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.

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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 where 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 misassembly, and the sole removal of the charges induces its assembly. A subsequent analysis of several other complexes targeted with alanine mutations suggested that negative design against mis-assembly and mislocalization is common. These results highlight that minimal perturbations in protein surfaces’ physicochemical properties can frequently drive assembly and localization changes in a cellular context.

SummaryPostnatal refinement of neuronal connectivity shapes the mature nervous system. Pruning of exuberant connections involves both cell autonomous and non-cell autonomous mechanisms, such as neuronal activity. While the role of neuronal activity in the plasticity of excitatory synapses has been extensively studied, the involvement of inhibition is less clear. Furthermore, the role of activity during stereotypic developmental remodeling, where competition is not as apparent, is not well understood.Here we use the Drosophila mushroom body as a model to show that regulated silencing of neuronal activity is required for developmental axon pruning of the γ-Kenyon cells. We demonstrate that silencing neuronal activity is mechanistically achieved by cell autonomous expression of the inward rectifying potassium channel (irk1) combined with inhibition by the GABAergic APL neuron. These results support the Hebbian-like rule ‘use it or lose it’, where inhibition can destabilize connectivity and promote pruning while excitability stabilizes existing connections.