Short linear motifs in cell death pathways under the lens of structural biology

A long-standing paradigm in biochemistry inextricably tied a protein function to the threedimensional protein structure. However, the foundations of this view have been shaken with the discovery of proteins and domains violating the structure-function relationship.
These proteins and regions are known as Intrinsically Disordered Proteins (IDPs) and Intrinsically Disordered Regions (IDRs), respectively, and their investigation prompted the adoption of a new concept incorporating dynamical behavior into a new structure-dynamics-function triad. IDPs and IDRs are highly represented in the proteomes of higher eukaryotes and often harbor disease-causing mutations. Furthermore, IDRs are often involved in protein-protein interactions.
For example, one interaction mechanism based on IDRs involves small sequence stretches known as Short Linear Motifs (SLiMs). SLiMs are characterized by few conserved positions spaced by non-conserved ones, generally low binding affinities, cooperativity, and layers of regulation through Post-Translational Modifications (PTMs). Their sequence degeneration and post-translational regulation hinder the identification and characterization through computational approaches. At the same time, the low affinities coupled with the possibility of cooperating with other SLiMs to enhance the binding make them elusive targets for experimental studies.
In this context, the aim of this Ph.D. project was twofold. On the one hand, we aimed at developing a suite of computational tools to tackle the identification and characterization of SLiMs and SLiM-mediated interactions. On the other hand, we focused on investigating two SLiMs, the BH3 motif and the LIR motif, crucial in the cellular processes of apoptosis and autophagy, respectively. BH3 motifs mainly interact with proteins belonging to the Bcl-2 family, whose members participate in a delicate interplay that ultimately decides the cell's commitment to apoptosis. In contrast, proteins belonging to the ATG8 protein family perform a critical role in the autophagic process by interacting with LIR-containing proteins.
In this manuscript, we first present an updated perspective on the most relevant literature and open questions regarding the Bcl-2 family and the BH3 motifs, as well as the ATG8 family and LIR-mediated interactions (Manuscript 1 and Manuscript 2).
Then, we dive into the details of a novel pipeline to discover instances of the LIR motif regulated by phosphorylation (Manuscript 3) and a structure-based workflow to annotate putative SLiM candidates (Manuscript 4).
We then focus on the tools we developed to analyze protein-SLiM complexes and, more in general, protein-protein interactions. In this context, we describe a new interface facilitating the usage of protocols to predict the impact of mutations on the structural stability of proteins and protein-protein interactions and present an example of its capabilities on a SLiM-mediated interaction of interest (Manuscript 5). 
Furthermore, we present two suites for building and analyzing interaction networks in protein conformational ensembles (Manuscripts 6-8).Finally, we show how we applied these approaches to investigate specific BH3- and LIRmediated interactions (Manuscripts 9 and 10) and the structure and dynamics of the ATG8 protein family (Manuscript 11).