsically disordered proteins (IDPs), and within the past 20 years, researchers have established that the IDPs are not just curiosities but play crucial roles in biology and in many diseases, including cancer and neurodegenerative diseases such as Alzheimer’s and Parkinson’s.

Researchers will have to learn to understand IDPs to conquer certain diseases.

“We will probably not be able to treat certain diseases until we find out how IDPs behave, and we are not there yet. But we have come a long way in the past 20 years. Early on, when I lectured on my research on IDPs, people asked many critical questions because their understanding of proteins was still based on conventional textbook wisdom. Fortunately, this is not the case today, and in the vast majority of fields, researchers recognize that some proteins behave very differently from others and that we simply do not understand why they behave the way they do and how they affect both poor and good health,” says Birthe B. Kragelund.

The Novo Nordisk Foundation awarded Birthe B. Kragelund a major Challenge Programme grant in 2018 to expand the understanding of the 30% of our proteins about which we currently know very little.

Proteins that do not behave in a structured way

IDPs differ from the rest of our proteins by being stunningly indifferent to structure and order.

For a century, textbooks described proteins folding into beautiful and precise structures.

The structures of proteins are directly linked to their functionality such that various active regions in a protein can interact with the environment by being precisely organized in a three-dimensional structure.

Enzymes are some of the most thoroughly studied proteins. For them to interact with other molecules in the cell, their three-dimensional structure must be organized such that their chemical side chains can link to the given binding partners in exactly the right places for the subsequent chemical reaction to take place.

In other words, a lock will only open if the protein key fits precisely.

“The beautiful thing is that when we know the structure of a protein, we can determine its functionality with relative certainty. It is like a chair. When we see a chair, no matter how it is designed, we figure out that we can sit on it,” explains Birthe B. Kragelund.

Unlike other proteins but still function

The process of “normal” proteins folding into structures is more or less automatic, at least for small proteins.

Proteins are large biomacromolecules comprising one or more chains of amino acid residues. These chains ensure that the proteins fold up and form a predetermined structure.

The chemical properties of the amino acids are key to this process. They include affinity for water or fat. When the amino acids move toward the centre or outside of the protein, the overall protein forms a structure in which the lipophilic (affinity for fat) amino acids predominantly face toward the centre of the protein and the lipophobic (fat-repelling) amino acids face outward.

Christian B. Anfinsen even received a Nobel Prize by showing that unfolding a protein from its structure makes the protein lose its functionality, but leaving the protein alone and allowing it to fold up again restores its lost functionality. The fold was encoded in the order of the amino acids in the chain.

“The initial assumption for the IDPs was therefore that they must be non-functional proteins, because how could they have function without structure? Early on, however, it turned out that these proteins have functionality, and that a loss of that function plays a role in the development of various diseases. It also turned out that IDPs can be very dynamic in binding to a binding partner, but how can they be both dynamic and bind as strongly as usual? And how do we explain specificity? The IDPs shattered our whole protein paradigm,” says Birthe B. Kragelund.

People have more IDPs than bacteria do

Researchers do not yet have a complete picture of what IDPs are and what they can do, but this is exactly what Birthe B. Kragelund is examining in her research.

First, the initial research into the IDPs has shown that they may well become more or less well-defined structures if they bind to another protein. In that case, part or all of the protein may fold up – not by itself, but by binding.

IDPs often bind to another protein through a motif comprising a short amino acid sequence of 3–11 residues in the IDP chain. This sequence binds to a surface or an indentation on a folded protein. The IDPs can have many motifs and thus arrange many other proteins in a larger overall protein complex.

Further, research has shown that the IDPs can interact much more with the environment than the structured proteins do.

“This means that we should consider the IDPs as a very special type of protein that serves very special roles in our cells. A protein with a well-defined structure can typically do one thing, and this might be equivalent to a bread knife in a kitchen drawer that only cuts bread. The IDPs, in contrast, can do many things and therefore correspond to a Swiss Army knife, with many possible functions depending on the binding partner and the needs of the cell,” explains Birthe B. Kragelund.

Research has also shown that higher organisms have more IDPs and therefore probably also need more proteins with multifunctionality.

