Intrinsically disordered proteins are a special type of proteins that challenge scientists’ understanding of what proteins can and cannot do. Researchers have now discovered more about them, providing new insight into the origins of life. The proteins also open the possibility of designing more effective drugs in a novel way.
Intrinsically disordered proteins do not appear to follow the rules all other proteins follow. They have baffled scientists for years because they are so different.
Researchers have now discovered that intrinsically disordered proteins can bind to other proteins regardless of whether they are left- or right-handed.
The discovery provides insight into the very earliest life on Earth and suggests how to design better drugs.
“At some point in the distant past, selection took place that preferred left-handed proteins over right-handed proteins, and we think that this is related to intrinsically disordered proteins. Because of this difference, we can use this new insight to work towards designing new types of drugs with a much longer half-life without breaking down,” explains principal investigator behind the study, Birthe B. Kragelund, Professor at the Department of Biology, University of Copenhagen, Denmark.
The research, which was carried out in collaboration with Estella Newcombe, Amanda Due, Johan Olsen and others, has been published in Nature.
Behaving unlike other proteins
Most known proteins behave in a specific way: for example, by folding up into very precise structures that give them a shape reflecting their function.
This very well-organised and precise structure is the entire basis for how proteins interact with their molecular surroundings. Drugs in general act by recognising and binding a protein target. Side-effects often arise when a drug binds to other proteins. That is why drug design and the detailed structure of target proteins are intimately connected to ensure high affinity and high specificity. This reduces side-effects and boosts efficiency.
Researchers have only become aware of intrinsically disordered proteins in the past 25 years. Unlike all other proteins, they are like cooked spaghetti in a hot tub. They have no well-defined three-dimensional structure but are a dynamic morass, and researchers have been puzzled about how they interact with their molecular surroundings.
Research on intrinsically disordered proteins over the past 20 years has revealed that they are not just curiosities but take care of many regulated cellular processes and have crucial roles in developing cancer and neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease. And they make up more than 25% of your proteome.
“Some of these proteins behave very differently from other proteins, and we have no idea why and what the influence is on disease and health,” says Birthe B. Kragelund.
All life has handedness – proteins are left-handed
Birthe B. Kragelund and colleagues launched the new study to understand the difference between ordered proteins and intrinsically disordered proteins. How are molecular interactions involving disordered proteins affected by chirality?
Proteins are built from amino acids – both l-amino acids and d-amino acids – and this determines handedness. The l means levorotatory and the d means dextrorotatory.
Proteins built from l-amino acids have one form, and proteins built from d-amino acids take the mirror-image form. The right-handed and left-handed “biological worlds” they each represent cannot interact. Or so we thought.
All life on Earth is chiral, meaning that all molecules that cannot overlap with their own mirror image (just like human hands) exist in only one form. Amino acids are always left-handed (l).
The left-handedness analogy corresponds to life having decided that all proteins shake hands with the left hand. Imagine a world in which half of us shook hands solely with the right hand and the other half solely with the left!
The fact that proteins have handedness makes them picky: when they interact with other chiral molecules (be it proteins or sugars or whatever), they can only bind one handedness and are completely blind to the other.
“However, we did not know how sensitive intrinsically disordered proteins are to chirality, since they do not fold into ordered three-dimensional structures. In nature, some protein complexes consist of pairs of disordered proteins, and in this study, we aimed to elucidate how chirality affects these interactions between disordered proteins,” explains Birthe B. Kragelund.
Investigating how chirality affects intrinsically disordered proteins
The researchers carried out many experiments investigating how intrinsically disordered proteins, built from either l-amino acids or d-amino acids, bind to proteins built from l-amino acids.
Equal amounts of d- and l-amino acids are formed in abiotic (non-living) nature, such as meteorites. But proteins in life on Earth – and thus also in the human body – are only built from l-amino acids.
Birthe B. Kragelund explains that the researchers expected that proteins built from l-amino acids can easily bind to other proteins built from l-amino acids, but they were not sure what would happen if they mixed protein binding-partners built from d- and l-amino acids, respectively.
