A group of investigators has taken a significant step toward determining what controls the switching on and off of genes that carry out all of life’s functions. The team switched out Escherichia coli CRP to use that from Mycobacterium tuberculosis instead, helping them begin to define how proteins bind to cAMP on one end, then attach to and activate DNA at the other end.
Believing that proteins somehow change their overall shape after binding cAMP, researchers had been working on this problem for over two decades with E. coli proteins, but had been unable to crystallize the protein in its inactive state. The tuberculosis CRP has been captured in that state, and now the team is working backwards to crystallize it in its active state.
“We know that many pathogenic bacteria use cAMP as a signal for activating genes that keep the microbes thriving in adverse conditions, and therefore, remaining virulent,” says Dr. Travis Gallagher, Ph.D., lead author of the study. “Blocking these processes might provide ways to shut down infections and save lives.”
In addition to obtaining crystals in the off state, the group believes that learning how this specific protein switch works may provide insight into how genes in general are regulated, and examined the crystals with x-ray diffraction.
“Although the M. tuberculosis protein in the ‘off’ state consists of two subunits that are genetically identical, we were surprised to see that the subunits were not structurally symmetrical as well,” says Dr. Gallagher. “In most two-subunit proteins, each subunit has the same conformation as the other.”
The team theorizes that it is the asymmetry in the absence of cAMP that prevents the protein from attaching to DNA. This, in turn, keeps CRP from activating genes when they are not needed.
“Our next step is to crystallize M. tuberculosis CRP in the active state and define its structure,” Gallagher says. “When that is accomplished, we’ll be able to see the identical protein from the same organism in both states, which may give us the means to explain how CRP switches from its asymmetric form to its symmetrical form.”
The report was published online recently in the Journal of Biological Chemistry.