Steps Toward a New Biological Canon: Directed Evolution of a Structurally Unique de novo ATPase

Abstract

Throughout time evolution has proven to be a powerful tool for advancements in biology. From these advancements come patterns in form and function for both effector molecules like proteins and substrates like nucleoside triphosphates (NTPs). NTP binding itself is one of the earliest identified binding motifs in naturally occurring proteins with one binding motif, the Rossmann fold (an  fold dubbed the  sandwich), present within a sizable portion of all known proteins found in the Protein Data Bank. Associated with the Rossmann fold is the phosphate loop (P-loop) motif (Consensus: GXXXXGK[ST]), a motif found in all naturally occurring ATPases. Alternative ATPase (AltTPase) is a de novo, 4-helix bundle ATPase discovered by Wang and Hecht that exhibits no characteristic  fold commonly found in NTP binding proteins nor the presence of any P-loop. However, AltTPase shows poor catalytic ability when compared to naturally occurring enzymes. We demonstrate that this catalytic rate can be improved qualitatively through directed evolution with selection done on a life-or-death basis with survival being linked to improved ability. Despite undergoing evolution and increasing in catalytic ability, S7-55 still shows no  fold, nor any -sheet identity either. Furthermore, S7-55 still exhibits no signs of evolving a traditional P-loop motif.