A major focus of our research is a class of proteins known as tandem-repeat proteins. They comprise small structural units repeated multiple times in tandem to form non-globular, mostly elongated structures that present extended scaffolds for molecular recognition. There is a large variety of repeat types ranging from purely helical or beta-sheet to a mix of both. Hence, they give rise to many different geometrical shapes, which in turn confer different biochemical and biophysical properties and functions.
In our lab we use a battery of techniques spanning single molecule methods, biophysics, biochemistry and analysis in cellulo and in silico. Most techniques allow us to probe a mechanism or process and find out whether there are overarching, fundamental laws that govern it. As such they provide a bottom-up approach: learning more about the molecular characteristics of a given system will subsequently enable us to guide traditional drug design and disease treatments. At the same time we are also investigating novel approaches to disease treatment, which are based on and motivated by what we know about the underlying mechanisms of repeat protein function we and other groups have uncovered.
Structure, function and mechanics of repeat proteins
One aim of our current research is to define the conformational transitions that repeat proteins undergo during the different stages of their life cycle - biosynthesis, folding, localisation, assembly and degradation - and how they are guided by the cellular machinery. Due to their modular architecture and Slinky spring-like shapes, it is proposed that repeat arrays utilise much more dynamic and elastic modes of action than globular proteins. For example: stretching and contraction motions to regulate the activity of a bound enzyme; acting as reversible nanosprings to operate ion channels; proteins that wrap around their cargoes to transport them between the nucleus and cytoplasm.
Our group and others have shown that the simple modular, one-dimensional-like architecture of tandem repeat proteins gives them distinctive properties compared with globular proteins and makes it uniquely straightforward to map the energetics of their structures and to rationally redesign their stability, folding and molecular recognition. This class of proteins is thus an exceptionally sensitive and versatile tool that we are now in a position to exploit to dissect otherwise intractable cellular mechanisms.
Molecular recognition between repeat proteins and IDP binding partners
Protein-protein interactions underpin all functions in organisms; therefore, to completely understand these processes and the changes that occur leading to a disease state we need to study the molecular level. We are taking a biophysical approach to elucidate the thermodynamics of interactions between binding partners.
One particular group of interactions we are interested in is what drives the binding of an intrinsically disordered segment to the extended surface area offered by a repeat protein; is it driven by a few key interactions or can binding be initiated from a number of different sites giving rise to parallel binding pathways? A paradigmatic example is the armadillo repeat protein β-catenin and its IDP ligands e.g. axin, adenonmatus polyposis coli (APC), e-cadherin and TCF-4.
Once we have elucidated the effects of individual mutations found in the disease state on a protein’s thermodynamics and ligand binding properties we can then try to connect this look in stability or binding to loss of function in the cell. We have shown that destabilising mutations in BRCA1 BRCT domain that commonly occur in breast cancer can be buffered by the cell allowing the protein to maintain its activity (Rowling et al 2010, Gaboriau et al 2015). The information obtained about interactions between binding partners in vitro and in vivo can then be used in a rational drug design effort, as exemplified by the gankyrin research program.
Protein design and molecular therapeutics
We are focusing on repeat proteins that play important roles in disease, in particular cancer, and we are using the insights we obtain to develop therapeutic strategies for targeting them. Moreover, ankyrin repeats have also been identified as targets in a range of diseases in addition to cancer. For example, antagonists of ankyrin repeat ion channels have shown potential in pain relief and in treating respiratory diseases, chronic acid reflux and esophageal hypersensitivity.
Traditionally, small molecules are used to target proteins and interactions with their binding partners. However, more recently the focus has shifted to alternatives that are more specific for a given protein or interaction. In our group we investigate three different approaches to target our proteins of interest: a) constrained peptides, b) designed repeat proteins, and c) nanobodies. In most cases, the proteins that we are targeting are repeat proteins or repeat domains that are very central to cellular function, organism development and homoeostasis.
The advantage of using peptides, repeat proteins and nanobodies instead of small molecules is that they have the potential to confer much greater specificity and reduced toxicity.
Repeat protein assembly and nano-materials
Repeat proteins have been observed to associate with binding partners having a variety of structures: some bind IDPs or short linear motifs, others bind globular structures or other repeat proteins. The properties of the individual units of tandem repeat proteins can be tailored by design and they can then be combined in a modular fashion to create artificial proteins with predictable properties (stability, binding etc.). Such a degree of rational engineering is not possible with globular proteins. We are interested in exploiting this extraordinary design-ability and functional variety with the ultimate goal of developing scaffolds that can bind and deliver e.g. peptides or small molecular compounds. We also envision a scaffold that could perform a single function or simple biological process in itself. Different approaches are used in our group to create these scaffolds and nano-materials; they include CuAAC (“click”) chemistry, protein-protein interactions and metal binding systems.