Periodic Reporting for period 1 - SYN-CHARGE (Novel avenues of action for a hallmark disordered protein of Parkinson's disease)
Berichtszeitraum: 2022-02-01 bis 2024-01-31
The study of protein-protein interactions is central to protein biochemistry with proteins being the functional workhorse of biological systems. Studying protein structure helps us to understand how proteins function, how they interact and their role within the context of the cellular machinery. A subset of proteins lacks persistent three-dimensional structure and are highly dynamic – the so-called intrinsically disordered proteins (IDPs). Historically, all protein interactions were thought to rely heavily on structure, for example: the interactions of IDPs may induce the formation of structure. This view changed upon the discovery of a high affinity, completely disordered, dynamic interaction characterized by a collaborative effort between the Kragelund, Best and Schuler research groups. The presence of interacting partners that remain disordered upon binding was unexpected, and challenged the ways in which we think about protein interactions. It is possible that more proteins can interact in this way, but researchers have lacked understanding of the ways in which they can be observed.
The goal of this research program was to identify a new ways of understanding disorder in protein-protein interactions.
Initially we aimed to do this using the interaction between the disordered region of a calcium pump (plasma membrane calcium ATPase) and alpha-synuclein, however upon careful investigation the link between the two proteins was found to be via an indirect mechanism, therefore disqualifying it as a good candidate for this investigation. Instead, we focused on the disorder-order continuum of interactions, substituting one disordered binding partner for its enantiomer. Enantiomers are chemically identical to each other, but are non-superimposable mirror images, meaning that proteins have a “handedness”. We hypothesized that in completely disordered interactions, enantiomers could interact with their binding partner regardless of their handedness, whereas in structured interactions, they could not. In this way, we could test the degree of disorder and reliance on protein charge within a protein-protein interaction.
The goal of this research program was to identify a new ways of understanding disorder in protein-protein interactions.
Initially we aimed to do this using the interaction between the disordered region of a calcium pump (plasma membrane calcium ATPase) and alpha-synuclein, however upon careful investigation the link between the two proteins was found to be via an indirect mechanism, therefore disqualifying it as a good candidate for this investigation. Instead, we focused on the disorder-order continuum of interactions, substituting one disordered binding partner for its enantiomer. Enantiomers are chemically identical to each other, but are non-superimposable mirror images, meaning that proteins have a “handedness”. We hypothesized that in completely disordered interactions, enantiomers could interact with their binding partner regardless of their handedness, whereas in structured interactions, they could not. In this way, we could test the degree of disorder and reliance on protein charge within a protein-protein interaction.
The overarching goal of this program was to understand further the charged, disordered interactions that intrinsically disordered proteins (IDPs) can undergo.
We started by addressing the interaction of alpha-synuclein with the disordered loop of the plasma membrane calcium ATPase (PMCA). We found, using nuclear magnetic resonance (NMR) that the interaction between these two proteins was not direct, and therefore not governed by their charges. We then characterized the PMCA loop, which had previously been difficult due to its disorder. The manuscript describing this is currently in preparation.
Our next strategy for probing disordered protein interactions was to test the disorder-order continuum of interactions, carefully choosing a set of protein interacting pairs from being very disordered to ordered. For each pair, we made a peptide ligand binding partner from either L-amino acids (the left-handed, naturally occurring enantiomer) or D-amino acids (the right handed, rare enantiomer). Although having the same amino acid sequence, D-peptides are the mirror image of L-peptides and therefore will fold in the opposite direction to L-peptides. This makes the likelihood of their interactions with an L-protein binding partner very low, if structural rearrangement and adaption is necessary for the peptide to fit into a binding pocket. We hypothesized that this might not be true for proteins that remain disordered upon binding.
We first tested this using a disordered protein binding pair, finding that if there is no structure formed upon binding, a D-peptide can interact equally well as an L-peptide. Following this finding, we moved onto a protein pair in which the peptide is required to fold upon binding. In this case, the D-peptide could not interact with its binding partner, while the L-peptide interacted as expected. We therefore observed a clear distinction between a disordered protein interaction, versus one that required the formation of structure.