For example, bacteria have very few IDPs, whereas researchers estimate that 30% of our proteins are multifunctional Swiss Army knives.

“Thus, this is not a small exceptional corner of our biology but a very large and significant part,” says Birthe B. Kragelund.

Well-known proteins are actually IDPs

The role of IDPs in making us human starts with transcription.

When DNA is translated into functional proteins, a major molecular machinery is initiated largely comprising proteins without a well-defined structure. Even the proteins that hold our DNA together, the histone proteins, have disordered regions.

“We are still determining why the transcription contains so many IDPs and why this is important” says Birthe B. Kragelund.

Another major role of IDPs is in relation to biological membranes.

Many membrane protein complexes comprise a protein with a well-defined structure on the cell surface that interacts with signal molecules from the environment. The part of the protein that penetrates the membrane also has a well-defined structure, but the part of the protein inside the cell is often wholly or partly an unstructured IDP.

Some of the best examples of this are the growth hormone receptors, which tell cells whether it is time to grow or not, and the prolactin receptor, which is important for developing breast tissue and producing breast-milk.

“We humans therefore delegate some absolutely essential functions to the IDPs, and we do this for a reason we do not yet fully understand,” says Birthe B. Kragelund.

Chemical modifications give proteins varied functions

A clue to greater understanding may lie in how cells regulate the functionality of IDPs.

Research has shown that the IDPs are very often tightly regulated through chemical modifications, including phosphorylation and methylation.

Phosphorylation and methylation are very simple phosphorus and methyl groups that are attached to a protein by enzymes to control its functionality.

They can usually be considered as the on and off buttons of the protein, but in the IDPs they can also act as gearboxes.

For example, phosphorylation can shut down an active area of the protein or turn it on so that the protein can bind to certain substances or other proteins, but the IDPs can have many more phosphorylation sites and thus more gears.

“This has to be very tightly regulated to ensure that misinformation and mis-signalling do not occur inside the cells. However, this also makes the IDPs multivalent, so the same protein can serve many functions depending on the levels of phosphorylation or methylation,” says Birthe B. Kragelund.

Mutations in one IDP can cause many types of cancer

An interesting example of a multifunctional IDP is tumour protein p53, which contains long disordered areas. These contain several small unstructured binding areas, and just one of these can create various structures, depending on which of the many partners the protein binds to.

Research on IDPs may well be clinically relevant. Birthe B. Kragelund also says that mutations in the disordered parts of p53 can cause many types of cancer.

Another example is that mutations in the disordered regions of the BRCA2 protein can lead to breast cancer development. Similarly, mutations in other parts of various intracellular IDPs are also linked to the development of breast cancer.

Mutations in the unstructured regions of the growth hormone receptor protein can also create unforeseeable health problems and lead to the development of cancer. A protein usually binds to the growth hormone receptor and signals that it needs to be sent for degradation so that the cells do not grow during the periods when growth is not needed.

However, a mutation in the receptor alters the dynamics of the disordered region so that the signalling molecules cannot bind so well to the receptor, and then the growth hormone receptor continues to signal growth, which is one of the clear characteristics of cancer. Research has shown that this can lead to the development of lung cancer.

However, cancer is just one negative effect. Neurodegenerative diseases such as Alzheimer’s and Parkinson’s can also be linked to problems with IDPs. The accumulation of amyloid beta is a characteristic of the development of Alzheimer’s and the accumulation of alpha-synuclein for Parkinson’s. Both are IDPs.

“Many diseases are caused by mutations in IDPs or defects in disordered parts of proteins. But developing medicine to treat the diseases is difficult if you do not know how the proteins behave, you don’t have a lock to block with an inhibitor” says Birthe B. Kragelund.

Major role in decoding the genome

Research on IDPs is an extensive field, since it affects about 30% of our proteins. Birthe B. Kragelund has therefore chosen several focus areas.

One is the proteins involved in translating the genome into functional proteins.

The DNA of the genome is wrapped around large protein complexes, including histones, and histones and DNA are linked by the linker histone H1 family of proteins.

