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Enzymatic mechanism decoded

Findings at the MFPL: enzyme BRE1 "shoots" protein ubiquitin to nucleosome
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(Vienna, 12-09-2016) Alwin Köhler’s group at the Max F. Perutz Laboratories of the MedUni Vienna and the University of Vienna has been investigating a „murder case“ centered on a “molecular bullet“ called Ubiquitin. This is a highly versatile protein, used in many cellular “guns“ for signaling purposes, including the nucleosome. In one example, ubiquitin is fired onto one particular nucleosome residue during the transcription process, which is equivalent to firing a shot while traveling on a train. The alleged „murderer“, which shoots the ubiquitin bullet, is an enzyme called Bre1.

There are about 20.000 protein-coding genes in humans, however, only a small number are activated in any given cell. To manage genetic information, nature has evolved a sophisticated system that facilitates access to specific genes relying on a DNA-histone protein complex called the nucleosome. These efficiently package the genetic information into a fiber called chromatin, which upon modification can open or close, activating or silencing genes. Countless human diseases are linked to the mutation, loss, and aberrant regulation of genes, making it important to understand chromatin biology.

The „crime“ (histone H2B ubiquitination on Lysine 123) had already been described in 1980, but the molecular mechanism had remained unclear. In their recent publication in PNAS, Alwin Köhler’s group could show how Bre1 (the murderer) recognizes the nucleosome (the victim), how Bre1 aims at the victim’s heart (histone H2B Lysine 123) and how the bullet (ubiquitin) is fired with such precision. This mechanistic understanding was the result of cross-linking and mass spectrometry studies that permitted biochemical capture of the transient Bre1-nucleosome interaction and elucidation of the precise topology at the interface.

Footprints at a crime scene can reveal direction, speed, the number of people, sex, and whether the individual knows that he/she is being tracked. Bre1 left a defined footprint at the „crime scene“, which allowed the investigators to reconstruct precisely how the „bullet“ was fired into the „victim’s heart“, or put scientifically, the enzymatic mechanism of the reaction. Besides taking a snapshot of the footprint by cross-linking and mass spectrometry, this reconstruction was the result of a painstaking reenactment of the crime. Laura Gallego, a PhD student and first author of the study, explains that the biochemical reconstitution of the enzymatic mechanism with purified protein components, was the bottleneck of the entire investigation. This reconstitution allowed the team to define what was necessary and sufficient for the crime to happen, which could only be done outside of the complicating environment of the cell. Close collaborations with other experts at the Gene Center in Munich, the neighboring Institute of Molecular Pathology, the Max F. Perutz Laboratories and the Howard Hughes Institute at the University of Washington in Seattle, USA, were essential for the success of the investigation.
Solving the mechanism behind the perfect ubiquitin shot on the nucleosome is a key step forward, but the case is not yet closed. Bre1 is likely assisted by several accomplices so the team is now looking for further telling footprints on chromatin.

Structural mechanism for the recognition and ubiquitination of a single nucleosome residue by Rad6–Bre1
Laura D. Gallego, Medini Ghodgaonkar Steger, Anton A. Polyansky, Tobias Schubert, Bojan Zagrovic, Ning Zheng, Tim Clausen, Franz Herzog, and Alwin Köhler
PNAS 2016; published ahead of print September 6, 2016, doi:10.1073/pnas.1606863113