Project Summary/Abstract PI/PD: Pryciak, Peter M. The proper function of cells depends on an enormous number of interactions between different proteins. Interactions that are weak and transient are especially important in controlling molecular events that are rapid and dynamic. In many cases these transient interactions are mediated by three-dimensionally folded protein domains in one partner protein that bind to short peptide sequences in the other partner. These peptide sequences are known as Short Linear Motifs, or SLiMs, and among human proteins there are over 200 distinct families of SLiM-binding domains and many hundreds of examples. SLiM-mediated interactions serve critical roles in subcellular localization, assembly of dynamic multi-protein complexes, and substrate recognition by post-translational modification enzymes such as kinases, phosphatases, ubiquitin ligases, etc. While some examples have been studied intensively, for the vast majority of SLiM-binding domains the key sequence features that govern recognition of their target motifs are poorly defined. Moreover, over 3 million residues of the human proteome are predicted to be structurally disordered and hence are likely to contain many as-yet undiscovered SLiMs. This proposal seeks to illuminate the molecular basis of SLiM-mediated interactions and fill current knowledge gaps. The experiments will develop a method for rapid quantification of relative binding strength for thousands of variant peptide motif sequences, and a systematic interrogation of SLiM sequence features that control their recognition and potency. The approach will use an intracellular functional assay that can define SLiM recognition rules for a wide variety of globular domains, provide a relative affinity ranking for large numbers of candidate motif sequences, and even identify competitive inhibitor peptides that can inform drug design. One goal will be to validate the methodology by establishing the correlation between functional potency and biochemical affinity, and the dependence of SLiM residue preferences on the surrounding peptide context or strength. These experiments will also seek to expand the range of binding affinities that can be resolved by the method. Another goal will be to apply the method toward a large number of SLiM-binding domains to characterize their sequence preferences and independently confirm large numbers of binding peptides identified in separate screens. A third goal will be to develop additional adaptations of the method that will allow for the design and optimization of peptide-based inhibitors and tethering molecules for linking together distinct domains inside cells. Overall, these studies will contribute to our general understanding of protein-protein interactions, with relevance to the mechanisms underlying normal cell function as well as disease states including the hijacking of SLiM-binding domains by human pathogens.