When the DNA is wrapped tightly around the histones, the genetic machinery of the cells cannot easily access the genetic material, and then no proteins are expressed.

However, the cell has several possibilities to initiate the expression of proteins. One is that a small IDP, prothymosin-alpha, can pull the linker histone H1 away from the nucleosome, thus increasing the availability of the DNA.

The linker histone H1 has a very long tail of disorder and is extremely positively charged, whereas prothymosin-alpha is completely unstructured and extremely negatively charged.

Research from Birthe B. Kragelund’s group and her collaborators has shown that In addition, that prothymosin-alpha and histone H1 bind to each other with extreme affinity but maintain disorder while bound.

“Together with our collaborators in Switzerland and the United States, we published our results in Nature in 2018, and here we showed that both proteins remain completely unstructured and disordered, even when bound in a tight complex. We have also performed further analysis showing that, to be able to release each other again, the unstructured proteins use competitive substitution, in which two prothymosin-alpha proteins alternate between binding to one histone H1 protein. This is a way of regulating binding affinity and is important to bear in mind to understand the complex interactions between proteins in connection with translation,” explains Birthe B. Kragelund.

Developing new techniques to study IDPs

Overall, Birthe B. Kragelund’s research is three-legged.

• The role of dynamics in highly charged protein complexes and how they are regulated.

• The role of IDPs in transcription, in which DNA is translated first into RNA and then into proteins.

• The significance of disordered regions in membrane proteins.

To study the role of IDPs, the researchers use nuclear magnetic resonance spectroscopy, in which they use isotopes of various atomic nuclei to visualize the proteins and thus determine whether they are structured or unstructured and monitor their dynamics. Thus, they can follow the dance of the proteins.

The researchers can do this with the proteins when they are alone and do not form complexes with other proteins but also when they interact and perform their function.

Nuclear magnetic resonance spectroscopy has turned out to be an invaluable tool for studying IDPs, but the research project must also optimize and develop new experiments to investigate IDPs so that the researchers can get all the answers they want.

A separate problem is that the researchers cannot use many of the techniques that researchers normally use to study proteins structurally. These include X-ray crystallography and cryogenic electron microscopy, both of which require the proteins to be structured, which is not possible with these dynamic IDPs.

“We face the problem that we have to illustrate something that does not have a well-defined structure but is not random either. We will use nuclear magnetic resonance spectroscopy for this but also combine it with other methods, including single-molecule spectroscopy and computer simulation, to enable us to illustrate and understand how the chains behave both alone and when they interact with other proteins,” says Birthe B. Kragelund.

Expanding Denmark’s role as a leading country for protein research

The research is scheduled to last 6 years (2019–2024), and at the end of the project, Birthe B. Kragelund hopes that the researchers will have obtained such good understanding of the mechanisms behind the functionality of the IDPs that they can also determine what happens when things go wrong.

The very long-term perspective is being able to develop drugs to combat diseases, with drug development incorporating the knowledge obtained by Birthe B. Kragelund and her colleagues.

Some drugs may be able to target the IDPs themselves, and others can set up a scaffold around the protein complexes with which the IDPs engage.

“We will not develop the drugs ourselves, but we will create a foundation onto which drug manufacturers can build to combat diseases caused by errors in IDPs,” says Birthe B. Kragelund.

Birthe B. Kragelund adds that the research is also establishing Denmark as a leading actor in this field, which will attract more and more attention in the future.

“In Denmark, we now have a large body of research in this field, and we will inevitably train many talented researchers who can influence the agenda not only in Denmark but also internationally. The students we are educating right now are obtaining knowledge and expertise that they cannot get by simply reading textbooks. The major molecular biology textbook for universities mentioned IDPs for the first time about 5 years ago on just one page. This is remarkably little, since the IDPs comprise about 30% of our proteins. This research project gives us the opportunity to strongly influence research, because with this large grant we can achieve the critical mass to move things forward,” concludes Birthe B. Kragelund.

In 2018, the Novo Nordisk Foundation awarded a Challenge Programme grant to Birthe B. Kragelund for the project REPIN – Rethinking Protein Interactions.