By studying the structure of the cyclic AMP receptor protein (CRP) found in Mycobacterium tuberculosis, researchers at the National Institute of Standards and Technology (NIST) and Brookhaven National Lab are unlocking the mystery of how genes turn on and off.
From a statement by NIST:
The biochemical puzzle surrounding the CRP switch is the mechanism by which the protein binds cAMP at one end, then attaches to—and activates—a gene (DNA) at the other end. Believing that the protein somehow changes its overall shape after binding cAMP, researchers set out 25 years ago to study the structure of CRP in both its active state (with cAMP bound to it) and inactive state (without bound cAMP) to document where the morphing occurs.
Unfortunately, the task proved to be extremely difficult. Using CRP from the bacterium Escherichia coli, researchers were able to crystallize the protein in its active ("on") state and examine the structure using a technique called X-ray diffraction. However, the structure of the inactive ("off") E. coli CRP eluded them as attempts to crystallize it repeatedly failed. With only the structure of the "on" state defined, the genetic switching mechanism remained a mystery.
The breakthrough was achieved when Gallagher; NIST colleagues Prasad Reddy, Natasha Smith and Sook-Kyung Kim; and BNL’s Howard Robinson substituted the CRP from Mycobacterium tuberculosis [the pathogen that causes tuberculosis] for the E. coli protein.
The team’s initial success—obtaining crystals of CRP in the "off" state—was dramatic given that no one had accomplished the feat in nearly three decades of trying with E. coli. But the real excitement came when the crystals were examined 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," Gallagher says. "In most two-subunit proteins, each subunit has the same conformation as the other."
Gallagher says that the NIST/BNL 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 [inactive state] to its symmetrical [active state] form."
Here’s video of NIST biochemist Travis Gallagher describing the study:
Press release: Long-Sought Protein Structure May Help Reveal How ‘Gene Switch’ Works NIST, Brookhaven Researchers Use Tuberculosis Bacteria to End 25-Year Quest
Abstract in J. Biol. Chem…
Image: Top: Computer model of the defined structure for the “off” state of the cyclic AMP receptor protein (CRP) found in Mycobacterium tuberculosis. The two subunits of the protein (colored purple on the left and green on the right) are genetically identical but, surprisingly were found to be asymmetric (different in shape) for the areas shown in white (top). This is the state of the CRP that is unable to activate genes necessary for the microbe’s survival. Bottom: After binding cyclic AMP molecules (the yellow bodies in the center), the CRP is believed to change its structure so that the two subunits (colored purple on left and green on right) become symmetrical (identical in shape). It is this state of the CRP that binds a gene (DNA) and activate it to carry out functions necessary for the microbe’s survival.