Negative Epistasis and Evolvability in XXXX b-Lactamase-The Thin Line between an Enzyme's Conformational Freedom and Disorder.

@entity2734 @entity74 @entity72974 @entity48627 @entity25054
UNASSIGNED: Epistasis is a key factor in evolution since it determines which combinations of mutations provide adaptive solutions and which mutational pathways toward these solutions are accessible by natural selection. There is growing evidence for the pervasiveness of sign epistasis-a complete reversion of mutational effects, particularly in protein evolution-yet its molecular basis remains poorly understood. We describe the structural basis of sign epistasis between @entity48627 and @entity72974 , two adaptive mutations in @entity25054 b-lactamase- an enzyme that endows antibiotics resistance. Separated by 10  , these mutations initiate two separate trajectories toward increased hydrolysis rates and resistance toward second and third-generation cephalosporins antibiotics. Both mutations allow the enzyme's active site to adopt alternative conformations and accommodate the new antibiotics. By solving the corresponding set of crystal structures, we found that @entity72974 causes @entity74 whereas @entity48627 induces discrete conformations. When combined, the mutations in 238 and 164 induce @entity74 whereby nonproductive conformations that perturb the enzyme's catalytic preorganization dominate. Specifically, @entity2734 170 that coordinates the deacylating water molecule is misaligned, in both the free form and the inhibitor-bound double mutant. This @entity74 is not restored by stabilizing global suppressor mutations and thus leads to an evolutionary cul-de-sac. Conformational dynamism therefore underlines the reshaping potential of protein's structures and functions but also limits protein evolvability because of the fragility of the interactions networks that maintain protein structures.
