On the Road to Discovery
CHORI Scientist Elucidates Structure and Mechanism of the Bacillus stearothermophilus RecU Holliday Junction Resolvase
The crystal structure of RecU Holiday junction resolvase, the protein responsible for DNA repair and recombination in B. stearothermophilus bacteria, has just been reported by the group of Mark Jedrzejas, PhD, in CHORI’s Center for Immunobiology and Vaccine Development. This new high resolution crystal structure provided enough detail to reveal the critical location of two ions, which led to the ability to discover the protein’s entire mechanistic function.
“The earlier data didn’t have a scientific basis for how it worked,” says Dr. Jedrzejas. “We obtained this crystal structure, and from there were able to identify critical ions and model a complex with DNA substrate that shows how this protein actually works.”
The critical ions that provided the key to understanding were two magnesium ions on what Dr. Jedrzejas refers to as a mushroom-like structure with a positively charged stalk that appeared to be designed to fit exactly into mobile, four-strand, DNA structures called Holliday junction DNA.
Using the charge distribution on the surface of the protein as reference, Dr. Jedrzejas and his colleagues were able to fit together the RecU and Holliday junction DNA to obtain a complex.
"The moment we modeled the complex, we knew we were home," Dr. Jedrzejas says.
This was due to the location in the model of two water molecules and how they coordinated with the two previously elusive magnesium ions as well as to significant amino acid residues necessary for catalysis. All of these players were positioned in the model exactly where they needed to be in order to cleave DNA, the catalysis for which RecU is responsible.
"The model is consistent with one of the water molecules acting as a nucleophile and the other as a general acid," explains Dr. Jedrzejas. "The catalytic amino acid residues act as the general base, the general acid, and the Lewis acid. Structural modifications, or dynamic behavior, of both the protein and DNA are essential for catalysis."Knowing how the molecule functions is the first step to understanding potential ways of interfering with bacterial functionality involved in its reproduction.
Monday, May 16, 2011 11:33 PM