Altered expression of a quality control protease in E. coli reshapes the in vivo mutational landscape of a model enzyme

Abstract

Protein mutational landscapes are shaped by the cellular environment, but key factors and their quantitative effects are often unknown. Here we show that Lon, a quality control protease naturally absent in common E. coli expression strains, drastically reshapes the mutational landscape of the metabolic enzyme dihydrofolate reductase (DHFR). Selection under conditions that resolve highly active mutants reveals that 23.3% of all single point mutations in DHFR are advantageous in the absence of Lon, but advantageous mutations are largely suppressed when Lon is reintroduced. Protein stability measurements demonstrate extensive activity-stability tradeoffs for the advantageous mutants and provide a mechanistic explanation for Lon’s widespread impact. Our findings suggest possibilities for tuning mutational landscapes by modulating the cellular environment, with implications for protein design and combatting antibiotic resistance.

Structurally Distributed Surface Sites Tune Allosteric Regulation

“…Secondly, DMS of native DHFR -under experimental conditions designed to resolve mutational effects near WT -revealed many beneficial (activating) mutations [27]. There are two explanations for the relative paucity of beneficial and neutral mutations in the present dataset.…”

“…THF then serves as a one-carbon donor and acceptor in the synthesis of thymidine, purine nucleotides, serine, glycine, and methionine. Because of these critical metabolic functions, DHFR activity is strongly linked to growth rate, and under appropriate conditions, E. coli growth rate can be used as a proxy for DHFR activity [21, 27]. Prior work found that the modest in vitro allosteric effect of DL121 conferred a selectable growth rate advantage in vivo : when an E. coli DHFR deletion strain (ER2566 ΛfolAΔthyA ) was complemented with DL121, the resulting strain grew 17% faster in the light than in the dark [21].…”

“…As in prior work, we measured the growth rate of many E. coli strains in parallel by using next generation sequencing (NGS) to monitor the frequency of individual DHFR mutants over time in a mixed culture ( Figure 2 ) [21, 27]. Allele frequencies ( f a ) at each time point (t) were normalized as follows: where N a and N WT are the number of mutant and wildtype (WT) counts at a given time point.…”

“…A cluster of beneficial mutations was observed just before the LOV2 insertion site at position 121 in both conditions, suggesting some potential to compensate for the inserted LOV2 ( Figure S4 ). The overall distribution of fitness effects shows some differences relative to prior DMS studies of natural proteins including native E. coli DHFR [27, 45]. First, the distribution of fitness effects for mutations in natural proteins is often centered around neutral, implying a certain degree of mutational robustness [44, 46].…”

“…First, the distribution of fitness effects for mutations in natural proteins is often centered around neutral, implying a certain degree of mutational robustness [44, 46]. Secondly, DMS of native DHFR – under experimental conditions designed to resolve mutational effects near WT – revealed many beneficial (activating) mutations [27]. There are two explanations for the relative paucity of beneficial and neutral mutations in the present dataset.…”