Abstract

A high-throughput mutagenesis study in a PDZ domain shows that biochemical function and adaptation primarily originate from a collectively evolving amino acid network within the structure termed a protein sector. Statistical analysis of protein evolution suggests a 'design' for natural proteins in which sparse networks of coevolving amino acids comprise the essence of three-dimensional structure and function. To better understand the relationship of sector-based architecture to these properties, the authors performed a comprehensive single-mutation study of a PSD95pdz3 — a typical PDZ family protein — in which each position is substituted independently of every other amino acid. PDZ domains, which are made up of tens of amino acids, are conserved in many signalling proteins in animals, plants and other organisms. Mutational analysis showed that sector positions are functionally sensitive to mutation, whereas non-sector positions are much more tolerant to substitution, and that adaptation to a new binding specificity initiates exclusively through variation within sector residues. These results show how proteins can be robust yet also capable of rapid functional change when conditions of selection change. Statistical analysis of protein evolution suggests a design for natural proteins in which sparse networks of coevolving amino acids (termed sectors) comprise the essence of three-dimensional structure and function1,2,3,4,5. However, proteins are also subject to pressures deriving from the dynamics of the evolutionary process itself—the ability to tolerate mutation and to be adaptive to changing selection pressures6,7,8,9,10. To understand the relationship of the sector architecture to these properties, we developed a high-throughput quantitative method for a comprehensive single-mutation study in which every position is substituted individually to every other amino acid. Using a PDZ domain (PSD95pdz3) model system, we show that sector positions are functionally sensitive to mutation, whereas non-sector positions are more tolerant to substitution. In addition, we find that adaptation to a new binding specificity initiates exclusively through variation within sector residues. A combination of just two sector mutations located near and away from the ligand-binding site suffices to switch the binding specificity of PSD95pdz3 quantitatively towards a class-switching ligand. The localization of functional constraint and adaptive variation within the sector has important implications for understanding and engineering proteins.