The decades-old question of how biological expression is controlled -- how genes are switched on and off has some surprising new answers from high-resolution x-ray structural studies carried out at UMBI's Center for Advanced Research in Biotechnology (CARB), in a partnership involving collaborating scientists from the National Institute of Standards and Technology (NIST) and Brookhaven National Labs (BNL). Results of this study were recently published online in the Journal of Biological Chemistry (http://www.jbc.org/cgi/content/abstract/C800215200v1 ).

The team reports that it has defined----for the first time----the structure of the off state of a metabolic switch found inside most bacteria----the cyclic AMP (cAMP) receptor protein, or CRP. CRP is the binding site (attachment point) for cAMP, a small molecule, that once attached, serves as the signal to throw the switch. When the protein is switched to its on state, the CRP-cAMP complex can bind to specific DNA sequences, activating the expression of genes to which it binds. These genes control the microbe's ability to metabolize sugars other than glucose, when the quantity of that nutrient is insufficient to keep the cell alive.

A key to this control mechanism has been revealed by a newly determined structure of the CRP protein from the pathogen Mycobacterium tuberculosis, the bacterium which causes tuberculosis (TB). Since such control mechanisms often affect the virulence of pathogens, these findings may have impact in the fight against TB, which infects about 1/3 of the world's population, killing 5,000 people per day. The researchers hope that once the switching mechanism is understood, the data can be used to develop new methods for preventing tuberculosis and other pathogenic bacterial diseases.

(Diagram showing the unusual asymmetry in the 2 subunits with identical amino acid sequences of the CRP protein from M. Tuberculosis)

For example, many pathogenic bacteria use cAMP as a signal for switching on genes for virulence factors and toxins, or for enhancing survival in a human host, says Dr. Travis Gallagher, who is first author on the Journal of Biological Chemistry paper. Blocking this process might provide ways to shut down infections and save lives.

The biochemical puzzle surrounding CRP is the mechanism by which the protein binds cAMP at one end, then attaches to----and activates----the gene (strand of DNA) at the other end. Believing that the protein somehow changes its overall shape during the process, 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 shape change occurs. Unfortunately, the task proved to be extremely difficult.

Using CRP from the bacterium Esherichia coli, the standard model system for bacterial studies, early researchers were able to crystallize and examine the structure in only its active on state, with cAMP bound to it. However, the structure of the inactive E. coli CRP (without bound cAMP) eluded the researchers, as attempts to crystallize it repeatedly failed. With only the structure of the active state defined, the genetic switching mechanism remained a mystery.

The breakthrough was achieved when NIST researchers Gallagher, Prasad Reddy, Natasha Smith and Sook-Kyung Kim, in collaboration with BNL's Howard Robinson, substituted the CRP from Mycobacterium tuberculosis (the pathogen that causes tuberculosis) for the E. coli protein. By turning to M. tuberculosis instead of the usual bacterial workhorse E. coli, the scientists, working at UMBI, were able to crystallize the protein structure----in its inactive state-- that had defied investigators for 25 years. Once crystallized, the protein in its inactive state was subjected to X-ray diffraction at the powerful synchrotron of the Brookhaven Institute, a particle accelerator that is onekilometer in circumference. The resulting measurements could then be analyzed, leading to determination of the shape of the CRP protein in its inactive off state.

The transcription factor CRP (cyclic-AMP receptor protein) activates many genes in response to binding cyclic-AMP (cAMP), in a way that is similar to hundreds of similar transcription factors in other organisms. CRP from E. coli has for decades been the best-studied model system for widespread research on how these proteins function. While it is well known that CRP binds cAMP at one end and then attaches to (and activates) DNA at the other, how the cAMP binding signal is transferred structurally from one end of the protein molecule to the other has remained a mystery.The prevailing hypothesis is that cAMP binding somehow changes the shape and structure of the protein so that it is able to bind to target sequences of DNA.

When the images from the inactive CRP of M. tuberculosiswere obtained and analyzed, a surprising result was found. Although the protein consists of two subunits with identical amino acid sequences, the two subunits adopt strikingly different shapes when they are in the inactive state, making the protein sharply asymmetric. This was a very unusual observation, as proteins like CRP that contain two identical sequences are nearly always very symmetric. In this case, the asymmetry in the inactive state is most likely related to how the protein carries out its function, i.e., the asymmetric shape appears to prevent CRP from binding DNA when it is in its off state, in the absence of bound cAMP.

To further this work, and reveal the details of how CRP switches from its asymmetric/inactive state to its symmetric/active conformation, current efforts are now focused on imaging the same M. tuberculosis CRP protein in its active state, so that the two states can be compared. The goal is to understand the dynamics of the shape change that takes place during the switching process.