Structural reverse genetics study of the PI5P4Kβ-nucleotide complexes reveals the presence of the GTP bioenergetic system in mammalian cells.

Reverse genetic analysis can connect a gene and its protein counterpart to a biological function(s) by knockout or knockdown of the specific gene. However, when a protein has multiple biochemical activities, the conventional genetics strategy is incapable of distinguishing which biochemical activity of the protein is critical for the particular biological function(s). Here, we propose a structural reverse genetics strategy to overcome this problem. In a structural reverse genetics study, multiple biochemical activities of a protein are segregated by mapping those activities to a structural element(s) in the atomic resolution tertiary structure. Based on the structural mapping, a mutant lacking one biochemical activity of interest can be produced with the other activities kept intact. Expression of the mutant by knockin or ectopic expression in the knockout strain along with the following analysis can connect the single biochemical activity of interest to a biological function. Using the structural reverse genetics strategy, we have dissected the newly identified GTP-dependent activity of a lipid kinase PI5P4Kβ from its ATP-dependent activity. The GTP-insensitive mutant has demonstrated the existence of the GTP bioenergetic sensor system in mammalian cells and its critical role in tumorigenesis. As structural reverse genetics can identify in vivo significance of individual biochemical activity, it is a powerful approach to reveal hidden biological functions, which could be a novel pharmacological target for therapeutic intervention. Given the recent expansion of choices in structural biological methods and advances in genome editing technologies, the time is ripe for structural reverse genetics strategies.