Following this, we investigated three different protein binding pairs, each with varying degrees of structure and disorder. We found that, instead of enantiomeric binding being black and white in disordered and ordered interactions, there were intermediate interactions that could occur depending on the extent of remaining disorder in the interaction.
In addition to this major finding, we also published a review on IDP phosphorylation and the implications of adding charges to an IDP post-translationally, and two research studies on charge distribution in IDPs and IDP size prediction.
Dissemination achievements:
• Three research papers published (The EMBO Journal, Biomolecules, Biophysical Journal)
• One review published (Essays in Biochemistry)
• One manuscript under review
• Three international conferences (one oral presentation: USA, two poster presentations: Australia, Switzerland)
We started by addressing the interaction of alpha-synuclein with the disordered loop of the plasma membrane calcium ATPase (PMCA). We found, using nuclear magnetic resonance (NMR) that the interaction between these two proteins was not direct, and therefore not governed by their charges. We then characterized the PMCA loop, which had previously been difficult due to its disorder. The manuscript describing this is currently in preparation.
Our next strategy for probing disordered protein interactions was to test the disorder-order continuum of interactions, carefully choosing a set of protein interacting pairs from being very disordered to ordered. For each pair, we made a peptide ligand binding partner from either L-amino acids (the left-handed, naturally occurring enantiomer) or D-amino acids (the right handed, rare enantiomer). Although having the same amino acid sequence, D-peptides are the mirror image of L-peptides and therefore will fold in the opposite direction to L-peptides. This makes the likelihood of their interactions with an L-protein binding partner very low, if structural rearrangement and adaption is necessary for the peptide to fit into a binding pocket. We hypothesized that this might not be true for proteins that remain disordered upon binding.
We first tested this using a disordered protein binding pair, finding that if there is no structure formed upon binding, a D-peptide can interact equally well as an L-peptide. Following this finding, we moved onto a protein pair in which the peptide is required to fold upon binding. In this case, the D-peptide could not interact with its binding partner, while the L-peptide interacted as expected. We therefore observed a clear distinction between a disordered protein interaction, versus one that required the formation of structure.
Following this, we investigated three different protein binding pairs, each with varying degrees of structure and disorder. We found that, instead of enantiomeric binding being black and white in disordered and ordered interactions, there were intermediate interactions that could occur depending on the extent of remaining disorder in the interaction.
In addition to this major finding, we also published a review on IDP phosphorylation and the implications of adding charges to an IDP post-translationally, and two research studies on charge distribution in IDPs and IDP size prediction.
Dissemination achievements:
• Three research papers published (The EMBO Journal, Biomolecules, Biophysical Journal)
• One review published (Essays in Biochemistry)
• One manuscript under review
• Three international conferences (one oral presentation: USA, two poster presentations: Australia, Switzerland)
Overall, this action substantially improved our understanding of intrinsically disordered proteins (IDPs), their properties, and the ways in which they can interact, especially in charged, disordered interactions. Reflecting on the independence of stereochemistry in disordered protein interactions challenges the way that we think about protein-protein interactions. Further, on a basic scientific level, the lack of a demand for stereochemistry in a disordered protein interaction is unique because it raises questions about the possibility that there was a time before a preference for L-amino acids developed. This adds to what we know to be possible in protein evolution.
On a pharmaceutical level, this finding makes D-peptides a viable option for treatment of diseases that are caused by IDPs. IDPs have been historically difficult to target, and issues with peptide therapies often arise due to the body’s capability to degrade proteins. D-peptides, however, are invisible to normal cellular proteolytic machinery, increasing peptide half-life significantly.
On a pharmaceutical level, this finding makes D-peptides a viable option for treatment of diseases that are caused by IDPs. IDPs have been historically difficult to target, and issues with peptide therapies often arise due to the body’s capability to degrade proteins. D-peptides, however, are invisible to normal cellular proteolytic machinery, increasing peptide half-life significantly.