Mutant libraries reveal negative design shielding proteins from supramolecular self-assembly and relocalization in cells

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 119; no. 5
• Main Authors: Garcia Seisdedos, Hector, Levin, Tal, Shapira, Gal, Freud, Saskia, Levy, Emmanuel D
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 01-02-2022
• Subjects: Alanine; Biological Sciences; Design; Fibers; Genotype; Genotypes; Localization; Mutants; Mutation; Mutation - genetics; Perturbation; Phenotype; Phenotypes; Physicochemical properties; Protein structure; Proteins; Proteins - genetics; Residues; Self-assembly; Shielding; Structure-function relationships; Surface charge; Yeasts; protein interactions; protein evolution; genotype–phenotype map
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2101117119

Understanding the molecular consequences of mutations in proteins is essential to map genotypes to phenotypes and interpret the increasing wealth of genomic data. While mutations are known to disrupt protein structure and function, their potential to create new structures and localization phenotypes has not yet been mapped to a sequence space. To map this relationship, we employed two homo-oligomeric protein complexes in which the internal symmetry exacerbates the impact of mutations. We mutagenized three surface residues of each complex and monitored the mutations' effect on localization and assembly phenotypes in yeast cells. While surface mutations are classically viewed as benign, our analysis of several hundred mutants revealed they often trigger three main phenotypes in these proteins: nuclear localization, the formation of puncta, and fibers. Strikingly, more than 50% of random mutants induced one of these phenotypes in both complexes. Analyzing the mutant's sequences showed that surface stickiness and net charge are two key physicochemical properties associated with these changes. In one complex, more than 60% of mutants self-assembled into fibers. Such a high frequency is explained by negative design: charged residues shield the complex from self-interacting with copies of itself, and the sole removal of the charges induces its supramolecular self-assembly. A subsequent analysis of several other complexes targeted with alanine mutations suggested that such negative design is common. These results highlight that minimal perturbations in protein surfaces' physicochemical properties can frequently drive assembly and localization changes in a cellular context.