[SCAN] A structural overview of Pontin, Reptin and their complex(es)

Scan Seminar

Title: A structural overview of Pontin, Reptin and their complex(es)

Speaker: Pedro Matias

From: Industry and Medicine Applied Crystallography Lab, ITQB
 

Abstract:

Pontin, also known as RuvBL1 and Reptin, also known as RuvBL2 are highly conserved eukaryotic proteins belonging to the AAA+ family of ATPases, and closely related to the bacterial DNA helicase RuvB. They are ubiquitously expressed and have been associated with many cellular functions. In addition, a link was established between Pontin, Reptin and cancer, making these proteins interesting targets for anticancer drugs.

The crystal structure of human Pontin was determined in our lab in 2006. It is hexameric, formed by ADP-bound monomers, and each monomer contains 3 domains: domains I and III are involved in ATP binding and hydrolysis and are structurally similar to equivalent domains in the bacterial homolog RuvB. A sequence insertion that is intercalated into domain I folds into a novel domain II, unique among AAA+ proteins.

3D structures of Pontin/Reptin complexes were determined by other groups using electron microscopy. In the human and yeast complexes, Pontin and Reptin form a dodecameric complex consisting of two structurally distinct hexameric rings. It was suggested that both rings interact via domain II, but neither study settled the issue of whether the rings are homo- or hetero-oligomeric. However, a third EM study reported a single hetero-hexameric ring structure for the yeast Pontin/Reptin complex.

The first crystal structure of a Pontin/Reptin complex was published in 2011 by our lab. For crystallization purposes, domain II was truncated in both proteins. The structure revealed a dodecameric ring, formed by two heterohexamers interacting via the retained part of domain II. More recently, heterohexamers of Pontin and Reptin have been identified in yeast chromatin remodelling complexes INO80 and SWR1. It now appears that the conflicting structural results may reflect a true flexibility in complex formation by these proteins.

Pontin and Reptin are molecular machines that use the chemical energy of ATP hydrolysis to drive a mechanical motion to unwind double-stranded DNA. However, in vitro, isolated Pontin and Reptin show low ATPase and even lower helicase activities. Surprisingly, the domain II truncation resulted in a significant increase in the activity of both proteins.