Leader: Patricia Faisca, University of Lisbon, Portugal

Co-leader: Joanna Sułkowska, University of Warsaw, Poland

General overview. With about 1% of all structures stored in the Protein Data Bank (PDB) found to have knots, it is now evident that proteins are a prime example of a natural, self-entangling system. The main aspects of interest in the study of knotted proteins are the functional role of the knot, the evolutionary pathways by which knotted protein structures have evolved, and the specific features of how these proteins fold in the presence of self-entanglements. In fact, if it is already challenging to determine how “regular” unknotted proteins fold, it is even more formidable to do so for knotted proteins. Computational studies have helped shed light on the processes that lead to the formation of a knot in some, typically small knotted proteins. Considerable help has also been provided by central repositories like the PDB and the dedicated web-servers that have made the challenging task of identifying knots in proteins accessible to non-experts. Recently, a new class of topologically entangled proteins, dubbed lasso proteins, has been identified, in which the entanglement originates from a loop stabilized by a covalent link in the form of a disulfide bridge. Furthermore, supramolecular entanglements among proteins have been detected, which cover a non-negligible fraction (~9%) of the multimeric structures stored in the PDB. In the experimental area, it has been shown that the structural complexity associated with the knotting process is responsible for slow folding rates, and the first artificially knotted protein has been created by fusing a homo-dimer. The latter represents an impressive achievement in terms of protein engineering, and paves the way for the pharmaceutical application of knotted proteins.

General objectives of WG3. To date, a number of in silico studies have been performed on knotted proteins, based on a diverse set of theoretical models and computational algorithms.
However, most of these studies have focused on proteins displaying knots with the simplest trefoil topology. Researchers involved in EUTOPIA are committed to push this investigation further, by exploring the folding mechanism of proteins with knots with more complex topology. A few questions to be addressed are in order: Are non-native interactions essential to drive the chain towards a complex topology? Is it possible to predict the changes in the protein primary sequence which are likely to lead to more complex topological structures? While most studies performed so far focussed on the knotted structure of native proteins, a number of related yet largely unexplored issues, which will be addressed within the proposed COST Action, concern the emergence and the implication of topologically non-trivial structures in protein misfolded states and in natively unstructured proteins. Tackling these and many other problems requires the development and application of advanced algorithms in order to overcome the underlying outstanding computational efforts, and of more accurate coarse-grained models of globular intrinsically disordered proteins to allow for larger statistics, longer time scales, and thorough studies of their topological properties. The EUTOPIA COST Action will aim at the following goals:

1. The development and application of computational models with systematically increasing detail to predict the folding of knotted proteins from a molecular viewpoint.
2. The computer-aided design of self-entangled proteins and their in vitro validation.
3. In silico studies moving forward from single knotted proteins to multiple entangled proteins.
4. The development of accurate coarse-grained models of intrinsically disordered proteins to allow for larger statistics, longer time scales, and thorough studies of their topological properties.

Specific tasks of WG3. The central elements of this WG are the entanglements that can be found in proteins – their origin, formation, role and manipulation. The investigation of the broad variety of knots and other kinds of entanglements in single as well as among multiple protein chains requires the combined effort of researchers with different expertise, and provides an ideal platform for the development of interdisciplinary activities and cross-fertilisation among traditionally distinct fields. The main objectives of this WG are the following:

1. Develop and apply novel techniques to overcome the intrinsic limitations of current in silico protein folding, and push the boundaries of our understanding of the protein knotting process.
2. Characterise the emergence and topological properties in intrinsically disordered proteins and multiple entangled proteins. In collaboration with WG1.
3. Push the integration of simulation and experiments to guarantee the exchange of ideas between the two approaches.
4. Establish a joint and coordinated effort to employ knotted protein design for pharmaceutical applications. Develop computational strategies to design proteins with target topologies and structures, and to validate the most successful candidates in vitro through the partnership with experimental groups.

Deliverables: STSMs within the WG and to other WGs. At least one article with 3 international members per each task. Identification and establishment of possible industrial partnerships.