Interacting chaperones: NMR and X-ray combine to unravel combined relationship

Combined effort

Researchers in the US have combined nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography to gain new insights into the way in which a member of the histone chaperone family of specialized proteins functions. The resulting three-dimensional structure of the histone chaperone Rtt106 and interactions could have applications under understanding gene silencing and the way DNA responds to damage.

"The interactions we described are important for gene silencing and the DNA damage response," explains structural biologist Georges Mer. "This is exciting because our newfound knowledge will help us better understand these fundamental cellular processes."

DNA is wound up in our cells in a structure called chromatin, which is comprised of proteins, the majority of which are histones. Associated with the histones are the histone chaperones, which work to control the assembly or disassembly of chromatin when DNA needs to be duplicated in cell division or repaired following damage. Aging, cancer and other diseases are thought to be linked to the histone chaperones going awry.

Researchers already knew that the histone chaperone Rtt106 assists in depositing histones - more specifically, a complex of the histones H3 and H4 - on to the replicating DNA. However, what was not clear was exactly how Rtt106 could do this, especially given that it does not possess any of the known functionality. Histone H3 is a protein that exists in a modified form in which one of its amino acid residues, the lysine 56, has been acetylated in its amino-terminal alpha-helix. Apparently, despite being the chaperone, Rtt106 does not have the requisite domain to "read" the acetylated lysine on histone H3. In other words, the chaperone lacks the necessary structure to recognise the histone and take it by the proverbial arm like any good chaperone would.

Data collection

The NMR spectra were collected at 25 Celsius on a Bruker Avance 700 MHz spectrometer, the team says, the solution NMR structure of the Rtt106 homodimerization domain (Rtt106DD) was determined using a simulated annealing-based protocol.

The NMR and X-ray data from the well-known "lab" yeast Saccharomyces cerevisiae, revealed to Mer and colleagues Dan Su, Qi Hu, Qing Li, James Thompson, Gaofeng Cui, Ahmed Fazly, Brian Davies, Maria Victoria Botuyan and Zhiguo Zhang, that Rtt106 possesses two novel domains that take on this role. "The 3D structure of Rtt106DD, determined using NMR spectroscopy, shows a previously undiscovered fold with each protomer adopting a V-shaped conformation consisting of two alpha-helices separated by a trans-proline residue," the team says.

The histone chaperone's homodimerization domain was then found to allow two molecules of Rtt106 to be linked so they can cooperate in binding histones H3 and H4. The other, the so-called PH domain, is responsible for sensing the acetylated lysine of H3 and further reinforces the interaction. The two domains thus work synergistically to allow Rtt106 to perform the role of chaperone efficiently. This is the first description of the specific association between a histone chaperone and a modified histone complex, the team says. Indeed, "This mode of specific association with a modified histone is fundamentally different from that of so-called histone mark reader domains [the more well known interaction]," the team says.