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.

Proteins display the capacity for adaptation to new functions, a property critical for evolvability. But what structural principles underlie the capacity for adaptation? Here, we show that adaptation to a physiologically distinct class of ligand specificity in a PSD95, DLG1, ZO-1 (PDZ) domain preferentially occurs through class-bridging intermediate mutations located distant from the ligand-binding site. These mutations provide a functional link between ligand classes and demonstrate the principle of "conditional neutrality" in mediating evolutionary adaptation. Structures show that class-bridging mutations work allosterically to open up conformational plasticity at the active site, permitting novel functions while retaining existing function. More generally, the class-bridging phenotype arises from mutations in an evolutionarily conserved network of coevolving amino acids in the PDZ family (the sector) that connects the active site to distant surface sites. These findings introduce the concept that allostery in proteins could have its origins not in protein function but in the capacity to adapt.

BACKGROUND More than 30 million children worldwide suffer from moderate acute malnutrition (MAM). Current treatments have limited effectiveness and much remains unknown about pathogenesis. Children with MAM exhibit perturbed development of their gut microbiota. METHODS Slum-dwelling Bangladeshi children, aged 12 to 18 months, with moderate acute malnutrition (n=124) received a microbiota-directed complementary food (MDCF-2) or an existing ready-to-use supplementary food (RUSF), twice daily for three months followed by a 1-month period of monitoring. We obtained weight-for-length, weight-for-age, and length-for-age Z-scores and mid-upper arm circumference at baseline and fortnightly, through four months. We compared the rate of change of these related phenotypes between baseline and three months, and between baseline and four months. We also measured levels of 4,977 proteins in plasma plus 209 bacterial taxa in fecal samples. RESULTS 118 children completed the intervention (n=59/arm). The rate of change in weight-for-length Z-score (β-WLZ), weight-for-age Z-score, and mid upper arm circumference is consistent with a benefit of MDCF-2 on growth over the course of the study including the one-month follow-up. Receipt of MDCF-2 was linked to the magnitude of change in levels of 70 β-WLZ-positively correlated plasma proteins including mediators of bone growth, neurodevelopment and inflammation (gene set enrichment analysis [GSEA];p<0.001) and the abundances of 23 WLZ-associated bacterial taxa (GSEA;p<0.001). CONCLUSIONS These findings provide support for further clinical investigation of MDCF-2 as a dietary supplement for young children with MAM and provide insight into mechanisms by which this targeted manipulation of microbiota components may be linked to growth. (Supported by the Bill and Melinda Gates Foundation and the NIH; ClinicalTrials.gov identifier: NCT04015999 )