The Internal Allosteric Architecture of Dihydrofolate Reductase

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Dihydrofolate reductase (DHFR) is a model metabolic enzyme for understanding the connection between long range conformational change and catalysis. In prior work, we showed that DHFR contains a co-evolving network of amino acids, termed a sector, which coincides with positions undergoing millisecond conformational exchange associated with catalysis. Moreover, we found that inserting a light sensing domain from plants (LOV2) at sector connected surfaces led to novel allosteric regulation, while never leading to allosteric regulation at non-sector connected surfaces. This suggests that sector connected edges are hotspots for engineering regulation, and that sector positions might provide the basis of allosteric communication within DHFR. To test this, we constructed a total saturation mutagenesis library of an allosterically active DHFR-LOV2 fusion. We then measured the DHFR activity of every library member in the light and dark, using a next-generation sequencing based assay in which DHFR activity is coupled to growth rate. From this data, we determined the coupling of the individual mutations in DHFR to allostery. We found, to high significance, that mutations positively affecting allostery are depleted within the sector and are enriched in surface exposed residues. Interestingly, there was no correlation between allosteric disrupting mutations and the sector or surface. This global map of allosteric contributions inside the enzyme provides insight into how allostery can be evolutionarily optimized, and suggests an especially important role for surface positions in tuning allostery. To corroborate these results, we are characterizing individual mutants biochemically, in vivo, and crystallizing the chimera.