“Interestingly, biology is based on interactions between proteins built from l-amino acids, but before this study we had no idea how stereochemistry would affect interactions between intrinsically disordered proteins,” she observes.
Chirality irrelevant for intrinsically disordered proteins
The results sent the researchers into a spin because they found that whether intrinsically disordered proteins were built from l-amino acids or d-amino acids is irrelevant to their interactions. The proteins bind equally well to each other.
Nevertheless, the researchers also found that an intrinsically disordered protein built from d-amino acids could not bind to a structured protein built from l-amino acids if the intrinsically disordered protein itself folded into a well-defined 3D structure upon binding.
However, intrinsically disordered proteins built from l-amino acids and structured proteins built from l-amino acids could bind to each other, as also happens in nature.
“This means that some complexes at one end are completely indifferent to the stereochemistry, whereas it means everything and is necessary for some complexes at the other end. We then investigated how more or less disordered proteins bound to ordered proteins and found a continuum. The more ordered a protein is in the complex, the more the chirality affects the binding efficiency. Our study also shows that the more dynamic and disordered a complex is, the less sensitive it is to stereochemistry. In retrospect, one can perhaps intuitively see that this is a consequence of disorder, but this is the first time anyone has demonstrated this experimentally,” explains Birthe B. Kragelund.
Insight into the origins of life
How does this new understanding affect the quest for the origins of the earliest life on Earth and the potential for designing drugs?
The very first common ancestor of all organisms on Earth was also homochiral, which means it also preferred proteins built from l-amino acids over proteins built from a mixture of l- and d-amino acids.
When amino acids are made abiotically, the quantities of l-amino acids and d-amino acids are equal.
The new study suggests that the fact that intrinsically disordered proteins can easily interact with proteins built from l- and d-amino acids affects how researchers could view the earliest forms of life on Earth.
“New technologies, insight into abiotic biochemistry and the exploration of planets and exoplanets have meant that research into the origins of life has transformed from a narrow and somewhat tentative and philosophical subject to a flourishing field. One profound mystery is homochirality. We can logically trace life back to the last universal common ancestor, a homochiral cell that was the starting-point for bacteria, archaea and later eukaryotes like us,” says Johan Olsen, Assistant Professor at the Department of Biology, University of Copenhagen, Denmark.
But there must have been something before: living systems without genetic material, without membranes, something that was chemically constructed from molecules drawn from the non-living pool of biochemical molecules such as amino acids.
"We have shown that right- and left-handed proteins can interact, so why does life use only one part of the mirror image? And why l- rather than d-amino acids? We plan to address this question in the future,” explains Johan Olsen.
Birthe B. Kragelund elaborates that researchers can now begin to think about the role of intrinsically disordered proteins in the origins of life on Earth.
“Perhaps the first rudimentary proteins were intrinsically disordered proteins, and from them life has since evolved towards being homochiral, or perhaps life was homochiral to begin with, and the intrinsically disordered proteins then evolved to function based on this chirality,” adds Birthe B. Kragelund.
Designing better drugs
Intrinsically disordered proteins play a role in numerous diseases, and peptide-based medicines based on small fragments of protein may be relevant for treating some diseases. However, the human body is full of enzymes developed to break down all proteins, and this limits the half-life of drugs based on peptides.
However, the interaction between enzymes and the proteins they break down is the same as between all other proteins, meaning that the body’s enzymes built from l-amino acids exclusively bind to and break down proteins and peptides built from l-amino acids.
In contrast, the enzymes have great difficulty in binding to and breaking down drugs built from d-amino acids.
This type of drug could therefore potentially have a much longer half-life in the body and thus be effective longer.
“The same amino acid sequence can be used with the same effectiveness, and the chirality means that the body’s enzymes cannot break down the peptides. This holds great potential for designing new types of drugs,” concludes Birthe B. Kragelund.
